Embed Size (px)
Citation preview
Synthesis and Applications of Polyfunctional Hydrogenated
Pyridines and other Thiolated Ligands Stabilized Metal
based Nanoparticles
Submitted by
Ayaz Anwar
Dissertation for the Partial Fulfilment of the Degree of
Doctor of Philosophy
H. E. J. Research Institute of Chemistry
International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270,
Pakistan 2016
i
Certificate
To whom it may concern
It is certified that the thesis entitled, Synthesis and Applications of Polyfunctional
Hydrogenated Pyridines and other Thiolated Ligands Stabilized Metal Based
Nanoparticles, submitted to the Board of Advance Studies and Research, University of
Karachi, by Mr. Ayaz Anwar, satisfies the requirements for the Ph.D. degree in
Chemistry.
Dr. Muhammad Raza Shah,T.I. Research Supervisor
H. E. J. Research Institute of Chemistry,
International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan.
ii
iii
DEDICATED TO
MY PARENTS AND MY FAMILY WHO ABETTED
ME IN THE BAFFLING COURSE OF MY
RESEARCH CORDIALLY AND EARNESTLY.
Acknowledgement
iv
ACKNOWLEDGEMENTS
I am thankful to Almighty Allah who grants me the power and courage tofulfil all my tasks. I
would like to dedicate and acknowledge this dissertation to my parents, and my siblings for
their constant prayers, encouragement and everlasting patience.
I am very thankful to Prof. Dr. Saleemuz Zaman Siddiqui (Late) founder HEJRIC, Patron in
Chief ICCBS, Prof. Dr. Atta ur Rahman FRS, (N.I, H.I., S.I., T.I.) and Director ICCBS, Prof. Dr. M.
Iqbal Choudhary(H.I., S.I., T.I.), for their wonderful leadership.
It is indeed my greatest pleasure to further extend my gratitude to my mentor and supervisor,
Dr. Muhammad Raza Shah (T.I.), for his constant support, encouragement and advices
throughout my Ph.D. studies as well as in my personal issues. He is a source of inspiration for
me to accomplish this task and no doubt my role model. His valuable efforts in encouraging me
to achieve my goals with a helping and kind heart made it possible for me. I cannot thank him
enough for his support throughout every day of my stay in his wonderful research group.
I would like to extend my thanks to all the collaborating groups. Dr. Massimo F. Bertino of
Virginia Commonwealth University USA, Dr. Yousuf Aqeel, and Dr. Naveed Ahmed Khan of
Aga Khan University, Dr. Fatima Basha, and extremely thankful to Dr. Abdul Hameed of
ICCBS for his guidance and mentorship in the synthesis of dihydropyridine derivatives for their
valuable support in my research. Very special thanks to all the faculty members of ICCBS who
laid my research foundation during the course work. I would also like to acknowledge all the
technical and non-technical staff of ICCBS.I am very much thankful to Mr. Amir for his
support in timely arrangements of research materials.
I would like to acknowledge and pay my special thanks to all my lab fellows and friends in
particular, Dr. Nadeem, Dr. Qamar, Dr. Akhtar, Dr. Zahid, Dr. Farrukh, Kiramat, Burhan, Dr.
Ateeq, Mehdi, Mumtaz, Tahir, Ambreen, Anwar Shamim, Farid, Ishfaq, Afifa, Wasia, Noor,
Azeem, Saira, Faiza, Dania, Sadia, Sadia Khan, Madiha, Imkaan, Imdad.
Special thanks to my friends Hamid, Umair, Afifa, Farid, Ishfaq, Zahid, M. Salman, Mateen,
Junaid, Saquib, Comail, Ateeq, Shabbir, Nusrat, all class fellows, all LPO members, and my
cousins Waqar, Danish, Uzair,and many more for their moral supports and appreciations.
At last but not the least, I would like to thanks all the members of Mass, NMR, and Analytical
instrumental lab of ICCBS.
Ayaz Anwar, Karachi, 2015
CV
v
CURRICULUM VITAE
I was born in a small extended town of Karachi, Pakistan called Landhi on the 4th of
January 1988. I gained my school education from a nearby school named Sohail
Academy. After Matric, I joined Urdu Science College Gulshan e Iqbal for F. Sc. and
somehow managed to pass the intermediate education. I then took my studies a little
more serious, and I realized that Chemistry is the subject which fascinates me and I
decided to learn the Chemistry of the highest levels right then. So I graduated in
Chemistry, Mathematics and Physics from Jamia Millia Government Degree College
Karachi in 2007. Later on, I joined the University of Karachi for postgraduate studies in
Organic Chemistry, where received M.Sc. degree with distinction on 2010. After the
exams, I had a great opportunity to join the summer internship program of ICCBS, where
I had my first exposure of Chemistry research. In March 2011, I joined the H.E.J.
Research Institute of Chemistry, International Centre for Chemical and Biological
Sciences, University of Karachi, for Ph.D. studies in Chemistry. I enjoyed research
activities in the field of Organic Synthesis and nanotechnology under the supervision of
Dr. Muhammad Raza Shah, mainly devoted to the nanoparticles based studies of metal-
coated synthetic ligands for enhanced photophysical and biological applications. I feel
myself very lucky to reach this day. It has been an extraordinary life so far, I enjoy
playing and watching football, watching movies and listening music.
Table of Contents
Certificate ...................................................................................................................... i Dedication ................................................................................................................... iii Acknowledgement ...................................................................................................... iv Personal Introduction ................................................................................................... v List of figure ................................................................................................................ x List of schemes ........................................................................................................... xv List of tables ............................................................................................................... xvi List of abbreviations ................................................................................................. xvii Summary ................................................................................................................. xviii Khulasa ..................................................................................................................... xix
PART I ........................................................................................................................ 1
1.1.1 Dihydropyridines ............................................................................................... 1
1.1.2 Porphyrin............................................................................................................ 5
1.1.2.1 Nomenclature .................................................................................................. 6
1.1.2.2 Synthetic Methodologies of Porphyrins.......................................................... 7
1.1.3 Thiolated Cationic Heterocyclic Ligands for Stabilization of Metal Nanoparticles ............................................................................................................ 12
1.2 RESULTS AND DISCUSSION ........................................................................... 13
1.2.1 Synthesis and Applications of Polyfunctional Hydrogenated Pyridines from Acetophenone Derivatives ........................................................................................ 13
1.2.1.1 Urease Activity and Structural Activity Relationship .................................. 15
1.2.1.2 Anticancer Activity and Structural Activity Relationship ............................ 17
1.2.1.3 DPPH Radical Scavenging Activity ............................................................. 17
1.2.2 Synthesis of Mannose Conjugated Porphyrin .................................................. 20
1.2.2.1 Synthesis of Tetraphenylporphyrin (TPP) (7)............................................... 20
1.2.2.2 Nitration of TPP (27) .................................................................................... 21
1.2.2.3 Mechanism of Nitration of Meso-H2TPP ..................................................... 22
1.2.2.4 Reduction of nitro group of MNPP ............................................................... 23
1.2.2.5 Mechanism of Nitro group of Reduction ...................................................... 24
1.2.2.6 Conjugation of MAPP with Mannose ........................................................... 25
vi
1.2.2.7 Antibacterial Study of Mannose Conjugated Porphyrin via Photochemotherapy................................................................................................... 26
1.3 EXPERIMENTAL ................................................................................................ 33
1.3.1 General Procedure for the Synthesis of Polyfunctional DHP Derivatives ...... 33
1.3.2 Urease Inhibition Assay (In vitro) ................................................................... 38
1.3.3 Anticancer Activity Assay (In vitro) ................................................................ 38
1.3.4 DPPH (1,1-Diphenyl-2-Picryl Hydrazyl) Free Radical Scavenging Assay (In vitro) .......................................................................................................................... 39
1.3.5 Acanthamoeba castellanii and Microvascular Endothelial Cells Cultures ...... 40
1.3.6 Synthesis of Mannose Conjugated Porphyrin .................................................. 40
1.3.7Amoebicidal Assays.......................................................................................... 41
1.3.8 Cytotoxicity assays of Host Cells .................................................................... 42
1.3.9 Excystation Assays .......................................................................................... 42
1.3.10 Synthesis of thiolated heterocyclic ligands .................................................... 43
References ................................................................................................................... 47 PART II ..................................................................................................................... 55
SYNTHESIS OF METAL NANOPARTICLES AND THEIR APPLICATIONS .... 55 2.1 INTRODUCTION ................................................................................................ 55
2.1.1 Introduction of Nanotechnology ...................................................................... 55
2.1.2 Fullerenes ......................................................................................................... 55
2.1.3 Carbon Nanotubes ............................................................................................ 56
2.1.4 Nanoparticles ................................................................................................... 57
2.1.5 Nanoclusters ..................................................................................................... 59
2.1.6 Synthesis of Nanoparticles and Nanoclusters .................................................. 60
2.1.6.1 Bottom-up Approach .................................................................................... 60
2.1.6.2 Top-down Approach ................................................................................... 60
2.1.7 Dispersity in Nanoscale Synthesis ................................................................... 60
2.1.8 Applications of Nanoparticles.......................................................................... 60
2.1.9 Nanoclusters in Biomedical Sciences .............................................................. 61
2.1.9.1 Biological Imaging........................................................................................ 61
2.1.9.2 Biodetection .................................................................................................. 62
vii
2.1.10 Chemosensing of Cu(I) .................................................................................. 63
2.1.11 Chemosensing of Fe(III) ................................................................................ 63
2.1.12 Chemosensing of Pd(II) ................................................................................. 64
2.1.13 Chemosensing of Antibiotics ......................................................................... 66
2.2 RESULTS AND DISCUSSION ........................................................................... 69
2.2.1.1 Synthesis and Characterization of Bisphosphine Gold Nanoclusters ........... 69
2.2.2 Atomic Force Microscopic guided detection of Copper in real samples and T-lymphocytes with Pyridinium Thioacetate capped Silver Nanoparticles ................. 82
2.2.2.1 Synthesis of 4-formylpyridiniumpropylthioacetate Coated Silver Nanoparticles (4-Py-AgNPs) .................................................................................... 82
2.2.2.2 Characterization of 4-Py-AgNPs .................................................................. 82
2.2.2.3 Chemosensing of Cu(I) by 4-Py-AgNPs....................................................... 85
2.2.2.4 Intracellular Cu(I) detection in T-lymphocytes: ........................................... 96
2.2.2.5 Chemosensing of 6-AminopenicillanicAcid by 4-Py-AgNPs .................... 104
2.2.2.6 Antimicrobial Activity of 4-Py-AgNPs ...................................................... 112
2.2.3 Synthesis of Pyrazinium Thioacetate Gold Nanoparticles Pyr-AuNPs and their Applications ............................................................................................................ 117
2.2.3.1 Synthesis of Pyr-AuNPs ............................................................................. 117
2.2.3.2 Characterization of Pyr-AuNPs .................................................................. 117
2.2.3.3 Photophysical Applications of Pyr-AuNPs ................................................. 118
2.2.4 A New Pefloxacin Chemosensor Based on Thioacetate Functionalized DMAP coated Gold Nanoparticles ...................................................................................... 135
2.2.4.1 Synthesis of Dimethylaminopyridinium Propylthioacetate (D2) Stabilized Gold Nanoparticles (D2-AuNPs) ............................................................................ 135
2.2.4.2 Stability of D2-AuNPs ................................................................................ 137
2.2.4.3 Exploration of Chemosensing Potential of D2-AuNPs .............................. 139
2.2.4.4 Effect of the pH on the Stability of ................... D2-AuNPs and Pef Response................................................................................................................................. 140
2.2.4.5 FT-IR Spectral Analysis ............................................................................ 141
2.2.4.6 AFM investigation of the interaction between D2-AuNPs and Pef ......... 141
2.2.4.7 Analytical Performance of the Pef Nanosensor .......................................... 142
viii
2.2.4.8 Interference Study ....................................................................................... 144
2.2.4.9 Analysis of Pef in Real Samples ................................................................. 145
2.3 EXPERIMENTAL .............................................................................................. 147
2.3.1 Materials ........................................................................................................ 147
2.3.2 Instruments ..................................................................................................... 147
2.3.3 Synthesis of Bisphosphines Coated Gold Nanoclusters ................................ 148
2.3.4 Synthesis of 1-(3-(acetylthio)propyl)-4-formylpyridin-1-ium (4-Py) Coated Silver Nanoparticles (4-Py-AgNPs) ........................................................................ 148
2.3.5 Synthesis of 1-(3-(acetylthio)propyl)pyrazin-1-ium (Pyr) Coated Gold Nanoparticles (Pyr-AuNPs) .................................................................................... 148
2.3.6 Synthesis of 1-(3-(acetylthio)propyl)-4-(dimethylamino)pyridin-1-ium (D2) Coated Gold Nanoparticles (D2-AuNPs) ................................................................ 149
2.3.7 Synthesis of Chlorhexidine (CHX) Coated Gold Nanoparticles (CHX-AuNPs)................................................................................................................................. 149
2.3.8 Synthesis of Neomycin (Neo) Coated Silver Nanoparticles (Neo-AgNPs)... 149
2.3.9 Synthesis of Guanidinobutyric Acid (GBA) Coated Silver Nanoparticles (Neo-AgNPs) .................................................................................................................... 149
2.3.10 Cell Incubation ............................................................................................. 150
2.3.11 Cell Treatment ............................................................................................. 150
2.3.12 MTT Based Cytotoxicity Assay................................................................... 150
2.3.13 Minimum Inhibitory Concentration by Agar Well Diffusion Method ........ 151
2.3.14 Atomic Force Microscopy Imaging ............................................................. 151
REFERENCES ......................................................................................................... 153 Conclusion ............................................................................................................... 177 Publications .............................................................................................................. 178
ix
x
List of Figures
Figure: 1.1 Structures of Nifedipine (1) and Lercanidipine (2) 1
Figure: 1.2 Comparative structures of our DHPs and some reported analogues
with drugs
4
Figure: 1.3 The tautomerization of porphyrins 6
Figure: 1.4 Typical structure of simplest porphyrin- porphyne 6
Figure: 1.5 Fischer (left) and IUPAC (right) nomenclature systems for
porphyrins
7
Figure: 1.6 X-rays crystallographic structures of compounds 9 and 11 14
Figure: 1.7 NMR spectrum of MNPP (27) 22
Figure: 1.8 EI-MS spectrum of MAPP (28) 24
Figure: 1.9 NMR spectrum of mannose conjugated porphyrin (29) 25
Figure: 1.10 Mannose-conjugated porphyrin (mann. por.) exhibited
amoebicidal effects against A. castellanii.
27
Figure: 1.11 A. castellanii was incubated with and without 50 µM mannose
conjugatedporphyrin, and representative images were obtained at 100 x (A)
and at400 x (B) magnification using a phase-contrast inverted microscope.
28
Figure: 1.12 (A). A. castellanii (5 x105) preincubated with and without 50
µM mannose-conjugated porphyrin (mann. por.) and exposed to light. (B). A.
castellanii (5 x105) were coincubated with human BMEC and 50µM
mannoseconjugatedporphyrin and exposed to light.
30
Figure: 1.13 Mannose-conjugated porphyrin inhibited excystation of A.
castellanii cysts. A. castellanii cysts were scraped from nonnutrient agar
plates, incubated with 50µM mannose porphyrin, and exposed to light.
31
Figure: 2.1 Structure of a Fullerene 56
xi
Figure: 2.2 Depiction of CNTs 57
Figure: 2.3 (A) 1,6-bis(diphenylphosphino)hexane denoted by L6, (B)
Xantphos denoted by LAr, (C) Binap. LBn, (D) Biphenyl denoted by LBp,
(e) Diphenylphosphinoacetylene denoted by LAc
69
Figure 2.4 UV-Visible Spectrum of L6 Au Clusters in all solvents 71
Figure: 2.5 MALDI spectra of L6 obtained in all solvents 72
Figure: 2.6 UV-Vis spectrum of LAr Au Clusters in all solvents used 73
Figure 2.7 MALDI Spectral Ananlysis of LAr Ligated Au Clusters in
different solvents
75
Figure: 2.8 UV-Vis Spectra of LAc Au Clusters in all Solvents 76
Figure: 2.9 MALDI Spectral Ananlysis of LAc Ligated Au Clusters in
different solvents
77
Figure: 2.10 UV-Vis Spectra of LBn Au Clusters in all solvents 78
Figure: 2.11 MALDI Analysis of LBn Au Clusters in all Solvents 79
Figure: 2.13 MALDI Analysis of LBp Au Clusters in all Solvents 81
Figure: 2.14 UV-visible spectra of the AgNPs 83
Figure: 2.15 UV-visible spectra of the AgNPs solution at room temperature
and boiling point
84
Figure: 2.16 UV-visible spectra of salt addition stability of AgNPs 85
Figure: 2.17 A) UV-visible spectra of metal cations after titrations with
nanoparticles. B) Bar graph representation of metal cations after titrations
with nanoparticles at λmax
86
Figure: 2.18: A) FT-IR spectrum of 4-Py AgNPs 87
Figure: 2.18 B) FT-IR spectrum of 4-Py AgNPs after treating with Cu(I) 88
Figure: 2.19 a) Topographical AFM image of 4-Py AgNPs b) Topographical
AFM image of AgNPs after mixing with Cu(I)
89
xii
Figure 2.20 Kinetic studies after interaction of Cu(I) with4-Py AgNPs 90
Figure: 2.21: Effect of pH on the selective recognition of Cu(I) by AgNPs. 91
Figure: 2.22 A) Selective recognition of Cu(I) by 4-Py AgNPs at different
concentrations B) Linear regression curve obtained after treating varies
concentration of Cu(I) with 4-Py AgNPs.
92
Figure: 2.23 A) Absorption spectra of 4-Py AgNPs+Cu(I) in presence of
other competing metal cations B) bar graph representation Absorption spectra
of 4-Py AgNPs+Cu(I) in presence of other competing metal cations
93
Figure: 2.24 Complexation ratio between 4-Py AgNPs and Cu(I) 94
Figure 2.25 UV-visible spectra of 4-Py AgNPs recorded in presence Cu(I)
spiked in (A) Human plasma (B) Tap water
95
Figure: 2.26 A) T cell (Control) 98
Figure: 2.26 B) 0.5 μM 4-Py AgNPs treated T cell 98
Figure: 2.26 C) 0.5 μM 4-Py AgNPs treated T cell with 5 μMCu (I), cell
surface is shrinked due to interactions.
99
Figure: 2.26 D) 0.5 micromolar 4-Py AgNPs treated T cell with 10
micromolar Cu (I)
100
Figure: 2.27 A) 1 micromolar 4-Py AgNPs treated T cell with 5 micromolar
Cu (I)
101
Figure: 2.27 B) 1 micromolar 4-Py AgNPs treated T cell with 30 micromolar
Cu (I)
101
Figure: 2.28 A) 10 micromolar 4-Py AgNPs treated t cell 102
Figure: 2.28 B) 10 micromolar 4-Py AgNPs treated t cell with 5 micromolar
copper
102
Figure: 2.28 C) 10 micromolar 4-Py AgNPs treated t cell with 50 micromolar
copper
103
Figure: 2.29 Drugs screening study 106
xiii
Figure: 2.30 Morphological change in the nanoparticles after interaction with
6-APA
107
Figure: 2.31 Effect of pH on the stability of 4-Py AgNPs and accumulation of
nanoparticles with 6-APA
108
Figure: 2.32 A) UV-visible spectra of concentration study, B) Calibration
curve and C) Job’s plot
110
Figure: 2.33 Effect of interfering drugs on 6-APA quenching 111
Figure: 2.34 6-APA quenching in Human blood plasma sample 112
Figure: 2.35 MIC of ligand in comparison with ligand stabilized 4- Py-
AgNPs in the presence of Cu(I).
114
Figure: 2.36 A) E. coli Control. B) E. coli after treating Cu(I) 116
Figure: 2.37 Ligand treated E. coli at A) 4 hours. B) 8 hours 116
Figure: 2.38 4- Py-AgNPs treated E. coli at A) 4 hours. B) 8 hours 116
Figure: 2.39 UV-visible spectrum of Pyr-AuNPs 117
Figure: 2.40 UV-visible spectra of metal salts screening 119
Figure: 2.41 Buffer stability of AuNPs with and without Fe(III) 120
Figure: 2.42 Comparative FT-IR spectra of Pyr-AuNPs before and after
mixing of Fe(III)
121
Figure: 2.43 AFM images of AuNPs alone and of AuNPs treated with Fe(III) 122
Figure: 2.45 Effect of interfering metal salts on the Fe(II) sensing 124
Figure: 2.46 Fe(III) sensing in drinking water and human blood plasma 126
Figure: 2.47A) Pictorial display of colorimetric sensing of Pd(II), B) UV-
visible spectrum of salt screening
128
Figure: 2.48 pH stability of AuNPs with and without Pd(II) 129
Figure: 2.49 A) AFM image of AuNPs, B) AFM image of AuNPs after
mixing with Pd(II)
130
xiv
Figure: 2.50 A) UV-visible spectral profile of the concentration study, inset
represents calibration curve, B) Job’s plot
132
Figure: 2.51 Effect of interfering metal ions on Pd(III) sensing 133
Figure: 2.52 UV-visible spectra of Pyr-AuNPs after mixing with A) tap
water, B) Human blood plasma spiked with Pd(II)
134
Figure: 2.53 A) UV-Visible spectrum of the AuNPs stabilized with D2, B)
AFM image of the D2 capped AuNPs, C) Size distribution histogram of D2-
AuNPs
137
Figure: 2.54 A) Effect of heating on SPB, B) Aggregation of D2-AuNPs
induced by various concentration of NaCl
138
Figure: 2.55 UV-Visible spectral profile generated after mixing drugs with
D2-AuNPs
139
Figure: 2.56 Effect of pH on the interaction of D2-AuNPs with Pef 140
Figure: 2.57 AFM image of the D2-AuNPs -Pef 1:1 mixture 142
Figure: 2.58 A) Concentration study of Pef with D2-AuNPs, B) Calibration
curve at 700 nm C) Job’s plot for binding ratio
144
Figure: 2.59 Effect of competing drugs on Pef response towards nanosensor 145
Figure: 2.60 UV-visible spectra of Pef recognition in (A) tap water (B)
Human blood plasma
147
xv
LIST OF SCHEMES
Scheme 1.1 Synthesis of meso-H2TPP (7) by Alder-Longo Method. 9
Scheme 1.2 Synthesis of meso-H2TPP (7) by Lindsey Method. 10
Scheme 1.3 Synthesis of meso-H2TPP(8) by MacDonald method. 10
Scheme 1.4 Proposed Mechanism of synthesis of meso-H2TPP. 11
Scheme 1.5 Synthetic methodology of the target DHPs. 13
Scheme 1.6 Plausible mechanism for the dimerization of alkylidene
malononitrile to give DHP.
15
Scheme 1.7 Synthesis of meso-H2TPP (7). 20
Scheme 1.8 Synthesis of MNPP (27). 21
Scheme 1.9 Mechanism of nitration of meso-H2TPP. 22
Scheme 1.10 Reduction of MNPP into MAPP. 23
Scheme 1.11 Proposed mechanism of reduction. 24
Scheme 1.12 Synthesis of Mannose conjugated Porphyrin (29). 25
Scheme 1.13 Synthesis of 1-(3-(acetylthio) propyl)-4-formylpyridinium (4-
Py)(31)
43
Scheme 1.14 Synthesis of 1-(3-(acetylthio)propyl)pyrazin-1-ium (Pyr) (33) 44
Scheme 1.15 Synthesisof1-(3-(acetylthio)propyl)-4-(dimethylamino)
pyridin- 1-ium (D2) (34)
45
xvi
LIST OF TABLES
Table 1.1 In vitro urease activity of DHPs. 16
Table 1.2 Anticancer activity of DHPs against PC3 and HeLa Cell line. 18
Table 1.3 DPPH free radical scavenging activity. 19
Table 1.4 Structures and spectroscopic data of synthesized DHPs. 34
Table 2.1 Zone of inhibition data at multiple dose of Cu(I) accumulation. 113
xvii
LIST OF ABBREVIATIONS
AuNPs Gold Nanoparticles
AgNPs Silver Nanoparticles
SPB Surface Plasmon Band
AFM Atomic Force Microscopy
DHP Dihydropyridine
HMDS Hexamethyl disilazane
TBAF Tetra-n-butyl ammonium fluoride
4-Py 1-(3-(acetylthio)propyl)-4-formylpyridinium
Pyr 1-(3-(acetylthio)propyl)-4-(dimethylamino)pyridin-1-ium
D2 1-(3-(acetylthio)propyl)-4-(dimethylamino)pyridin-1-ium
MALDI Matrix Assisted Laser Desorption/Ionization
FTIR Fourier Transform Infrared
IR Infrared
ESI Electrospray Ionization
EI Electron Impact
MS Mass spectrometry
NMR Nuclear Magnetic Resonance
PE Petroleum Ether
EA Ethyl acetate
DMSO Dimethyl Sulfoxide
NaOH Sodium Hydroxide
nps Nanoparticles
APA 6-Aminopenicillanic acid
Pef Pefloxacine
xviii
SUMMARY
This Ph. D. dissertation, deals with the synthesis of heterocyclic thiolated ligands,
stabilization of metal nanoparticles and their photophysical and biological applications. The
ligands explored in this study include derivative of Pyridines, Dihydropyridines, Pyrazine,
and Porphyrin. Chemical synthesis are monitored and characterized by1HNMR, Mass
spectrometry, FT-IR spectrophotometry, while the nanomaterials were studied by UV-visible
spectrophotometry, and AFM. The antimicrobial potential of synthesized ligand and
nanoparticles were also studied comprehensively. As a summary of the research conducted
during the course of Ph. D., a series of novel dihydropyridine derivative was synthesized and
their cytotoxicity, enzyme inhibition and antioxidant activity were studied. Para-methoxy
dimer of alkylidene malononitrile is found to be most active in in vitrourease inhibition
assay, while para-bromo analogue is found to be significantly cytotoxic against two cell lines.
Moreover, 1-(3-(acetylthio) propyl)-4-formylpyridinium (4-Py), 1-(3-(acetylthio)propyl)
pyrazin-1-ium (Pyr), 1-(3-(acetylthio)propyl)-4-(dimethylamino)pyridin-1-ium (D2) were
synthesized as thiolated capping agents for stabilization of metal nanoparticles particularly
Silver and Gold. 4-Py coated AgNPs were successfully synthesized and used as chemosensor
for detection and quantification of Cu(I) ions and 6-Aminopenicillanic acid detection in
aqueous media in the presence of other interfering species. Pyr capped AuNPs were
synthesized, which colorimetrically sensed Pd (II)in the presence of other metal ions by
inducing hypochromic shift in the surface plasmon band, and Fe(III) ions by quenching of
SPR band. D2 stabilized gold nanoparticles were synthesized, which exhibited selective
recognition of Pefloxacin mesylate by showing a substantial red shift in the SPR band. The
antibacterial potential of 4Py-AgNPs was also explored and compared with uncoated ligand
against E. coli, and the morphological studies were carried out via AFM. Silver conjugated 4-
Py displayed two times enhanced anti-E. coli activity as compared to pure compound, while
the intracellular Cu(I) detection was also successfully achieved, which can be used to reduce
the copper poisoning in biological systems. In another study, a unique selective
Photochemotherapic agent mannose conjugated porphyrin was synthesized and used against
the Acanthamoeba via in vitro studies. Three antibiotics Chlorhexidine, Neomycin, and
guanidine butyric acid were also decorated on metal nanoparticles surface to study their
antibacterial activities.
xix
Part I
1
CHAPTER 1
SYNTHESIS OF HETEROCYCLIC ORGANIC LIGANDS AND
THEIR BIOLOGICAL APPLICATIONS
1.1 INTRODUCTION
1.1.1 Dihydropyridines
Alkylidenemalononitriles are important targets and intermediates compounds in synthetic
and bioorganic chemistry. These are synthesized from acetophenone and malononitrile
derivatives via Knoevenagel condensation (Barnes, Haight et al. 2006) and are often used
in the preparation of many medicinally vital heterocyclic compounds. Pyridines are also
an important class of the heterocyclic compounds, which are commonly used in many
fields including medicinal chemistry, agro chemicals etc. (Bogdanowicz-Szwed and
Krasodomska 2006). Pyridine ring is the core in many known natural products and
medicinally important compounds, for example, nifedipine and its different derivatives
(Johnson and Li 2007) (Fig.1.1). These compounds showed significant biological
activities against cardiovascular diseases and act as calcium antagonist (Lichitsky,
Dudinov et al. 2001). Many research groups are recently involved in the synthesis of
useful pyridine derivatives owning to their significant biological potential. Based on these
properties and recent research reports, we aimed to develop an efficient and cost-effective
method for the synthesis of potentially useful pyridine derivatives. It is predicted that
preparation of poly functional 5, 6-pyridine derivatives would carry significant biological
activities.
1 2
Figure: 1.1 Structures of Nifedipine (1) and Lercanidipine (2)
Part I
2
Readily available alkylidenemalononitriles were selected as suitable precursor due to the
reactive nature of the dicyanoolefin and the acidic nature of the methyl group for the
dimerization reaction to synthesize desired 5,6-dihydropyridine derivatives 9-26
(Table1.4). Previously, (Dunkel, Hess et al. 1997) reported the synthetic strategy to
produce different dicyano substituted dimers by using DIMCARB (dimethylamine
carbondioxide complex), while (Abdelrazek, Kassab et al. 2010) carried out the
dimerization of phenyl alkylidenemalononitrile in the presence of NaOEt in ethanol.
Alkylidenemalononitriles can be formed by knoevenagel condensation of acetophenones
and malononitrile. There are several reported methods for this purpose (Knoevenagel
1898; Barnes, Haight et al. 2006). For example:
However, we have developed a novel one-pot solvent-free methodology for the synthesis
of alkylidenemalononitrile and their subsequent conversion into DHP derivative via
dimerization. The reaction is carried out in neat TBAF.3H2O (Tetrabutylammonium
fluoride trihydrate) to prepare highly functionalized compound 9-26 (Table 1.4). These
conditions are green as no organic solvent is used, eco-friendly, facile and rapid. We have
explored this optimized method on a variety of different substituted alkylidenemalononitriles
analogues and synthesized 16 different dihydropyridine derivatives.
Part I
3
TBAF is commonly used for deprotection of silyl group from silyl ethers in organic
reactions (Corey and Snider 1972). However, recently several research groups have
explored many other applications of this reagent, for example, nucleophilic substitution
of fluoride to synthesize organo-fluoro compounds (Cox, Terpinski et al. 1984),
cyclization reactions for the synthesis of different heterocyclic rings systems (Hiroya,
Jouka et al. 2001; Yasuhara, Suzuki et al. 2002), oxidation reaction (Chung, Moon et al.
2006), click chemistry (Amantini, Beleggia et al. 2004) and [4+2]-cycloaddition (Alden-
Danforth, Scerba et al. 2008) etc. We for the first time explored TBAF.3H2O as a base for
the dimerization of alkylidenemalononitriles 9-26 (Table 1.4) under solvent-free
conditions. Since pyridine derivatives are an important class of N-heterocyclic
compounds which are reported to exhibit a wide range of biological activities, including
antifungal (Hargreaves, Pilkington et al. 2000), antibacterial (Malicorne, Bompart et al.
1991) and anticancer (Szökő and Tábi 2010). In this perspective, we evaluated a series of
DHPs as urease inhibitor, anticancer, and antioxidant agent.
The development of urease inhibitors is directly a step towards the medicinally important
antiulcer agents. Urease enzyme is clinically used for the diagnosis of pathogens in the
gastrointestinal and urinary tract. It has been associated with the several infectious
diseases, like stomach cancer, stones and peptic ulcer formation etc. (Burne and Chen
2000). Urease is also involved in other pathogenesis like urolithiasis, hepatic come, oral
cavity infections, and urinary catheter encrustation (Mobley, Island et al. 1995). Since
Urease metal part is consist of Nickle, our polyfunctional DHPs especially substituted
with electron donating groups are an ideal candidate to interact with the enzyme and
cause its inhibition.
On the other hand, Cancer, an unrestrained aggregation of abnormal cells is one of the
major causes of human mortality in the world. The available doxorubicin IV and related
compounds chemotherapies are encountered with some major concerns such as
cardiotoxicity (Menna, Gonzalez Paz et al. 2012) and multidrug-resistance (MDR). One
of the main causes of MDR mainly occur due to the over expression of multidrug
resistance protein efflux pump, which extrude the drugs from the cell (Volm, Mattern et
al. 1991; Talelli, Morita et al. 2011). Toyota et al., reported the synthesis of 1,4-
Part I
4
dihydropyridine, calcium channel antagonist, (Structure 3, Fig. 1.2) derivatives and used
them in combination with commercially doxorubicin IV. Because, multiple resistant cells
enhance the membrane transportation, in particular the excretion process which lower the
drug concentration in the cells. So, by increasing the anticancer drug concentration in
cancer cells through inhibiting extracellular excretion process of cell, their method
displayed quite interesting outcome (Toyota, Shinkai et al. 1993). Inspired by their work
and of (Abadi, Ibrahim et al. 2009) antitumor investigations in drug resistant cancer cells,
we screened our polyfunctional dihydropydridine compounds (Structure 5) for anticancer
activity (Fig. 1.2). And the results showed a wide range of both Urease inhibition and
cytotoxic activities depending upon the substituents present on the phenyl rings.
Figure: 1.2 Comparative structures of our DHPs and some reported analogues with drugs
Moreover, on the basis of very deep understanding of antioxidant research studies, it is
now a well-known fact that free radicals generated due to oxidative biochemical
processes cause many biological oxidative damages, including protein denaturing,
mutagenesis and several other degenerative pathological events (Beckman and Ames
Part I
5
1998; Cui, Kong et al. 2012). These damages are responsible for aging, diabetes, cancer
and asthma (Meyer, Heinonen et al. 1998; Hunt, Lester et al. 2001; Martínez-Martínez,
Razo-Hernández et al. 2012). A literature review shows that various examples of
antioxidant agents bearing heterocyclic aromatic rings are reported to inhibit cellular
oxidative damages (Rice-Evans, Miller et al. 1996; Villaño, Fernández-Pachón et al.
2007). The polyphenols are known to have a tremendous antioxidant potential, and
hundreds of species of polyphenols display antioxidant activity in vitro, but their
antioxidant role in in vivo studies are found to be significantly low and matter of concern
which give rise to the quest of more efficient antioxidant agents (Williams, Spencer et al.
2004; Ajitha, Mohanlal et al. 2012). Although all oxidative reactions are not less
important for living cell processes some of them are vital, but the main problem of these
are the formation of free radicals in such reactions, which due to their high reactivity
initiate many undesired chain reactions in cell, which can ultimately lead to cell damage
and/or death. The mode of action of an antioxidant compound can be generally regarded
as an electron contributor to free radical species to convert them into harmLess
molecules, so that the cells are prevented from oxidative damage (Holiman, Hertog et al.
1996; Moure, Cruz et al. 2001). So, the development of biologically benign radical
scavengers to inhibit unwanted oxidative reactions is an important field of interest for
antioxidant researchers.
1.1.2 Porphyrin
Porphyrins (Greekoriginated means “purple, scarlet”) are very significant class of natural
occurring molecules. Several biologically significant molecules for example, vitamin B-
12, heme, and chlorophylls contain porphyrin in their basic skeleton (Willstatter and Stoll
1928; Boucher and Katz 1967; Lemberg and Barrett 1973; Hodgkin 1979; Milgrom
1997). Porphyrin macrocyle contains 22 π-electrons, among which only 18 are found to
actually participate in any one delocalization pathway as shown in Fig. 1.3. It follows
Hückel’s [4n+2] rule for aromaticity, where n = 4. While the other four electrons are
situated outside the delocalization pathway are on the two double bonds opposite to each
other at pyrrole rings.
Part I
6
Figure: 1.3 The tautomerization of porphyrins
As a typical aromatic system, porphyrin is able to undergo electrophilic aromatic
substitution reactions (EAS), such as nitration, halogenation and formylation at any
unsubstituted meso- and/or β-pyrrolic positions (Smith and Falk 1975). However, the
quaternary α-pyrrolic carbons are almost inert, and do not give the substitution products.
While the two double bonds outsidethe delocalization pathway are susceptible to
reductions and oxidations, for example: catalytic hydrogenation, reduction with diimide,
and hydroboration.
Figure: 1.4 Typical structure of simplest porphyrin- porphyne
1.1.2.1 Nomenclature
According to the older nomenclature (Fischer system), the meso positions are labeled by
a Greek letters system, and the four pyrrolic sub-unit rings are also labeled with the
capital letters A, B, C, and D. However the more modern IUPAC system identifies every
carbon in the macrocyclic ring and it also numbered the carbon of the substituents in
more complex systems (Merritt and Loening 1979).
Part I
7
Figure: 1.5 Fischer (left) and IUPAC (right) nomenclature systems for porphyrins
1.1.2.2 Synthetic Methodologies of Porphyrins
Fischer synthesized and named protoporphyrin-IX in 1929 for the first time. Afterwards,
a large number of synthetic routes have been developed for the synthesis of porphyrin
and its derivatives for structural, mechanistic, and biological studies. Although
porphyrins can be generated from the total synthesis from monopyrrolic subunits, but the
types of porphyrins that can be generated by this scheme are very limited. Recently,
many improved synthetic routes have been reported, which now provide easy access to
useful porphyrins. Now a days one of following methods can be employed for the
synthesis of porphyrin:
Rothemund Method
Alder-Longo Method
Lindsey Method
Mc Donald (2+2 synthesis) Method
Rothemund Method
Rothemund was the first scientist who introduced monopyrrole tetramerization to tetra-
arylporphyrin synthesis (Rothemund 1939). His method involved the heating of
Part I
8
benzaldehyde with pyrrole in pyridine as solvent in a sealed tube, to producemeso-
H2TPP, this reaction is called the Rothemund reaction. Zn (OAc)2 was used to act as
template in the presence of high pressure resulting 11% yield. Due to the high
temperature and pressure the reaction occurs in lower yields. When the reaction was
carried out in a mixture of methanol/pyridine at atmospheric pressure, both meso-H2TPP
and the chlorin (dihydro) form of meso-H2TPP were obtained. The chlorin is then
converted to meso-H2TPP by oxidization with oxygen or DDQ. This method has
applications with only a limited number of aldehydes.
Alder-Longo Method
Adler and Longo modified the Rothemund methodin 1967 (Adler, Longo et al.
1967).They studied many solvent systems along with a variety of salts to enhance the
formation of meso-H2TPP. The reaction conditions were relatively mild, from which a
much higher yield was achieved as well as the rate of reaction, was much faster as
compared to Rothemund method. In the Alder-Longo method refluxing pyrrole,
benzaldehyde in propionic acid at atmospheric pressure is used to affordthemeso-H2TPP
in 20% yield (Scheme 2.1). Although this method have a significant advantage over the
Rothemund method, but the Adler-Longo methodology have certain vexing problems,
which can be stated as:
The reaction conditions are relatively less selective towards some reactive
functionalities.
The high level of tar produced presents purification problems in mixed aldehyde
reaction mixture.
The reaction has poor reproducibility.
But despite all these disadvantages, it was still the most efficient method for the synthesis
of meso-tetra-alkylporphyrins at that time.
Part I
9
7
Scheme 1.1 Synthesis of meso-H2TPP (7) by Alder-Longo Method.
Lindsey Method
In 1986, Lindsey optimized the method with improved synthesis of porphyrins, under so
called Lindsey conditions. This is by far the most effective route for synthesizing
symmetrical porphyrins i.e. porphyrins with the same substituents on all four meso-
positions, or all eight of β-pyrrolic positions, and/or a combination of them. Lindsey
method is two-step process which consists of the formation of porphyrinogen from
monopyrrole tetramerization and then a separate oxidation (Scheme 1.2) (Lindsey, Hsu et
al. 1986).
Lindsey demonstrated that the formation of meso-H2TPP could be achieved under
equilibrium conditions, in which many functional groups could survive. A colorless
porphyrinogen was first formed, followed by a subsequent oxidation step with p-chloranil
or DDQ. meso-H2TPP was formed by dissolving benzaldehyde and pyrrole in
dichloromethane (DCM) in the presence of acid catalyst BF3•Et2O or TFA. The yields of
product under these conditions were improved to around 30-40 %. He further
concludedthat p-chloranil afforded higher yield as an oxidizing agent as compared to
DDQ.
Part I
10
7
Scheme 1.2 Synthesis of meso-H2TPP (7) by Lindsey Method.
McDonald (2+2 synthesis) Method
Finally, McDonald developed a series of linear steps for the formation of porphyrin with
greater degree of regioselectivityas compared to Lindsey and Alder methods. The
McDonald method involves condensation of two molecules of dipyrromethane one of
which bears substitutions at α-position while the other having no such substitution
(scheme 1.3). Model studies of porphyrins occurring in nature were carried out to study
ferrochelate enzyme by using modified MacDonald approach (Hoare and Heath 1960).
8
Scheme 1.3 Synthesis of meso-H2TPP by McDonald method.
Part I
11
1.1.2.3 Mechanism of Synthesis of Meso-H2TPP
7
Scheme 1.4 Proposed Mechanism for synthesis of meso-H2TPP
Formation of meso-H2TPP occursvia electrophilic substitution between pyrrole and
benzaldehyde in the presence of an acid. While the presence of phenyl rings in the
tetrapyrrole, open chain transition state increased the probability of cyclization as
opposed to aliphatic chain, due to the hindrance in rotation about the bridge carbon
atoms.
The condensation of pyrrole with benzaldehyde was carried out by dropwise addition of
pyrrole in high dilution to the aldehyde solution. Under these conditions, the
concentration of co-polmers, especially tetrapyrryl becomes considerably high and the tar
formation is lower.
Part I
12
1.1.3 Thiolated Cationic Heterocyclic Ligands for Stabilization of Metal Nanoparticles
Pyridinium compounds are known to form ionic liquids (ILs) which have been
extensively used as stabilizing/capping agents for metal nanoparticles (Dupont, Fonseca
et al. 2002; Kim, Demberelnyamba et al. 2003). Reports show that these systems usually
tend to cause aggregation of nanoparticles due to instability and their phase transfer
characteristics (Cassol, Umpierre et al. 2005; Wang and Yang 2005). The uses of
pyridinium ligands are not only limited to ILs but their wide spread occurrence in nature
make them fascinating candidate for biological and photophysical evaluation. Several
pyridinium nanoconjugates and complexes have recently being developed for catalysis
(Tourneux, Gornitzka et al. 2007; Zhu, Jang et al. 2011), drug delivery, biocidal activities
(Patil, Kokate et al. 2011) and molecular recognitions (Saha, Agasti et al. 2012;
Bagherzadeh, Pirmoradian et al. 2014).
Part I
13
1.2 RESULTS AND DISCUSSION
1.2.1 Synthesis and Applications of Polyfunctional Hydrogenated Pyridines from
Acetophenone Derivatives
Commercially available substituted acetophenone derivatives were converted into
alkylidene malononitrile via Knoevenagel condensation by following Barnes et al.
protocol. Towards our proposed synthetic target, we test the TBAF mediated cyclization
conditions to form dicyano substituted dihydropyridine derivatives (Scheme 1.5). The
desired products 9-26 were obtained in good to excellent yield (50% to 95%).
Scheme 1.5 Synthetic methodology of the target DHPs
The successful preparation of alkylidenemalononitriles in the first step enabled us to
explore their dimerization potential under solvent-free conditions for the preparation of
DHPs. The next step was the structure elucidation of these new dimerized compounds.
There are few possibilities in which the alkylidenemalononitrile could be dimerized to
give different positional isomeric structures as shown in Scheme 1.5.
The two interesting 1H NMR signals were observed in the spectra of different analogues
which proved to be very useful in solving the structures of these novel compounds, those
are, two doublets for two non-equivalent protons of methylene group around 3 ppm, and
a broad singlet for NH group around 6.5 ppm in chloroform. Furthurmore, compounds 9
Part I
14
and 11 were successfully crystallized with little effort in the Ethanol/H2O mixture, and
the X-ray crystallographic structures confirmed the correct assignment of NMR peaks.
The crystal structure clearly showed the attachment of malononitrile group to the C-2 via
double bond, cyano group at C-3 position, phenyl ring at C-4 position and quaternary
center at C-6 position (Fig. 1.6).
Figure: 1.6 X-rays crystallographic structures of compounds 9 and 11
All the spectroscopic data which includes 1H NMR, 13C NMR, FT-IR and high resolution
mass spectra showed complete agreement with the structure determination. The
dimerization of ortho substituted ylidenes (entry 14, 20, and 25 of table 1.4) was
unsuccessful which could be possibly explained in terms of steric hindrance.
A probable mechanism of this dimerization has been outlined in Scheme 1.6. The
carbanion, generated by the abstraction of proton from methyl group of alkylidene
malononitrile by TBAF, may rearrange itself to keteneimine intermediate, which may
then attack on dicycano alkene of another molecule to afford another reactive
intermediate carbanion. This intermediate then might undergo rearrangement to afford
the final product by cyclization.
Part I
15
Scheme 1.6 Plausible mechanism for the dimerization of alkylidene
malononitrile to give DHP
1.2.1.1 Urease Activity and Structural Activity Relationship
The urease inhibition activity analysis of the synthesized DHPs showed that most of the
derivatives are inactive in the series. However, entry 11 (IC50 = 20.4 ± 0.66 µM) showed
comparable urease inhibitory potential with standard inhibitor thiourea (IC50 = 21.0 ±
0.01 µM). While entry 12 (IC50 = 84.8 ± 6.99 µM), demonstrated moderate and entry 7
(IC50 = 244.6 ± 9.37 µM) showed weak inhibition potential (Table 1.1).
The 4,6-dimethoxy-5,6-dihydropyridine derivative demonstrated excellent inhibitory
potential among the series with IC50 values 20.4 ± 0.66, while 4,6-dihydoxy-5,6-
dihydropyridine derivative with an IC50 value 84.8 ± 6.99 µM exhibited moderate
activity. The difference between these structural analog’s activities suggests that change
in position and electronic effects of substituents on phenyl rings affect the urease
inhibitory activity.
Part I
16
Table 1.1 In vitro Urease activity of DHPs
Entry
Ring substituents IC50 (μM ± SEMA)
1 p-Br NAb
2 p-Cl NAb
3 acetophenone NAb
4 naphtha. NAb
5 p-I NAb
6 m-Br NAb
7 p-Me 244.66 ± 9.37
8 m-OMe NAb
9 p-F NAb
10 m-F NAb
11 p-OMe 20.48 ± 0.66
12 p-OH 84.87 ± 6.99
13 m-OH NAb
14 p-NO2 NAb
Thioureac 21.0 ± 0.01
SEMa is the standard error of the mean, NAb Not active
Thioureac standard inhibitor
It can be observed in table 1.1 that most of the electron withdrawing substituents and
meta position of electron donating groups in the series of these compounds are inactive.
The 4,6-dimethyl-5,6-dihydropyridine derivative with IC50 value 244.66 ± 0.01 µM also
exhibit weak inhibition due to low electron donating effects of para methyl substituent on
the phenyl rings of DHPs. Thus, it can be summarized that electron releasing groups at
para positions on phenyl ring greatly affect the urease inhibitory activity. This study
identifies a potent compound 11 (Table 1.1) which may serve as lead compound for
future research.
Part I
17
1.2.1.2 Anticancer Activity and Structural Activity Relationship
We also explored our DHP series against two cancer cell lines; PC3 and HeLa cell line.
The results of the anticancer activity (Table 1.2) on both cell lines showed that most of
the compounds are cytotoxic and have a varying degree of anticancer potential having
IC50 values ranging between 4.40 ± 0.12 to >30 µM, in comparison with standard
doxorubicin (IC50 value 0.91 ± 0.12 and 3.1 ± 0.20µM). Entry 1, and 2 showed highest
cytoxicity among the series in both cell lines as compared to doxorubicin. Entry 3, 4, 5, 6,
7, 8, 9, 10, and 11 demonstrated moderate cytoxicity, while compounds 12, 13, and 14
exhibited a weak activity towards both Cells line with IC50> 30.
The structure activity relationship can be constructed by closely observing the trend in
cytoxicity results. The variation in the anticancer activity through the series mainly
depends upon the position and electronic nature of the substituents on the phenyl rings.
The higher cytoxicities of p-Bromo and p-Chloro derivative, as compared to other
compounds in the series, can be understood on taking into account the electronic effect,
leaving group ability and reactivity of the substituents on the aromatic rings. The DHPs
with electron withdrawing groups having good leaving group character contained highest
cytotoxicity through the series. While the positions of the substituents also plays a pivotal
role in the anticancer activity, since the activity of 3-bromo derivative is lower than that
of 4-bromo derivative, whilst in the case of 3-methoxy derivative, activity is slightly
higher than 4-methoxy derivative. Moreover, an addition of phenyl ring as in the casse of
6-methoxynaphthalene dihydropyridine derivative also showed significant activity. These
results can be concluded as; electron withdrawing group having good leaving ability, as
well as their position on the phenyl ring significantly enhanced the anticancer activity of
the compounds. This study identifies compounds two potent novel anticancer agents
which may serve as lead compounds for the future research.
1.2.1.3 DPPH Radical Scavenging Activity
Free radical scavenging activity results show that all the DHP derivatives 1-13 are
moderately active to low active with a wide range of IC50 values from 127.4 ± 3.5 to
284.5 ± 0.66 (Table 1.3).
Part I
18
Table1.2Anticancer activity of DHPsagainst PC3 and HeLa Cell line
Entry Ring
substituents PC3 Cell line HeLa Cell line
IC50 (μM ± SEMA)
IC50 (μM ± SEMA)
1 p-Br 4.40 ± 0.13 8.81 ± 0.25
2 p-Cl 6.85 ± 0.34 8.81 ± 0.63
3 acetophenone 8.03 ± 0.22 23.06 ± 0.10
4 naphtha. 8.36 ± 0.48 14.67 ± 0.75
5 p-I 8.87 ± 0.59 9.67 ± 0.20
6 m-Br 9.62 ± 0.55 11.26 ± 0.85
7 p-Me 10.48 ± 0.24 18.84 ± 0.87
8 m-OMe 11.51 ± 0.45 20.18 ± 0.39
9 p-F 13.10 ± 0.62 23.09 ± 0.03
10 m-F 14.02 ± 0.42 26.41 ± 1.97
11 p-OMe 14.55 ± 0.38 14.73 ± 0.14
12 p-OH > 30 > 30
13 m-OH > 30 > 30
14 p-NO2 > 30 > 30
Doxorubicinb 0.91 ± 0.12 3.1 ± 0.20
SEMa is the standard error of the mean Doxorubicinb standard anticancer drug for many type of cancer cell lines
The derivatives including 3'-fluoro phenyl, 3'-bromo phenyl, 3'-methoxy phenyl, 4'-
bromo phenyl, 4'-iodo phenyl and 4'-methoxy phenyl exhibited moderate activity, while
the other derivatives in the series displayed low activity in comparison with standard
inhibitors ascorbic acid (IC50 = 40.6 ± 1.2μM) and butylated hydroxyanisole (IC50 = 40.6
± 1.2 μM).
Part I
19
Table 1.3 DPPH free radical scavenging activity
Entry
Substituted group (R) IC50 (μM ± SEM A)
1 phenyl NA b
2 4'-methyl phenyl 284.5 ± 0.66
3 4'-hydroxy phenyl 228.2 ± 3.36
4 3'-hydroxy phenyl 223.8 ± 3.30
5 4'-methoxy phenyl 161.4 ± 2.81
6 3'-methoxy phenyl 142.2 ± 0.60
7 4'-fluoro phenyl 172.8 ± 1.72
8 3'-fluoro phenyl 127.4 ± 3.5
9 4'-chloro phenyl 164.6 ± 4.50
10 4'-bromo phenyl 144.7 ± 2.46
11 3'-bromo phenyl 132.5 ± 3.32
12 4'-iodo phenyl 153.7 ± 0.50
13 6'-methoxynaphthalenyl 196.3 ± 6.5
14 Ascorbic acid c 40.6 ± 1.2
15 Butylated hydroxyanisole (BHA) c 44.7 ± 0.67
a SEM is the standard error of the mean. b NA: Not active.c Standard inhibitor for DPPH
radical scavenging activity.
Part I
20
1.2.2 Synthesis of Mannose Conjugated Porphyrin
1.2.2.1 Synthesis of Tetraphenylporphyrin (TPP) (7)
7
Scheme 1.7 Synthesis of meso-H2TPP (7)
TPP was synthesized by a modified Alder method. Propanoic acid was heated at 90 oC,
and a solution of (2 g, 18.86 mmoles) benzaldehyde dissolved in propanoic acid 2.3 mL
was added very slowly in hot propanoic acid. Then after continuous heating of the
solution at reflux temperature, pyrrole (18.86 mmoles) solution in propanoic acid (1.3 mL
in 2.3 mL) was added in above mixture dropwise for half hour. The reaction was refluxed
and monitored by TLC. After completion (about 1.5hrs) the reaction mixture was cooled.
The reaction mixture was worked up by extracting the crude porphyrin in 50 mL ethyl
acetate thrice from 50 mL, 2.5M NaOH aqueous solution.
Solvent was evaporated and solid product was passed through silica gel column using
ethyl acetate and hexane as mobile phase (3:1 respectively). The yield of pure product
was 1.2 g, 10 %.
Part I
21
1.2.2.2 Nitration of TPP (27)
27
Scheme 1.8 Synthesis of MNPP (27)
TPP was nitrated with the help of NaNO2 in TFA. By controlling the amount of TFA and
reaction time mono nitrated phenyl porphyrin (MNPP) was obtained as a major product
along with dinitro phenyl porphyrin (Kruper Jr, Chamberlin et al. 1989). To a solution of
TPP (160 mg, 0.26 mmoles) in 10 mL TFA was added (90 mg, 1.30 mmoles) NaNO2 and
reaction mixture was stirred for an hour. Reaction mixture was quenched with 15mL
water, extracted with 25 mL DCM thrice. The combined organic phases were evaporated
under reduced pressure affording brown residue. MNPP was purified through silica gel
column chromatography (DCM, Hexane 3:1 to 1:1) to get 50 mg in 30% yield.
Part I
22
Figure: 1.7 NMR spectrum of MNPP (27)
1.2.2.3 Mechanism of Nitration of Meso-H2TPP
A solution of TFA and sodium nitrite was used as source of nitronium ion for the
regiospecific nitration of meso-H2TPP. The steps involved in the formation of nitronium
ion from TFA and sodium nitrite can be given as,
Scheme 1.9 Mechanism of nitration of meso-H2TPP
Part I
23
The basic mechanism involved in the mentioned reaction is electrophilic aromatic
substitution of meso- substituted phenyl ring of the meso-H2TPP.
1.2.2.4 Reduction of nitro group of MNPP
Scheme 1.10 Reduction of nitro group of MNPP
MNPP (100 mg, 0.15 mmoles) was dissolved in 0.5 mL HCl. Tin dichloride (100 mg,
0.52 mmoles) was added in the solution and was heated at 60 oC for 2.5 hrs. Reaction was
monitored by TLC. After 2.5 hours, ammonium hydroxide was added to neutralize the
reaction mixture and extracted with 25 mL DCM thrice to get crude product. Purification
was carried out by Silica gel Column chromatography (DCM, Hexane 1:3 to 3:1). Yield
of monoamino phenyl porphyrin (MAPP 28) was 50 mg, 53 %.
Part I
24
Figure: 1.8 EI-MS spectrum of MAPP (28)
1.2.2.5 Mechanism of Nitro group of Reduction
The nitro group in MNPP was successfully reduced into amino group with the help of tin
(II) chloride hence yielding MAPP. Different solvents were screened for the reduction of
nitroporphyrins, and HCl was found to be the best one as it was acting as solvent and
source of reduction. The conversion of nitrporphyrin to aminoporphrin takes place
through free radical mechanism as shown in scheme 1.11.
Scheme 1.11 Proposed mechanism for reduction of nitro group
Part I
25
1.2.2.6 Conjugation of MAPP with Mannose
Scheme 1.12 Synthesis of target molecule (29)
Mannose (15 mg, 0.083 mmoles) was dissolved in 10 mL ethanol, MAPP (50 mg, 0.079
mmoles) was added in the solution. Few drops of acetic acid were added and reaction
mixture was refluxed overnight (Ojala, Ostman et al. 2000). Precipitates formed were filtered
and washed with 20 mL each DCM and MeOH separately. The residue was dissolved in
DMSO and was subjected to NMR for characterization. Yield was 47 mg, 75 %.
Figure: 1.9 NMR spectrum of mannose conjugated porphyrin (29)
Part I
26
1.2.2.7 Antibacterial Study of Mannose Conjugated Porphyrin via Photochemotherapy
Severe infections are produced including sight intimidating keratitis and deadly
granulomatous by pathogenic Acanthamoeba sp. (Marciano-Cabral and Cabral 2003;
Visvesvara, Moura et al. 2007). Regardless of the improvements made in supportive care
and antimicrobial chemotherapy to treat pathogenic Acanthamoeba, one of the most
upsetting aspects is that the death rate connected with GAE has remained significant
(90%). The existing diagnosis for the treatments of Acanthamoeba keratitis are expensive
and not totally operational against all strains (Pérez-Santonja, Kilvington et al. 2003), due
to the ability of the amoebae to form a strong cyst (Turner, Russell et al. 2004), which
results in the reappearance of infection. Therefore the treatment of pathogenic
Acanthamoeba keratitis is challenging. Complete knowledge about the pathophysiology
and pathogensis of infections associated with Acanthamoeba keratitis will certainly guide
to the improvement of diagnostic tools as well as therapeutic mediations (Clarke and
Niederkorn 2006). Recently it is recognized that a mannose binding protein (MBP) which
play a role in adhesion, as an exogenous mannose-block is expressed on the
Acanthamoeba membrane surface to adhere the parasites to microvascular endothelial-
cells of primary human brain and corneal epithelial cells (Garate, Cao et al. 2004).
Remarkable improvements were observed in Acanthamoeba sp. in Chinese hamster
model through oral immunization using recombinant mannose binding protein (Garate,
Cubillos et al. 2005).
Photochemotherapy is a relatively new technology that can treat only the target pathogens
and thus can be used as a therapeutic means. The basic principle of this technique is that
the photo-sensitizing compound is activated by the electromagnetic radiations which
results in generation of the nascent oxygen which induce cell necrosis in the target
pathogen (Hamblin and Hasan 2004; Huang, Dai et al. 2010). On the other hand, it is also
possible that host cells can bind with the photo sensitizing compound and then followed
by irradiation results in cell necrosis. Therefore to target a pathogenic Acanthamoeba sp.,
has persisted as a key concern (Baig, Iqbal et al. 2013). The main focus of our study is to
synthesize a photo-sensitizing compound such as porphyrin derivative conjugated with
mannose moiety to target specific Acanthamoeba and evaluate its in vitro potential. In the
Part I
27
current study mannose conjugated porphyrin derivative was prepared and subjected to
irradiation to kill the microbe.
Mannose conjugated porphyrin inhibited viability of A. castellanii
The A. castellanii trophozoiteswere incubated with two different concentrationof
mannose conjugated porphyrin i.e. 10 µM and 50 µM, to determine the amoebicidal
potential of mannose conjugated porphyrin on A. castellanii trophozoites. Which showed
that 50 µM concentration of mannose conjugated porphyrin revealed a significant
amoebicidal potential (P<0.001) as clearly visible in Fig. 1.10. Microscopic analysis also
confirmed the amoebicidal potential mannose conjugated porphyrin (Fig. 1.11). While A.
castellanii incubated with unconjugated mannose and unconjugated porphyrin having
concentration 50 µM did not showed any amoebicidal potential. The negative solvent
control groups also showed no effect on the A. castellanii tropozoites.
Figure: 1.10 Mannose-conjugated porphyrin (mann. por.) exhibited amoebicidal effects
against A. castellanii.
Part I
28
Figure: 1.11 A. castellanii was incubated with and without 50 µM mannose conjugatedporphyrin, and representative images were obtained at 100 x (A) and at400 x (B) magnification using a phase-contrast inverted microscope.
DMSO
Chloroform: Methanol
Mannose conjugated porphyrin
Mannose RPMI Alone
Porphyrin
Part I
29
Mannose conjugated porphyrin inhibited A. castellanii mediated host cell cytotoxicity
The mannose conjugated porphyrin was initially treated with A. castellanii, followed by
cytotoxicity assays of the host cells. The model group showed 95% 8.5% BMEC cell
deaths within 1440 minutes while the pretreated group with mannose conjugated
porphyrin significantly reduces the cytotoxicity of the host cells to 4.9% 2.5% (P <0.01)
as shown in Fig. 1.12A. A. castellanii was pretreated with mannose conjugated
porphyrin, which was followed by the performance of host cell cytotoxicity assays.
Without mannose conjugated porphyrin, A. castellanii produced 95% 8.5% BMEC cell
deaths within 24 h. The pretreatment of A. castellanii with mannose conjugated porphyrin
reduced host cell cytotoxicity significantly to 4.9% (P <0.01) compared to that with
amoebae alone (Fig. 1.12A). While unconjugated mannose, porphyrin, and solvents
showed no cytotoxicity of the host cells when they were separately incubated with A.
castellanii, and silmilarly solvent has showed no cytotoxicity to the microvascular
endothelial cell of the human brain (Fig. 1.12A). A coincubation assays was also
performed with microvascular endothelial cell of the human brain, along with mannose
conjugated porphyrin having concentration around 50 µM, and A. castellanii which also
reduces cytotoxicity of the host cells i.e. 58% 4% (P <0.01) as shown in Fig. 1.12B.
A
Part I
30
Figure: 1.12 (A). A. castellanii (5 x105) preincubated with and without 50 µM mannose-conjugated porphyrin (mann. por.) and exposed to light. (B). A. castellanii (5 x105) were coincubated with human BMEC and 50µM mannoseconjugatedporphyrin and exposed to light.
Mannose conjugated porphyrin blocked excystation in A. castellanii
The A. castellanii cysts were incubated in two medium which were capable of growth,
one A. castellanii cysts groups were initially treated with mannose conjugated porphyrin
while the other group was not treated to mannose conjugated porphyrin. The results
showed that the group A. castellanii cysts which were not treated with mannose
conjugated porphyrin appeared as workable trophozoites (1.2 x 106 amoebae) as shown in
Fig. 1.13. While the group treated with mannose conjugated porphyrin having 50 µM
concentration significantly repressed the A. castellanii excystation within three days (P
<0.01). The negative control groups with unconjugated mannose, porphyrin and solvents
showed sustainable trophozoites which were appeared in the well plates having A.
castellanii cysts after three days (Fig. 1.13).
B
Part I
31
Figure: 1.13 Mannose-conjugated porphyrin inhibited excystation of A. castellanii cysts. A.
castellanii cysts were scraped from nonnutrient agar plates, incubated with 50µM mannose porphyrin, and exposed to light.
Different drugs, like neomycin,chlorhexidine, propamidine isethionate, and
polyhexamethylene biguanide, are used to cure Acanthamoebainfections, but the recurrence
of these infections after a year or so is the main issueassociated with drugs. So therefore
complete treatments of diseases associated with Acanthamoeba toxicities is a challenging
work in the current scenario. Ficker et al. reported a fascinating study by showing that during
the treatments of Acanthamoeba keratitis, results in the resistance developments of
propamidine which ultimately led into the reappearance of infections. Different research
groups have shown that Acanthamoeba sp. resist to the antimicrobial treatments via
chemotherapy. In the current studies, we demonstrated the possible application of
photochemotherapy for the treatment of Acanthamoeba infections. (Aqeel, Siddiqui et al.
2015), a photo activated compound was synthesized from porphyrin and then it was
conjugated with mannose, in order to target the Acanthamoeba selectively. The mannose-
conjugated porphyrin showed potential applications in amoebicidal assays and can inhibited
the excystation effectively. The basic principle of this technique is that the photo sensitizing
compound is activated by the electromagnetic radiations which results in generation of the
Part I
32
nascent oxygen which induce cell necrosis in the target pathogen. The specific delivery of
photochemotherapeutic compounds to the target site is the considered to be the major
challenge. It was reported that mannose was considered to be the sugar with which
pathogenic Acanthamoeba can binds specifically in the presence of other sugars; MBP is
identified as a main adhesin which is expressed on the Acanthamoeba membrane surface.
Therefore, mannose was conjugated to photoactivated compound, a step towards targeted
drug delivery.
Importantly, the co-treatments and pretreatments of mannose conjugated porphyrin with A.
castellanii showed different results towards cytotoxicities of the host cells that is 58% and
4% respectively. A possible explanation of the higher cytotoxicity of the host cells in the co-
treatments of mannose conjugated porphyrin is the ROS generation by the mannose
conjugated porphyrin, which is a photosentising compound and in presence of brain cells of
human may have be involved to increases the cytotoxicity of the host cells. The mannose
conjugated porphyrin has also inhibited the excystation and complete inhibition of the
excystation assay was observed at 50 µM concentration of mannose-conjugated porphyrin.
This result was fascinating, as it was believed cysts don’t adhered to the host cells as MBP
was not expressed in the membrane surface, but here it seems that Acanthamoeba cysts
actually expresses the MBP on the membrane surface, which needs further experiments. The
lengthy exposure of photoactivated compound like mannose conjugated porphyrin and lipid
soluble compound were considered to be responsible for the complete inhibition of
excystation assays (Ferro, Coppellotti et al. 2006; Chen, Xuguang et al. 2008) which results
the reappearance of tropozoites from the cyst stage. The molecular mechanism associated
with aggregation of photo-dynamic compound at the Acanthamoeba cyst, and the movement
of this compound via operculum membrane to the trophozoites of Acanthamoeba remain to
be investigated in future along with its in vivo evaluation. In future porphyrin will be
conjugated to Acanthamoeba specific antibody for treatments of Acanthamoeba infections for
enhanced targeted therapeutic potential. Furthermore, pulsed interval treatments can also be
introduced, which will be more effective for clinical treatments. This type of treatment will
be very useful, as light used in this therapy is in the visible range which will never damage
tissues. The photodynamic therapy has been also useful for diverse diseases (Chen, Keltner et
al. 2002; Selbo, Kristian et al. 2002; Silva, Filipe et al. 2006) to kill Acanthamoeba in a less
harmful way, and it should be further investigated for in vivo applications.
Part I
33
1.3 EXPERIMENTAL
All the chemicals (Solvents and reagents) used were analytical grade and used as
purchased, without any pre-treatment until stated. 4-Pyridine carboxaldehyde, Pyrazine,
and Dimethyl aminopyridine were purchased from Tokyo chemicals industry (TCI,
ChinA). Dibromo propane and potassium thioacetate were purchased from Alfa-Aesar
(MA, USA). Instruments used for the analysis are, UV-visible spectrophotometer
Evolution 300, FT-IRspectra were recorded on Shimadzu FTIR 8900 spectrometer and 1HNMR spectra were recorded on Bruker 300 MHz and 400 MHz spectrometers.Mass
spectra were obtained on JEOL MS Route 600 H spectrometer at positive mode by using
electron impact (EI+) technique, HRMS was carried out at Thermo Finnigan MAT 95XP
instrument, while Matrix assisted laser desorption ionization (MALDI) mass
spectrometer Bruker Ultra flex, TOF-TOF. Coupling constant were calculated in Hertz
(Hz). The Merck silica gel 60 was used for column chromatography with commercially
available double distilled ethyl acetate, DCM and hexane as eluent.
1.3.1 General Procedure for the Synthesis of Polyfunctional DHP Derivatives
An oven dried screw-capped vial having magnetic stirrer bead was charged with
alkylidenemalononitrile (1.0 equiv) and neat TBAF.3H2O (1.0 equiv). The reaction
mixture was heated at 85-90 °C with continuous stirring for 15 h. The reaction progress
was monitored by TLC analysis (ethyl acetate hexane 1:4). The reaction mixture was
cooled to room temperature, diluted with 3 mL dioxane and then 100 mL, 1.0 M HCl
aqueous solution was added to remove excess TBAF. The resulting precipitates were
filtered and washed with more 1.0 M HCl to give the pure compounds in excellent yield
(52% to 98%). The structures of these compounds were characterized by using different
spectroscopic techniques including 1H NMR, 13C NMR, FT-IR, and EI-HRMS
spectroscopy.
Part I
34
Table 1.4 Structures and spectroscopic data of synthesized DHPs
S. No.
Structures with HR-
EIMS
And Melting Points
%
Yield
1HNMR
(Chemical shift in ppm)
FT-
IR1/cm
9
m/z = 336.1355 193-195 oC
98
7.50-7.40 (6H, m, ArH), 7.35 (2H app d, J = 8.0 Hz, ArH), 7.24 (2H app d, J = 8.0 Hz, ArH), 6.58 (1H, brs, NH), 3.49 (1H, d, J = 18.0 Hz, CH2), 3.15 (1H, d, J = 18.0 Hz, CH2), 1.79 (3H. s, CH3)
3448, 2925,2215,1598,1546,1445
,1257
10
m/z = 364.1645 125-128 oC
95
7.32 (2H, d, J = 8.4 Hz, ArH), 7.25-7.18 (4H, m, ArH), 6.11 (2H, d, J = 8.0 Hz, ArH), 6.50 (1H, br s, NH), 3.46 (1H, d, J
= 18, CH2), 3.09 (1H, d, J = 18, CH2), 2.37 (3H, s, CH3), 2.33 (3H, s, CH3), 1.76 (3H. s, CH3)
3391, 3279, 2923, 2214, 1591, 1427, 1259
11
m/z = 372.166 120-122 oC
75
7.39 (2H, app dd, J = 8.8 Hz, 5.0 Hz, ArH), 7.23 (2H, app dd, J = 9.0 Hz, 5.0 Hz, ArH), 7.12 (4H, app dd, J = 16 Hz, 8.5 Hz, ArH), 6.41 (1H, br s, NH), 3.40 (1H, d, J = 18 Hz, CH2), 3.15 (1H, d, J = 18 Hz, CH2), 1.79 (3H, s, CH3)
3446, 2216, 1602, 1507, 1432, 1237
12
m/z = 372.1176
130-135 oC
75
7.45-7.39 (2H, m, ArH), 7.22-7.15 (2H, m, ArH), 7.07 (1H, app td, J = 8.0 Hz, 2.0 Hz, ArH), 7.03-6.99 (2H, m, ArH), 6.94 (1H, app dt, 7.6, 2.0, ArH), 6.15 (1H, br s, NH), 3.40 (1H, d, J = 18 Hz, CH2), 3.16 (1H, d, J = 18 Hz, CH2), 1.79 (3H, s, CH3)
3249, 2222, 2204, 1614, 1584, 1487, 1437, 1270
Part I
35
13
m/z = 404.0571 132-135 oC
90
7.42 (4H, app t, J = 8.8 Hz, ArH), 7.22 (2H, d, J= 8.7 Hz, ArH), 7.16 (2H, d, J = 8.7 Hz, ArH), 6.43 (1H, br s, NH), 3.39 (1H, d, J = 18 Hz, CH2), 3.15 (1H, d, J = 18 Hz, CH2), 1.78 (3H. s, CH3)
3449, 2218, 1593, 1542, 1489, 1260
14
No React-ion
--- ---
15
m/z = 492.9691 138-140oC
90
.56 (4H, app dd, J = 16 Hz, 8.4 Hz, ArH), 7.21 (2H, d, J = 8.4 Hz, ArH), 7.09 (2H, d, J = 8.4 Hz, ArH), 6.83 (1H, br s, NH), 3.38 (1H, d, J = 18 Hz, CH2), 3.15 (1H, d, J = 18 Hz, CH2), 1.77 (3H, s, CH3)
3438, 1591, 1544, 1429, 1259
16
m/z = 492.9691
148-150 oC
84
7.63 (1H, app dt, J = 6.4 Hz, 1.6 Hz, ArH), 7.51 (1H, d, J = 7.6 Hz, ArH), 7.40 (2H, app d, J = 6.4 Hz, ArH), 7.34-7.30 (H, m, ArH), 7.16 (1H, d, J = 7.6 Hz, ArH), 6.15 (1H, br s, NH), 3.39 (1H, d, J = 18 Hz, CH2), 3.14 (1H, d, J = 18 Hz, CH2), 1.79 (3H, s, CH3)
3447, 3285, 2220, 2190, 1585, 1560, 1433, 1408
17
m/z = 587.9291
89
7.77 (4H, app dd, J = 17 Hz, 8.8 Hz, ArH), 7.07 (2H, d, J =
8.4 Hz, ArH), 6.96 (2H, d, J =
8.8 Hz, ArH), 6.41 (1H, br s, NH), 3.37 (1H, d, J = 18 Hz, CH2), 3.14 (1H, d, J = 18 Hz, CH2), 1.77 (3H. s, CH3)
3441, 1589, 1548, 1429, 1261
Part I
36
18
m/z = 368.1273
253-255 oC
75
10.55 (1H, br s, NH), 9.55 (1H, br s, ArOH), 9.45 (1H, br s, ArOH), 7.50 (2H, d, J = 8.7 Hz, ArH), 7.17 (2H, d, J = 8.4 Hz, ArH), 6.87 (2H, d, J = 8.7 Hz, ArH), 6.73 (2H, d, J = 8.4 Hz, ArH), 3.75 (1H, d, J= 18 Hz, CH2), 3.14 (1H, d, J= 18 Hz, CH2), 1.65 (3H, s, CH3).
3382, 1575, 1510, 1435, 1261
19
m/z = 368.1273 270-272 oC
93
9.92 (1H, br s, NH), 9.65 (1H, br s, ArOH), 9.55 (1H, br s, ArOH), 7.29 (1H, app t, J = 7.8 Hz, ArH), 7.18 (1H, app t, J = 7.8 Hz, ArH), 6.92 (1H, d, J = 8.0 Hz, ArH), 6.85-6.78 (3H, m, ArH)), 6.70-6.65 (2H, m, ArH), 3.61 (1H, d, J= 18 Hz, CH2), 3.27 (1H, d, J= 18 Hz, CH2), 1.67 (3H, s, CH3).
3449, 3250, 2213, 1594, 1451, 1429
20
No React-ion
--- ---
21
m/z = 396.1515 119-121 oC
95
7.45 (2H, d, J = 9.0 Hz, ArH), 7.15 (2H, d, J = 8.7 Hz, ArH), 6.91 (4H, app t, J = 9.4 Hz, ArH), 6.73 (1H, br s, NH), 3.82 (3H, s, OCH3), 3.78 (3H, s, OCH3), 3.46 (1H, d, J = 18 Hz, CH2), 3.05 (1H, d, J = 18 Hz, CH2), 1.75 (3H, s, CH3).
3449, 2926, 2212, 1510, 1459, 1259.
Part I
37
22
m/z = 397.1638
133-135 oC
80
7.34 (2H, app t, J = 8.0 Hz, ArH), 7.01 (1H, app dd, J = 8.0 Hz, 2.0 Hz, ArH), 6.93 (1H, d, J = 8.0 Hz, ArH), 6.87-6.79 (3H, m, ArH), 6.73 (1H, app t, J = 2.1 Hz, ArH), 6.38 (1H, br s, NH), 3.80 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.45 (1H, d, J = 18 Hz, CH2), 3.11 (1H, d, J = 18 Hz, CH2), 1.77 (3H, s, CH3)
3449, 3284, 2212, 1598, 1427
23
m/z = 427.1130
52
9.68 (1H, br s, NH), 8.30 (2H, dd, J = 9.0 Hz, ArH), 8.26 (2H, d, J = 9.0 Hz, ArH), 7.73 (2H d J = 9.0 Hz, ArH), 7.62 (2H, d, J = 9.0 Hz, ArH), 3.70 (1H, d, J = 18 Hz, CH2), 3.53 (1H, d, J
= 18 Hz, CH2), 1.77 (3H, s, CH3).
3447, 3419, 2963, 2928, 2188, 2160, 1596, 1520, 1453
24
m/z = 426.1068.
80
9.84 (1H, br s, NH), 8.37 (1H, app dt, J = 8.0 Hz, 2.0 Hz, ArH), 8.33 (1H, br s, ArH), 8.26 (2H, app d, J = 9.6 Hz, ArH), 8.20-8.13 (3H, m, ArH), 8.07 (1H, d, J = 8.0, Hz, ), 7.94-7.89 (2H, m, ArH), 7.84 (1H, app d, J = 3.6 Hz, ArH), 7.80 (1H, d, J = 8.0 Hz, ArH), 7.74 (1H, d, J = 8.0 Hz, ArH), 7.70-7.62 (3H, m, ArH), , 3.90 (1H, d, J = 18 Hz, CH2), 3.46 (1H, d, J = 18 Hz, CH2), 1.78 (3H, s, CH3)
3447, 3382, 3266, 2219, 2202, 1612, 1523, 1437
25
No reacti-on
--- ---
Part I
38
26
m/z = 496.1912
148-150 oC
65
7.90 (1H, app d, J = 1.4 Hz, ArH), 7.81 (1H, app d, J = 8.8 Hz, ArH), 7.81 (3H, app d, J =
8.8 Hz, ArH), 7.60 (1H, app d, J = 1.4 Hz, ArH), 7.43 (1H, dd, J = 8.8 Hz, 2.0 Hz, ArH), 7.38 (1H, dd, J = 8.8 Hz, 2.0 Hz, ArH), 7.19 (2H, app td, J
= 8.8 Hz, 2.8 Hz, ArH), 7.12 (1H, d, J = 2.0 Hz, ArH), 7.08 (6H, d, J = 2.4, Hz, ArH), 6.49 (1H, br s, NH), 3.91 (6H, s, 2 x OCH3), 3.70 (1H, d, J = 18 Hz, CH2), 3.27 (1H, d, J = 18 Hz, CH2), 1.89 (3H, s, CH3)
3448, 2926, 2212, 1627, 1579, 1540, 1482, 1269.
1.3.2 Urease Inhibition Assay (In vitro)
The reaction mixture is consist of the solutions of 25 µL jack bean urease enzyme, 55 µL
of its substrate having 100 mM urea. Reaction mixture was incubated with 5 µL of DHPs
(0.5 mM concentration) at 30 ºC for 15 min in 96-well plates. Then, the urease inhibitory
activity was evaluated by determining ammonia production via indophenol method
(Weatherburn 1967). Briefly, 45 µL of phenol reagent (1% w/v phenol with 0.005% w/v
sodium nitroprussside), and 70 µL of alkali reagent (0.5% w/v NaOH with 0.1% active
chloride NaOCl) were added to each well. The change in absorbance per minute was
measured after 55 min at 630 nm using a microplate reader (Molecular Device, USA). All
the reactions were performed in triplicate in a final volume of 200 µL and the results
(change in absorbance per min) were processed by using softMax Pro software
(molecular Device, USA). The pH was maintained at 6.8 in all assays, while the
percentage inhibitions were calculated from the formula 100-(ODtestwell/ODcontrol) ×100.
Thiourea was used as the standard inhibitor of urease (Table 1.1).
1.3.3 Anticancer Activity Assay (In vitro)
Cytotoxicities of compounds were evaluated in 96-well flat-bottomed micro plates via
standard MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide)
Part I
39
colorimetric assay (Mosmann 1983). PC3 and HeLa cells (Prostrate Cancer) were
cultured in Dulbecco’s Modified Eagle Medium, which was supplemented with 5% fetal
bovine serum (FBS), 100 IU/mL penicillin, 100 µg/mL streptomycin in 75 cm2 flasks,
and kept in 5% CO2 incubator at 37ºC. The exponentially growing cells were harvested,
counted by using haemocytometer, and then diluted with a particular medium. Cell
culture with the concentration of 1x105 cells/mL was prepared and 100 µL/well of this
solution was introduced into 96-well plates. After overnight incubation, medium was
removed and 200 µL of fresh medium was added with multiple concentrations of
compounds ranging in 5-50 µM. After 48 h, 200 µL MTT (0.5 mg/mL) was added to
each well and incubated for 4 more hours. Subsequently, 100 µL of DMSO was added to
each well. The extent of MTT reduction to formazan within cells was calculated on a
micro plate reader (Spectra Max plus, Molecular Devices, CA, USA) by measuring the
absorbance at 570 nm. The cytotoxicity was recorded as IC50 for both cell lines. The
percent inhibition was calculated by using the following formula: % inhibition = 100-
((mean of O.D of test compound – mean of O.D of negative control)/ (mean of O.D of
positive control – mean of O.D of negative control)*100). Doxorubicin was used as the
standard anticancer drug (Table 1.2).
1.3.4 DPPH (1,1-Diphenyl-2-Picryl Hydrazyl) Free Radical Scavenging Assay (In vitro)
The antioxidant activity of DHPs against 1,1-diphenyl-2-picrylhydrazil (DPPH) radical
was determined by following reported literature protocol.(Morales and Jiménez-Pérez
2001; Shaheen, Ahmad et al. 2005) The test sample (5μL, 1mM in DMSO) was added to
95 μL of DPPH (Sigma, 300μM) in ethanol and the resulting mixture was introduced into
a 96-well plate. The reaction mixture was incubated for half an hour at 37º C, and then
absorbance was determined at 515 nm on micro plate reader (Molecular Devices, USA).
DPPH radical scavenging activity was measured by comparing with a DMSO containing
control. All the assays were performed in dark and analysed in triplicate. The IC50 values
± SD of each compound were recorded and presented in the Table 1.3. Ascorbic acid and
Butylated hydroxyanisole (BHA) were used as standard inhibitors for DPPH radical
scavenging activity.
Part I
40
1.3.5 Acanthamoeba castellanii and Microvascular Endothelial Cells Cultures
All chemical reagents were obtained from Sigma laboratories England. A clinical isolate
of Acanthamoeba castellanii belongs to strain ATCC-50492. Amoebae were routinely
grown as reported in literature (Baig, Iqbal et al. 2013). The medium was every time
revitalized 16 to 21 hours before the start of experiments. The tropozoite forms of
amoebae were adhered to the walls of the flask and which were then collected and used in
all experiments. The microvascular endothelial cells were exclusively from human brain
which was isolated, cultured and purified as reported in literature (Stins, Gilles et al.
1997). The microvascular-endothelial cells of human brain were developed in collagen-
coated tissues of rat tail. The cytotoxicity assay was carried out by using microvascular-
endothelial cells of human brain which were grown in 24 well plates by 106 cells per mL
per well. The cell-density, confluent monolayers were molded within 1440 minutes and
were then used in subsequent experiments.
1.3.6 Synthesis of Mannose Conjugated Porphyrin
For the synthesis of mannose conjugated porphyrin, propionic acid (40 mL) was heated
up to 95 ºC followed by slow addition of benzaldehyde solution into hot propionic acid
which is prepared from 1.9 mL of benzaldehyde (18.85 mmoles) into 2.5 mL of propionic
acid. Then 1.3 mL of the pyrrole solution (18.85 mmmoles) was gradually added
dropwise in 30 minutes to the above solution at reflux temperature and was monitored by
TLC (DCM and MeOH 1:1). The reaction was completed in 1.5 h and then the reaction
mixture was cooled and the product tetraphenylporphyrin along with by products were
obtained through extraction in ethyl acetate (3 x 50 mL) from 50 mL of NaOH aqueous
solution having molarity 2.5 M. After solvent evaporation the solid residue were
subjected to silica column chromatography by using hexane and ethyl acetate in 9:1 ratio
respectively to get pure tetrahydroporphyrin in 7% yield. In the next step, 160 mg of
tetraphenylporphyrin (0.26 mmoles) was nitrated by continuous stirring for 1 h by adding
90 mg of NaNO2 having molarity 1.3 mM in 10 mL of TFA. Mono-nitrated
phenylporphyrin was obtained as a major product simply by controlling the reaction time
and TFA amount. The reaction was stopped by addition of water and then the product
Part I
41
was extracted in DCM (3 x 25 mL). After vacuum evaporation the pure mono nitrated
derivative was isolated in 30% yield by TLC with hexane and DCM with 1:1 to 1:3 ratio
respectively.
Then 100 mg of mono nitrophenylporphyrin (0.15 mmoles) along with 100 mg of tin
dichloride dehydrate (0.44 mmoles), were added to 0.5 mL of HCl and then the
temperature of reaction mixture was increased up to 60°C and continued for 2.5 h and the
progress of reaction was monitored by TLC. After reaction completion, the reaction
mixture was neutralized by addition of NH4OH and mono aminophenylporphyrin was
obtained in 53% yield after extraction with DCM (3 x 25 mL) and purified by TLC by
using hexane and DCM in 1: 3 to 3:1 ratio respectively. In the final step, 15 mg mannose
(0.079 mmoles) was mixed with 50 mg of mono aminophenylporphyrin (0.079 mmoles)
and the mixture was dissolved in ethanol (8 mL). Then, only three drops of glacial
CH3COOH were added and the reaction mixture was refluxed for 24 h. The product was
precipitated which was then filtered and washed with methanol and DCM separately. The
solid residue was dissolved in DMSO and was then subjected to NMR spectroscopy for
characterization and determination of the product purity. The synthesized product absorbs
electromagnetic radiation in the blue light range around 450 nm.
1.3.7Amoebicidal Assays
The amoebicidal assays were performed for mannose conjugated porphyrin, (12).
Mannose-conjugated porphyrin having molarity 10 µMand volume about 4 µl along with
mannose and porphyrin alone were incubated with A. castellaniifor 1 h in RPMI-1640
(106 amoebae per mL per well) in 24 well plates. After centrifugation the amoebae were
collected at 1,000 x g for about 5 min. After filtration the residue was re-suspended in 0.5
mL of RPMI-1640. Finally, the PYG was used for the re-suspension of amoeba pellet. In
the next step, lids were opened to expose to it to blue light inabiosafety hood for1hat
room temperature using a blue-round LED. Then these plates were incubated at 37°C for
24 h. Then hemocytometer was used for the amoeba counts and a phase contrast inverted-
microscope was used for imaging. The effect of solvents were checked by solvent
controls in which in same volume of DMSO i.e. 4-µl and 20-µl were used for amoebae
Part I
42
incubation, along with 50% chloroform and 50% methanol alone i.e. 1.34-µl and 6.7-µl
volumes of RPMI-1640. While in the positive controls, antiacanthamoebic drugchlorhexidine
was used at 20 µM in RPMI-1640 for incubation of the amoebae which showed 100%
killing of the amoebae.
1.3.8 Cytotoxicity assays of Host Cells
The mannose-conjugated porphyrin was subjected to in vitro antiamoebic assays by using
microvascular endothelial cells of the human brain. Mannose conjugated porphyrin were
incubated with A. castellanii for 1 h in RPMI-1640 (106 amoebae per mL per well) in 24-
well-plates and was exposed to light. After centrifugation the amoebae were collected at
1,000 x g for about 5 min. After filtration the residue was re-suspended in 0.5 mL of
RPMI-1640. Finally, the amoeba was incubated in 24 well plates in a 5% CO2 incubator
at 37 0C on the monolayers of microvascular endothelial cells of the human brain. On the
following day, a cell free accustomed medium was together and measuring of LDH
release give the host cells cytotoxicity assays by cytotoxicity detection kit. The
microvascular endothelial cells of the human brain were used a negative control while
monolayers lysed with 1% Triton X-100 were used as a positive control. The absorbance
value was converted to percent cytotoxicity by using (sample value negative value /high
value negative value) x 100 = % cytotoxicity.
In addition to the pretreatment of amoeba, the antiamoebic potential of mannose
conjugated porphyrin was also evaluated by coincubating microvascular endothelial cells
of the human brain with mannose-conjugated porphyrin and A. castellanii, as described
above. In the next step, the plates were incubated in a 5% CO2 incubator at 37 °C for 24
h, and cell free accustomed medium was gathered and by determining the LDH release,
the host cell cytotoxicity was evaluated.
1.3.9 Excystation Assays
Non-nutrient agar-plates were used for the excystation assays, which were prepared by
using distil water with 3% oxoid agar technical. Trophozoites of A. castellanii
(2x106amoebae) were immunized on the non-nutrient agar plates and accustomed for 15
Part I
43
days at 37 0C in order to allow the differentiation of tropozoites into cysts. No
trophozoites were then observed in phase-contrast inverted-microscope. The agar plates
were then swamped in 15 mL of PBS and keptat room temperature in a shaker for about
30 minutes. The cysts were then scratched off in a 50 mL tube and then were subjected
to centrifuge at 1,500 x g for about 10 minutes. The solid residue was re-suspended in 10
mL PBS. A hemocytometer was used to count the A. castellanii cysts. The excystation
assays were then carried out in order to evaluate the potential of mannose-conjugated
porphyrin on A. castellanii cysts (Lakhundi, Khan et al. 2014). Briefly, A. castellanii
cysts (5 x 105 cysts per mL per well) were accustomed with mannose conjugated
porphyrin having molarity 10 µM, along with mannose and porphyrin alone for about 1
h. The amoebae were gathered, washedwith PBS, and re-suspended in RPMI 1640 in 24
well plates. In the next step, lids were opened in order to exposed plates to
electromagnetic radiations for 1 h. In the final step, the amoebae cysts were gathered and
were then re-suspended in the growth medium (0.5 mL of PYG medium per well) in 24
well plates. The well plates were then incubated for three days at 37°C, which results in
the appearance of growing trophozoites under microscopic. A hemocytometer was used
to counts trophozoites.
1.3.10 Synthesis of thiolated heterocyclic ligands
Scheme 1.13 Synthesis of 1-(3-(acetylthio) propyl)-4-formylpyridinium (4-Py) (31)
Synthesis of 1-(3-bromopropyl)-4-formylpyridin-1-ium (30)
Pyridine carboxaldehyde (0.9 mL, 1 mmole) was stirred in dibromopropane (3 mL as
solvent) at 60 ºC for 8 hours. The brown precipitates formed after 08 hours which were
Part I
44
filtered and washed with 10 mL of methanol. The methanol filtrate was evaporated under
reduced pressure to get the N-alkylated product in viscous liquid state and was
characterized by Mass and NMR spectroscopy without any further purification. Yield 195
mg, 85 %. mp 140-145 ºC ESI-MS 227.96 M+. 1H NMR in MeOD, 9.04 (d, 2H, Ar,
metA), 8.16 (d, 2H, Ar, ortho), 4.89 (t, 2H, N-CH2), 3.52 (t, 2H, Br-CH2), 2.56 (m, 2H,
C-CH2-C). 13C NMR in MeOD, 161.7 (Ar, ipso), 146.3 (Ar, metA), 127.20 (Ar, ortho),
59.0 (CH2-N), 34.7 (CH2-Br), 29.6 (CH2-CH2-CH2).
Synthesis of 1-(3-(acetylthio)propyl)-4-formylpyridin-1-ium (31)
To a stirred solution of 1-(3-bromopropyl)-4-formylpyridin-1-ium (229 mg, 1 mmole) in
ethanol (8 mL; HPLC grade) at room temperature, potassium thioacetate (137 mg, 1.2
mmole) was added and the progress of reaction was monitored with thin layer
chromatography (TLC; dichloromethane: methanol 9:1). After 10 hours the solvent was
evaporated under reduced pressure to obtain brown solid crystalline residue which was
washed with methanol. The 1-(3-(acetylthio) propyl)-4-formylpyridin-1-ium (4-Py)was
recovered from filtrate. Yield 206 mg, 92%. mp 160-165 ºC ESI-MS 223.06 M+. 1H-
NMR in MeOD, 8.96 (d, 2H, Ar, metA), 8.33 (d, 2H, Ar, ortho), 4.65 (t, 2H, N-CH2),
2.92 (t, 2H, Br-CH2), 2.27 (m, 2H, C-CH2-C), 1.89 (s, 3H, CH3CO). 13C-NMR in MeOD,
166.4 (Ar, ipso), 145.1 (Ar, metA), 128.7 (Ar, ortho), 61.4 (CH2-N), 59.2 (CH2-S), 33.5
(CH3-CO), 22.1 (CH2-CH2-CH2).
Scheme 1.14 Synthesis of 1-(3-(acetylthio)propyl)pyrazin-1-ium (Pyr) (33)
Part I
45
Synthesis of 1-(3-bromopropyl)pyrazin-1-ium (32)
Pyrazine (160 mg, 2 mmol) was added in stirring 5 mL of dibromopropane (used as
solvent), and the reaction mixture was heated at 60 0C for 06 hours. The progress of
reaction was monitored with thin layer chromatography (TLC; dichloromethane (DCM):
methanol 9:1). After 06 hours, the excess dibromopropane was evaporated under vacuum
and 1-(3-bromopropyl)pyrazin-1-ium is obtained in viscous liquid state, which was used
without any further purification (378 mg, 94 %). MS (ESI-TOF): m/z: 200.9680 M+. 1H
NMR (300 MHz, MeOD): 9.50 (s, 2H), 9.15 (d, 2H), 4.88 (t, 2 H), 3.56 (t, 2 H), 2.63 (m,
2H). 13C NMR (75 MHz, MeOD): 152.50, 145.18, 62.14, 34.30, 29.04.
Synthesis of 1-(3-(acetylthio)propyl)pyrazin-1-ium (Pyr) (33)
To a stirred solution of 1-(3-bromopropyl)pyrazin-1-ium (202 mg, 1 mmole) in ethanol (8
mL; HPLC grade) at room temperature, potassium thioacetate (137 mg, 1.2 mmole) was
added and the progress of reaction was monitored by TLC (dichloromethane: methanol 9:1).
After 10 hours the solvent was evaporated under reduced pressure to obtain solid residue,
which was washed with DCM and recrystallized with 1:1 mixture of DCM and methanol to
afford 1-(3-(acetylthio)propyl)pyrazin-1-iumas brown crystalline solid (190 mg, 96%).
Melting point 278-282 oC. MS (ESI-TOF) m/z: 197.0686 M+. 1H-NMR (300 MHz, MeOD):
9.47 (s, 2H), 9.13 (s, 2H), 4.74 (t, 2 H), 2.94 (t, 2 H), 2.32 (s, 3H), 2.29 (m, 2H).
Scheme 1.15 Synthesis of ligand 1-(3-(acetylthio)propyl)-4-(dimethylamino)pyridin-1-ium (D2)
Part I
46
Synthesis of 1-(3-(acetylthio)propyl)-4-(dimethylamino)pyridin-1-ium (D2)(34)
Dimethyl aminopyridine (245 mg, 2 mmole), and Dibromomethane (1.6 mL, 16 mmole)
were stirred in 5 mL acetone at 60 oC for 30 minutes. Solvent was evaporated under
vaccou to obtain white precipitates of the mono substituted product 1-(3-bromopropyl)-4-
(dimethylamino) pyridin-1-ium. The precipitates (243 mg, 1 mmole) were dissolved in 5
mL ethanol and potassium thioacetate (140 mg, 1.2 mmole) was added. The reaction
mixture was stirred at 60 oC, while reaction progress was monitored by thin layer
chromatography (TLC; dichloromethane: methanol 9:1). After 10 hours the solvent was
evaporated under reduced pressure to obtain brown solid crystalline residue which were
filtered and washed with 10 mL of methanol. Methanol filterate was evaporated to get the
N-alkylated product (D2) in white crystalline state and was characterized by mass and
NMR spectroscopy. Yield 442 mg, 92%. Melting point 240-242 oC. ESI-MS 277.07
KM+. 1H-NMR in MeOD, 8.16 (d, 2H, Ar, metA), 6.99 (d, 2H, Ar, ortho), 4.21 (t, 2H, N-
CH2), 3.29 (s, 6H, N-CH3), 2.86 (t, 2H, S-CH2), 2.10 (m, 2H, C-CH2-C), 1.89 (s, 3H,
CH3CO). 13C-NMR in MeOD, 180.30 (Ar, ipso), 143.21 (Ar, metA), 109.01 (Ar, ortho),
57.21 (CH2-N), 40.34 (CH2-S), 32.13 (CH3-CO), 31.46 (CH3-N), 24.14 (CH2-CH2-CH2).
Part I
47
References
Abadi, A. H., T. M. Ibrahim, et al. (2009). "Design, synthesis and biological evaluation
of novel pyridine derivatives as anticancer agents and phosphodiesterase 3
inhibitors." Bioorganic & Medicinal Chemistry 17(16): 5974-5982.
Abdelrazek, F. M., N. A.-L. Kassab, et al. (2010). "Synthesis of some novel pyridine and
naphthyridine derivatives." European Journal of Chemistry 1(4): 368-372.
Adler, A. D., F. R. Longo, et al. (1967). "A simplified synthesis for meso-
tetraphenylporphine." The Journal of Organic Chemistry 32(2): 476-476.
Ajitha, M. J., S. Mohanlal, et al. (2012). "DPPH radical scavenging activity of tricin and
its conjugates isolated from "Njavara" rice bran: a density functional theory
study." Journal of Agricultral and Food Chemistry 60(14): 3693-3699 and
references therein.
Alden-Danforth, E., M. T. Scerba, et al. (2008). "Asymmetric cycloadditions of o-
quinone methides employing chiral ammonium fluoride precatalysts." Organic
letters 10(21): 4951-4953.
Amantini, D., R. Beleggia, et al. (2004). "TBAF-catalyzed synthesis of 5-substituted 1 H-
tetrazoles under solventless conditions." The Journal of Organic Chemistry 69(8):
2896-2898.
Aqeel, Y., R. Siddiqui, et al. (2015). "Photochemotherapeutic strategy against
Acanthamoeba infections." Antimicrobial Agents and Chemotherapy: AAC.
05126-05114.
Baig, A. M., J. Iqbal, et al. (2013). "In vitro efficacies of clinically available drugs against
growth and viability of an Acanthamoeba castellanii keratitis isolate belonging to
the T4 genotype." Antimicrobial Agents and Chemotherapy 57(8): 3561-3567.
Part I
48
Barnes, D. M., A. R. Haight, et al. (2006). "New conditions for the synthesis of
thiophenes via the Knoevenagel/Gewald reaction sequence. Application to the
synthesis of a multitargeted kinase inhibitor." Tetrahedron 62(49): 11311-11319.
Beckman, K. B. and B. N. Ames (1998). "The Free Radical Theory of Aging Matures."
Physiological Reviews 78(2): 547-581.
Bogdanowicz-Szwed, K. and M. Krasodomska (2006). "Synthesis of aryl-and pyridinyl-
substituted 2-amino-6-thioxopyridine-3-carbonitrile derivatives by tandem
Michael addition and cyclization reactions." Monatshefte für Chemie/Chemical
Monthly 137(3): 347-355.
Boucher, L. J. and J. J. Katz (1967). "Aggregation of metallochlorophylls." Journal of the
American Chemical Society 89(18): 4703-4708.
Burne, R. A. and Y.-Y. M. Chen (2000). "Bacterial ureases in infectious diseases."
Microbes and Infection 2(5): 533-542.
Chen, J., L. Keltner, et al. (2002). "New technology for deep light distribution in tissue
for phototherapy." The Cancer Journal 8(2): 154-163.
Chen, Z., S. Xuguang, et al. (2008). "In vitro amoebacidal activity of photodynamic
therapy on Acanthamoeba." British Journal of Ophthalmology 92(9): 1283-1286.
Chung, K., B. Moon, et al. (2006). "Oxidation of aromatic aldehydes with
tetrabutylammonium fluoride: Competition with the cannizzaro reaction."
Bulletin of Korean Chemical Society 27(8): 1203.
Clarke, D. W. and J. Y. Niederkorn (2006). "The pathophysiology of Acanthamoeba
keratitis." Trends in Parasitology22(4): 175-180.
Corey, E. and B. B. Snider (1972). "Total synthesis of (+-)-fumagillin." Journal of the
American Chemical Society 94(7): 2549-2550.
Part I
49
Cox, D. P., J. Terpinski, et al. (1984). "" Anhydrous" tetrabutylammonium fluoride: a
mild but highly efficient source of nucleophilic fluoride ion." The Journal of
Organic Chemistry 49(17): 3216-3219.
Cui, H., Y. Kong, et al. (2012). "Oxidative Stress, Mitochondrial Dysfunction, and
Aging." Journal of Signal Transduction 2012.
Dunkel, S., U. Hess, et al. (1997). "Synthese von cyanosubstiuierten Di‐und
Tetrahydropyridinen in DIMCARB (Dimethylamin–CO2‐Additionsverbindung)."
Journal für Praktische Chemie/Chemiker‐Zeitung 339(1): 414-419.
Ferro, S., O. Coppellotti, et al. (2006). "Photosensitized inactivation of Acanthamoeba
palestinensis in the cystic stage." Journal of Applied Microbiology 101(1): 206-212.
Garate, M., Z. Cao, et al. (2004). "Cloning and characterization of a novel mannose-
binding protein of Acanthamoeba." Journal of Biological Chemistry 279(28):
29849-29856.
Garate, M., I. Cubillos, et al. (2005). "Biochemical characterization and functional
studies of Acanthamoeba mannose-binding protein." Infection and Immunity
73(9): 5775-5781.
Hamblin, M. R. and T. Hasan (2004). "Photodynamic therapy: a new antimicrobial
approach to infectious disease?" Photochemical & Photobiological Sciences 3(5):
436-450.
Hargreaves, S. L., B. L. Pilkington, et al. (2000). "The synthesis of substituted
pyridylpyrimidine fungicides using palladium-catalysed cross-coupling
reactions." Tetrahedron Letters 41(10): 1653-1656.
Hiroya, K., R. Jouka, et al. (2001). "Cyclization reactions of 2-alkynylbenzyl alcohol and
2-alkynylbenzylamine derivatives promoted by tetrabutylammonium fluoride."
Tetrahedron 57(48): 9697-9710.
Part I
50
Hoare, D. and H. Heath (1960). "Dipyrromethanes and the biosynthesis of porphyrins."
Biochimica et Biophysica Acta 39(1): 167-169.
Hodgkin, D. C. (1979). New and Old Problem in the Structure Analysis of Vitamin B12.
Vitamin B12, Proceedings of the Third European Symposium on Vitamin B12
and Intrinsic Factors, Walter de Gruyter: Berlin, Germany.
Holiman, P. C. H., M. G. L. Hertog, et al. (1996). "Analysis and health effects of
flavonoids." Food Chemistry 57(1): 43-46.
Huang, L., T. Dai, et al. (2010). Antimicrobial photodynamic inactivation and
photodynamic therapy for infections. Photodynamic Therapy, Springer: 155-173.
Hunt, E. J., C. E. Lester, et al. (2001). "Effect of St. John's wort on free radical
production." Life Sciences 69(2): 181-190.
Johnson, D. S. and J. J. Li (2007). The Art of Drug Synthesis. New Jersey, John Wiley &
Sons, Inc.
Knoevenagel, E. (1898). "Condensation von Malonsäure mit aromatischen Aldehyden
durch Ammoniak und Amine." Berichte der Deutschen Chemischen Gesellschaft
31(3): 2596-2619.
Kruper Jr, W. J., T. A. Chamberlin, et al. (1989). "Regiospecific aryl nitration of meso-
substituted tetraarylporphyrins: a simple route to bifunctional porphyrins." The
Journal of Organic Chemistry 54(11): 2753-2756.
Lakhundi, S., N. A. Khan, et al. (2014). "Inefficacy of marketed contact lens disinfection
solutions against keratitis-causing Acanthamoeba castellanii belonging to the T4
genotype." Experimental Parasitology 141: 122-128.
Lemberg, R. and J. Barrett (1973). "Cytochromes." Journal of Cell Science 13: 1.
Part I
51
Lichitsky, B., A. Dudinov, et al. (2001). "Reaction of 3-aminocyclohex-2-en-1-ones with
arylidenemalononitriles: synthesis of N-substituted 1,4,5,6,7,8-hexahydro-
quinolin-5-ones."Arkivoc 9: 73-79.
Lindsey, J. S., H. C. Hsu, et al. (1986). "Synthesis of tetraphenylporphyrins under very
mild conditions." Tetrahedron Letters 27(41): 4969-4970.
Malicorne, G., J. Bompart, et al. (1991). "Synthèse et activité anti-bactérienne d'acides 4,
7-dihydro-4-éthyl-7-oxothiéno (3, 2-B) pyridine-6-carboxyliques." European
Journal of Medicinal Chemistry 26(1): 3-11.
Marciano-Cabral, F. and G. Cabral (2003). "Acanthamoeba spp. as agents of disease in
humans." Clinical Microbiology Reviews 16(2): 273-307.
Martínez-Martínez, F., R. Razo-Hernández, et al. (2012). "Synthesis and in vitro
Antioxidant Activity Evaluation of 3-Carboxycoumarin Derivatives and QSAR
Study of Their DPPH• Radical Scavenging Activity." Molecules 17(12): 14882-
14898 and references therein.
Menna, P., O. Gonzalez Paz, et al. (2012). "Anthracycline cardiotoxicity." Expert
Opinion on Drug Safety11(S1): S21-S36.
Merritt, J. and K. Loening (1979). "Nomenclature of tetrapyrroles." Pure and Applied
Chemistry 51: 2251-2304.
Meyer, A. S., M. Heinonen, et al. (1998). "Antioxidant interactions of catechin, cyanidin,
caffeic acid, quercetin, and ellagic acid on human LDL oxidation." Food
Chemistry 61(1–2): 71-75.
Milgrom, L. R. (1997). The Colours of Life: An Introduction to the Chemistry of
Porphyrins and Related Compounds, Oxford University Press Oxford.
Mobley, H., M. D. Island, et al. (1995). "Molecular biology of microbial ureases."
Microbiological Reviews 59(3): 451-480.
Part I
52
Morales, F. J. and S. Jiménez-Pérez (2001). "Free radical scavenging capacity of Maillard
reaction products as related to colour and fluorescence." Food Chemistry 72(1):
119-125.
Mosmann, T. (1983). Journal of Immunological Methods 65: 55-63.
Moure, A., J. M. Cruz, et al. (2001). "Natural antioxidants from residual sources." Food
Chemistry 72(2): 145-171.
Ojala, W. H., J. M. Ostman, et al. (2000). "Schiff bases or glycosylamines: crystal and
molecular structures of four derivatives of d-mannose." Carbohydrate Research
326(2): 104-112.
Pérez-Santonja, J. J., S. Kilvington, et al. (2003). "Persistently culture positive
Acanthamoeba keratitis: in vivo resistance and in vitro sensitivity."
Ophthalmology 110(8): 1593-1600.
Rice-Evans, C. A., N. J. Miller, et al. (1996). "Structure-antioxidant activity relationships
of flavonoids and phenolic acids." Free Radical Biology and Medicine 20(7): 933-
956.
Rothemund, P. (1939). "Porphyrin Studies. III. 1 The Structure of the Porphine2 Ring
System." Journal of the American Chemical Society 61(10): 2912-2915.
Selbo, P., a. l. Kristian, et al. (2002). "Photochemical internalisation: a novel drug
delivery system." Tumor Biology 23(2): 103-112.
Shaheen, F., M. Ahmad, et al. (2005). "Alkaloids of Aconitum laeve and their anti-
inflammatory, antioxidant and tyrosinase inhibition activities." Phytochemistry 66
(8): 935-940.
Silva, J. N., P. Filipe, et al. (2006). "Photodynamic therapies: principles and present
medical applications." Bio-medical Materials and Engineering 16: S147-S154.
Part I
53
Smith, K. M. and J. E. Falk (1975). Porphyrins and Metalloporphyrins, Elsevier
Amsterdam.
Stins, M. F., F. Gilles, et al. (1997). "Selective expression of adhesion molecules on
human brain microvascular endothelial cells." Journal of Neuroimmunology
76(1): 81-90.
Szökő, É. and T. Tábi (2010). "Analysis of biological samples by capillary
electrophoresis with laser induced fluorescence detection." Journal of
Pharmaceutical and Biomedical Analysis 53(5): 1180-1192.
Talelli, M., K. Morita, et al. (2011). "Synthesis and characterization of biodegradable and
thermosensitive polymeric micelles with covalently bound doxorubicin-
glucuronide prodrug via click chemistry." Bioconjugate Chemistry22(12): 2519-
2530.
Toyota, K., H. Shinkai, et al. (1993). Anticarcinogenic agents; potentiation, Google
Patents.
Turner, N., A. Russell, et al. (2004). "Resistance, biguanide sorption and
biguanide‐induced pentose leakage during encystment of Acanthamoeba
castellanii." Journal of Applied Microbiology 96(6): 1287-1295.
Villaño, D., M. S. Fernández-Pachón, et al. (2007). "Radical scavenging ability of
polyphenolic compounds towards DPPH free radical." Talanta71(1): 230-235.
Visvesvara, G. S., H. Moura, et al. (2007). "Pathogenic and opportunistic free‐living
amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and
Sappinia diploidea." FEMS Immunology & Medical Microbiology 50(1): 1-26.
Volm, M., J. Mattern, et al. (1991). "Overexpression of P-glycoprotein and glutathione S-
transferase-pi in resistant non-small cell lung carcinomas of smokers." British
Journal of Cancer 64(4): 700.
Part I
54
Weatherburn, M. W. (1967). Analytical Chemistry 39: 971.
Williams, R., E. Spencer, et al. (2004). "Flavonoids: antioxidants or signalling
molecules?" Free Radical Biology and Medicine 36(7): 838-849.
Willstatter, R. and A. Stoll (1928). "Investigations on Chlorophyll: Methods and
Results."
Yasuhara, A., N. Suzuki, et al. (2002). "A concise synthesis of furostifoline by
tetrabutylammonium fluoride-promoted indole ring formation." Chemical and
Pharmaceutical Bulletin-Tokyo 50(1): 143-145.
Part II
55
CHAPTER 2
SYNTHESIS OF METAL NANOPARTICLES AND THEIR
APPLICATIONS
2.1 INTRODUCTION
2.1.1 Introduction of Nanotechnology
The concept behind nanoscience and nanotechnology started with a talk entitled “There’s
Plenty of Room at the Bottom” by physicist Richard Feynman at an American Physical
Society meeting held on December 29, 1959 at the California Institute of Technology
(CalTech). Feynman described the idea to manipulate and control individual atoms and
molecules. Over a decade later, Professor Norio Taniguchi coined the term
nanotechnology in his explorations of ultra-precision machining. Later on, with the
development of the atomic and electron microscopes in 1980s the modern
nanotechnology began.
Nanotechnology is emerging as a broad field in almost all field of science these days
especially in chemistry. It has the ability to produce materials, devices, and systems
having new highly controlled and fascinating properties by functioning at the atomic as
well as molecular levels. It utilizes exceptional physical and chemical properties that
appear when matter is constructed at the nanoscale (Blonder 2010).
The nanomaterials are distributed in vast variety according to their properties and
applications.Interface and colloid science has given rise to many materials which are
being usefully studied in nanotechnology, such as fullerenes, carbon nanotubes,
nanoparticles and nanoclusters.
2.1.2 Fullerenes
Fullerenes are molecules composed of carbons in the form of a hollow sphereand many
other shapes. Fullerenes are graphite analogues, which are composed of piled graphene
sheets of linked hexagonal rings, but the difference between graphene and fullerenes is
that fullerenes may also contain pentagonal or sometimes heptagonal rings. The first
Part II
56
fullerene, buckminsterfullerene or buckyball C60, was discovered in 1985 by Richard
Smalley, Robert Curl, James Heath, Sean O'Brien, and Harold Kroto. The encounter of
fullerenes greatly stretched the number of known carbon allotropes, which were limited
to graphite, diamond, and amorphous carbon. Buckyballs and buckytubes have been the
subject of concentrated research, both for their unique chemistry and for their
applications in materials science, electronics, and nanotechnology. The other known
natural fullerenes are C70, C76, C82 and C84 molecules, which are formed by lightning
discharges in the atmosphere from soot in trace amounts.
Figure: 2.1 Structure of a Fullerene
2.1.3 Carbon Nanotubes
Carbon nanotubes (CNTs) are carbon allotropes having a cylindrical nanostructure. The
graphite layer appears somewhat like a rolled-up chicken wire with a continuous
unbroken hexagonal mesh and carbon molecules at the apexes of the hexagons. Carbon
Nanotubes have several structures, which differ in length, thickness, and in the type of
number of layers. There are two main types of CNTs those are simply based on number
of walls, single walled carbon nanotubes (SWCNT) and multi walled carbon nanotubes
(MWCNT). Although they are formed from essentially the same graphite sheet, their
electrical characteristics differ depending on these variations, acting either as metals or as
semiconductors. As a group, Carbon Nanotubes typically have diameters ranging from <1
nm up to 50 nm. Individual nanotubes naturally align themselves into "ropes" held
together by van der Waals forces, more specifically, pi-stacking. These supramolecular
Part II
57
forces and unique structural features of CNTs make them ideally strong and excellent
conductor of both charge and heat.
Figure: 2.2 Depiction of CNTs
The recent literature is extremely rich with the examples and applications of CNTs. For
example, the H-bonding ability of cyclic peptides makes them striking candidate for the
formation of supramolecular nanotubes. Peptide nanotubes exist as tubular beta-sheet-like
structures which are formed by the self-association of flat, ring-shaped peptide subunits
made up of alternating D- and L-amino acid precursors. Peptide self-assembly formed by
extensive network of intersubunit hydrogen bonds (Ghadiri, Granja et al. 1993; Kim, Suh
et al. 2002).In another example, Calix[4]hydroquinone also self-assemble by H-bonding
and form nanotubes. In additon to hydrogen bonding, aromatic rings in Calix[4]
hydroquinone also displayed pi-pi stacking(Semetey, Didierjean et al. 2002).
2.1.4 Nanoparticles
Nanoparticles are nanodimensional materials (in the 1–100 nm size domains) which act
as a bridge between atomic and bulk materials and have been shown to exhibit a variety
of unique chemical, physical and electronic properties (Matsukawa, Watase et al. 2006).
As nanoparticles are essentially finely divided bulk materials, they are typically
thermodynamically unstable with respect to agglomeration. Consequently, they need to
Part II
58
be kinetically stabilized and this is typically done using a protective stabilizer. The
stabilization is achieved by electrostatic or steric forces or a combination of the two
known as “electrosteric forces”. The stabiliser is typically introduced during the
formation of the nanoparticles, the stabilizer may be organic ligands, surfactants,
polymers and dendrimers (Cookson 2012).
These NPs can be composed of a variety of materials including noble metals e.g. Au
(Boisselier and Astruc 2009) , Ag (Lu, Tung et al. 2008) (Podsiadlo, Paternel et al. 2005)
, Pd (Ung, Tung et al. 2009), semiconductors e.g. CdSe, CdS, ZnS (Boisselier and Astruc
2009) (Jamieson, Bakhshi et al. 2007), TiO2 (Drbohlavova, Adam et al. 2009), PbS
(Rogach, Eychmüller et al. 2007), InP (Rogach, Eychmüller et al. 2007), Si (O'Farrell,
Houlton et al. 2006). Metal nanoparticles can be synthesized by use of reducing agent
(Sun and Zeng 2002). Nanoparticles can be synthesized both in organic and aqueous
solution. Nano-structure materials including Au (Turkevich, Stevenson et al. 1951)
(Wang, Sato et al. 2003), Ag (Podsiadlo, Paternel et al. 2005), Co(Lu, Tung et al. 2008),
Nix(Verma, Giri et al. 2010), Fe2O3, FeO (OH) (Gnanaprakash, Mahadevan et al. 2007) ,
and CdTe (Bao, Wang et al. 2006) (Rogach 2000) have been synthesised in aqueous
solution. NPs composed of noble metals, transition metals, oxides and semiconducting
materials (Lu, Tung et al. 2008) (Brust, Walker et al. 1994) (Gittins and Caruso 2001)
(Sun, Murray et al. 2000) (Murray, Norris et al. 1993) have been synthesized in organic
solvents. Whilst much research has focussed on nanomaterials of the coinage metals
(especially those of gold) (Daniel and Astruc 2004). The most important of these metal
based nanoparticles are of noble metals such as gold and silver due to their
biocompatibility and photophysical properties.
Gold is the subject of interest in science from long time ago. Several gold catalysts and
compounds are formed in recent years that entertain chemistry to a variety of uses.
Recently a trend of gold compounds formation and their applications is developed in the
context of emerging nanosciences and nanotechnology with both nanoparticles and self-
assembled monolayers (SAMS) (Daniel and Astruc 2003). Gold nanoparticles are formed
by reduction of gold salts, conventionally of gold (III) derivatives. Turkevitch in 1951
reduced HAuCl4 in water using citrate (Turkevich, Stevenson et al. 1951). It leads to
Part II
59
AuNPs of size 20 nm. Size controlled methods consist of maintaining the ratio between
reducing/stabilizing agents (Frens 1973). One of the methods for gold nanoparticles
production is the Brust-Schiffrin two phase synthesis, the ratio between thiol/gold
determines the average size of forming nanospheres again. Larger ratios along with faster
addition of the reducing agent in cooled solutions give smaller Nanospheres (Brust,
Walker et al. 1994). AuNPs stabilized with thiols are of significant current interst due to
their ease in formation and substantial stabilities (Hutchings, Brust et al. 2008). Jadzinsky
et al., studied the structural features of AuNPs by X-Rays diffraction (Ackerson,
Jadzinsky et al. 2005; Jadzinsky, Calero et al. 2007).
Silver is another metal that has been widely used for nanoparticles synthesis since it is
relatively cheaper than the gold. Silver nanoparticles have been synthesized by a range of
different physical procedures such as evaporation/condensation technique (Kruis et. al
2000) and by using laser (Mafune et. al 2001), while chemical methods involve the
reduction of silver salts (Tao et. al 2006., Wiley et. al 2005).
Other metals based nanoparticles include zinc(Hattori, Mukasa et al. 2011), palladium
(Corthey, Rubert et al. 2012) etc. Metal oxide nanoparticles are also an important
paradigm in the nanotechnology; common examples are Fe (III) oxide (Rosen, Chan et al.
2012), titanium oxide (Shiraishi, Ikeda et al. 2011), zinc oxide etc. Some rare earth doped
nanoparticles are also reported (Bouzigues, Gacoin et al. 2011).
2.1.5 Nanoclusters
A group of atoms when reaches a size just too big to be called a molecule and too small
to show characteristic bulk properties and ranges between sub nm to 10 nm is termed a
nanocluster. Ideally the number of atoms is less than 100. Nanoclusters exhibit a
completely different behaviour from that predicted by scaling. There unique geometric
structure and quantized electronic states give them an exclusive set of properties. The
increase in number of atoms and consequent size play a vital role in controlling other
parameters responsible for the behaviour of these clusters. Individual clusters can be
assembled to build sub units for novel functional materials. The behaviour of
Part II
60
nanoclusters can be individually studied only when they are protected by a ligand to that
separated each cluster to avoid coalescence.
2.1.6 Synthesis of Nanoparticles and Nanoclusters
There are two main approaches which are most commonly employed to synthesize the
nanomaterials.
2.1.6.1 Bottom-up Approach
In this approach some chemical modifications utilized to arrange the smaller molecules
into complex molecular assemblies. These molecular self-assemblies basically operate on
the basic supramolecular interactions mainly so that a single molecule may arrange into a
useful conformations for the formation of larger assemblies.
2.1.6.2 Top-down Approach
Top-down approach is based on getting larger components to construct self-assembled
smaller molecular assemblies. In this methodology, physical methods for example laser
ablation etc. are mainly utilized to obtain the nanomaterials.
2.1.7 Dispersity in Nanoscale Synthesis
The vast applications and straightforward synthesis of nanostructures has made such a
fast growing area of research and synthesis is carried on every scale in every field.
Although the synthesis is simple there exist some limitations when it comes to size
selectivity and precision. Clusters of a limited size distribution and controlled nuclearity
have been obtained through different methods of separation as well as target oriented
synthesis but a technique that could yield monodisperse nanoclusters in bulk quantities is
yet to be devised.
2.1.8 Applications of Nanoparticles
Nanoparticles are explored in a variety of ways like their assessment of the in vivo
toxicity is also studied (Chen, Hung et al. 2009). Their use in in vitro assays, in vitro and
Part II
61
in vivo imaging, cancer therapy, and drug delivery are the hot topics in research these
days (Chen, Huang et al. 2008). Gold nanoparticles are very efficient drug delivery
systems because gold can easily be fabricated(Liu, Zhang et al. 1998). Other properties of
different metal based nanoparticles are surface Plasmon band (SPB) (Craig F. Bohren
1983), Fluorescent biological labelling (Bruchez, Moronne et al. 1998), Electrochemistry
(Miles and Murray 2003), DDSs (Mah C 2000), used for probing of DNA structures
(Mahtab, Rogers et al. 1995), used in hyperthermia (Shinkai, Yanase et al. 1999), used as
MRI contrast enhancers (Weissleder, Elizondo et al. 1990) etc. While their applications
in catalysis, supramolecular chemistry (Tshikhudo, Demuru et al. 2005; Ohyama, Hitomi
et al. 2009), molecular recognition (Xiao, Gao et al. 2012), biomarker detectors (Hong,
Huh et al. 2012), as sensors for example biosensors in food analysis (Pérez-López and
Merkoçi 2011), gas sensing (Sun, Liu et al. 2012), environmental applications include the
heavy metals removal from water (Hua, Zhang et al. 2012), automobile catalytic
convertor (Zhang, Cattrall et al. 2011), and their remarkable use in clinical diagnostic
(Larguinho and Baptista 2012).
2.1.9 Nanoclusters in Biomedical Sciences
2.1.9.1 Biological Imaging
Flourescence imaging technique offers certain advantages over other imaging techniques
like sensitivity and cost. Flourescent metal nanoclusters are excellent flourophores for
biological imaging due to their remarkably biocompatibile size and bright emission.
Human mesenchymal stem cells have been imaged by a 2 photon imaging technique
using dextran encapsulated Au nanoclusters as flourophores.
In vivo fluorescence imaging has also been reported in animals with bovine serum
albumen stabilized ultra-small Au clusters. High stability and low toxicity of the Au
nanoclusters have qualified them for use in continuous imaging. The nanoclusters were
intravenously injected into the mice then subjected to fluorescence imaging. The
fluorescent nanoclusters were soon visibly accumulated in the tumour sights where
permeability and retention have been enhanced. The nanoclusters stay in the living body
for 24 hours and afterwards are easily excreted through kidney filtration.
Part II
62
2.1.9.2 Biodetection
Good solubility in water, high emission, low toxicity are some of the reasons that make
nanoclusters so suitable for biodetection of different analytes in living systems.
Detection of Nucleic Acids
Among the other factors scaffolding is a main factor that effect the synthesis and
properties of nanoclusters. The emission properties of DNA-Ag nanoclusters have been
studied. As the nucleotide sequence changes, the fluorescence properties of nanoclusters
has also been changed this has led to the development of a new assay to determine single
nucleotide modification in DNA. In another assay wherein dark DNA-Ag clusters can
light up to bright red fluorescent clusters when placed in proximity of guanine rich
sequence of DNA.
Detection of Proteins
Metal nanoclusters have been proven to be excellent fluorescent protein sensors.
PAMAM encapsulated Nanocluster has been established as a detector of various protein
structures like, polyclonal goat driven antihuman antibody IgG, concanavalin A,
immunoglobulin G platelet drived growth factor AA, glutathione S-transferase tagged
protein etc.
Detection of other Biomolecules
Some other bio molecules with thiol functional group such as cysteine, homocysteine and
glutathione are very important biological analytes. In a certain bioassay Ag nanoclusters
are used for determination of cysteine among other amino acids. Fluorescence of Ag
nanoclusters can be successfully quenched with cysteine and not by other amino acids.
BSA-stabilised gold nanoclusters have been reported to detect glucose. The mechanism
of glucose oxidation involves production of H2O2 that can quench fluorescence of the
BSA-Au nanoclusters.
Part II
63
2.1.10 Chemosensing of Cu(I)
Since copper is one of the most abundant transition metal and is used in biological and
industrial systems. Selective recognition of copper has attained tremendous attention due
to its key role in several fundamental physiological processes including enzyme function
and transcriptional events. However elevated concentrations of copper ions in biological
systems can cause adverse health issues such as cytotoxicity and diseases like kidney and
liver damage and some neurodegenerative diseases are also caused by copper ions
imbalance (Scheinberg and Sternlieb 1996; Uauy, Olivares et al. 1998). Copper (I) ions
usually disproportionate rapidly in aqueous media and the most common example of
Cu(I) compound is cuprite ore (Cu2O) from which metallic copper is extracted. Due to
relative instability and low solubility of Cu(I) as compared to Cu(II), most of the
detection systems of copper are of Cu(II), which includes electrochemical (Yantasee,
Hongsirikarn et al. 2008), mass spectrometric (Siripinyanond, Worapanyanond et al.
2005), atomic absorption spectroscopic methods (Xiang, Zhang et al. 2010). Some
colorimetric assays are also recently reported (Kirubaharan, Kalpana et al. 2012; Deng,
Li et al. 2014) along with other optical systems as well.
2.1.11 Chemosensing of Fe(III)
Fe(III) is one of the most abundant metals on planet Earth and plays a significant role in
many biological and environmental systems, while it is the most abundant, vital transition
metal ion in the human body. As an essential part of heme groups, several proteins use
Fe(III) for oxygen and electron transportation, as well as for a variety of enzymatic
reactions (Thomson, Rogers et al. 1999). Fe(III) distribution in the human body is needed
to be critically regulated, due to its high potential for biological toxicity and many other
reasons. The cellular toxicity of Fe(III) ions has been associated with severe diseases,
including Alzheimer’s, and Parkinson’s disease (Andrews 2000). A lack of Fe(III) can
lead to anaemia, a disorder in which there are too few red blood cells, moreover, Fe(III)
deficiency limits oxygen delivery to cells, which results in fatigue, and decreased
immunity (Umbreit 2005). Conversely, excess of Fe(III) ions level in cells can catalyse
the production of reactive oxygen species (ROS), which can ultimately damage proteins,
Part II
64
nucleic acids and lipids (Tripathy, Woo et al. 2013). Furthermore, Fe(III) is a common
contaminant often found in the waste water released from the industrial sites. Fe(III) is
usually found in its more stable oxidized form that is Fe(III), which is nondegradable,
however the low solubility of Fe(III) does not give the necessary sensitivity for detection
in water.
Drinking water is a common source of heavy metal intake for humans. The toxicological
and carcinogenic effects of ultra-trace heavy metals can upset the central nervous system,
kidney, liver, skin, lungs, and bones (Kaplan, Yildirim et al. 2011). The detection and
removal of trace amounts of toxic metals in drinking water is an important factor in
monitoring environmental pollution and their adverse outcomes (El-Safty and Shenashen
2013).
Based on above discussion, the monitoring of metal ions traces in waters, soil surfaces,
and living beings, has become more and more important in recent times. Thus a variety of
methods have been developed for quantification of Fe(III) concentrations (Wang, Hai et
al. 2010; Wang, Yu et al. 2012; Mehta, Kailasa et al. 2014), flame atomic absorption
spectroscopy (FAAS) (Ajlec and Štupar 1989), ICPMS (Huang and Lin 2001), and
spectrophotometric detection using organic dyes or quantum dots(Wu, Li et al. 2009; Ju
and Chen 2014). To date, most Fe(III) sensing protocols depend on the fluorescence
quenching mechanism because of the paramagnetic character of ionic Fe(III), which
unavoidably give high background signals (Piao, Lv et al. 2014). The general drawbacks
of these techniques are that they often difficult to synthesize fluorescent probe, tedious
sample pretreatment, and sophisticated instruments (Annie Ho, Chang et al. 2012). The
selective optical sensing strategy has been striking due to its ultra-sensitivity, rapid
response, non-destructive nature and cost-effective manner (Bindhu and Umadevi).
2.1.12 Chemosensing of Pd(II)
Palladium contamination comes in environment mainly from two sources; one is by the
use of palladium in the catalytic converters of automobiles that release palladium, and the
other is by the residual palladium of synthetic intermediates in active pharmaceutical
products (Garrett and Prasad 2004; Magano and Dunetz 2011; Prichard and Fisher 2012).
Part II
65
Literature reveals that traces of palladium species in body organs interrupt a variety of
cellular processes and cause severe physiological disorders which includes loss of
memory, dizziness, migraine, immune system impairment, and paralysis (Kielhorn,
Melber et al. 2002). Therefore, the threshold level of palladium in pharmaceutical
products is strictly limited to be 5–10 ppm, while the maximum daily human dietary
intake of palladium is restricted to 1.5–15 mg (Carey, Laffan et al. 2006). It plays an
essential part in the manufacture of dental and medicinal devices, jewelry and automobile
(Iwasawa, Tokunaga et al. 2004). Palladium is known to bind with thiol containing
proteins, DNA and vitamin, then interferes the normal cell metabolism (Kielhorn, Melber
et al. 2002).
Palladium is a heavy transition metal that plays a critical role in synthetic materials and
chemical reactions. Palladium catalyzed reactions such as the Heck, Sonogashira, and
Suzuki- Miyaura reactions are turning increasingly important because of the influence of
making difficult covalent bonds. However, even after purification, residual palladium is
often found in the final product, which may cause health hazard (Song, Garner et al.
2007). Palladium compounds which are excessively used in the organic synthesis
reactions are Pd (PPh3)4, PdCl2, and Pd (OAC)2, while among these, PdCl2 is the most
toxic (Liu, Lee et al. 1979). Because of its toxicity but importance, the monitoring of Pd
traces in water, soil, plants, and physiologically important samples has become
increasingly important. Thus, the more traditional methods including AAS, ICP AES,
ICPMS, polarography, and voltammetry has been utilized for Pd(II) analysis over the past
few years (Wang and Varughese 1987; Zhao and Gao 1988; Locatelli 2006; Locatelli
2007; Gao, Koshizaki et al. 2009). But since all of these techniques in general require
expensive and dedicated equipments, tedious sample pretreatment, the use of toxic
reagents, and highly analytical expertise. Therefore, producing a facile, robust,
inexpensive, and sensitive method that permits real-time detection of Pd(II) ions is still a
challenging goal.
In recent years, several colorimetric and fluorimetric methods for detection of palladium
are reported because they are relatively more simple and nondestructive in nature
(Huang, Wang et al. 2004; Tang, Zhang et al. 2004; Schwarze, Müller et al. 2007; Song,
Part II
66
Garner et al. 2007; Duan, Xu et al. 2008; Jiang, Jiang et al. 2011; Zhou, Zhang et al.
2012). But most of the fluorescent sensors for palladium are based on the coordination
mechanism which are partially quenched due to the paramagnetic nature of the palladium
ion, and would suffer from varying degrees of interference from other transitional metal
ions. Among them, the reaction-based fluorescent sensing approaches have shown great
advantages in selectivity and sensitivity. However, these sensing systems have been
suffering from the demerits like synthesis of multi steps and expensive fluorescence
scaffolds, long response time, laborious test conditions etc., which greatly restrict their
practical applications. Therefore, the designing of some novel and effective chemosensor
for palladium detection with rapid response and high selectivity and sensitivity under
mild conditions is required for further developments (Ren, Pradhan et al. 2012).
2.1.13 Chemosensing of Antibiotics
6-Aminopenicillanic Acid (6-APA)
6-APA is the core of Beta lactam antibiotic class of compound penicillins. The penicillins
are being used commonly for their antimicrobial activity against both gram positive and
gram negative organisms (Wishart 1984). However, pencillins have limited stability, and
are susceptible to form different degradation products (Tyczkowska, Voyksner et al.
1992). The presence of degradation and related substances may cause the reduced activity
of the drug, as well as some side effects in humans. These antibacterial agents can
inactivate penicillin-binding proteins (PBP), which are used in the synthesis of bacterial
cell walls through the formation of stable covalent acyl complexes. As an outcome of this
irreversible substrate binding, the cell-wall-synthesizing activity of PBP is strongly
inhibited, which results into cell death. (Matagne, Lamotte-Brasseur et al. 1998; Fisher,
Meroueh et al. 2005). On the other hand, the occurrence of these antibiotics applied for
human or veterinary use in lakes and streams all over the world has created an alarming
situation due to the toxicity of these drugs for aquatic creature (Wilson, Smith et al. 2003;
Sanderson, Johnson et al. 2004). The common sources of their entry into the environment
include the excretion in urine, the wash off of topical treatments of livestock animals and
the pharmaceutical industrial unsafe waste. Hence, during our recent work of chemical
analyses of the drinking water system of Karachi, Pakistan we found a large number of
Part II
67
pharmaceuticals drugs in considerably high concentrations (Scheurell, Franke et al. 2009;
Selke, Scheurell et al. 2010).
Currently the most common analytical procedures which are employed for the detection
and/or determination of beta lactam antibiotics involve spectrometry (Abdalla 1991;
Devani, Patel et al. 1992), voltammetry (Ali and Mohammad 1994; Leszczynska, Koncki
et al. 1996), high-performance liquid-chromatographic (HPLC)(Pinto, Pavon et al. 1995),
(LC-MS) ( a -Cruz, López de Alda et al. 2003; Poole 2003), chemiluminescence
method(Yang, Zhou et al. 1998), and NMR (Shamsipur, Talebpour et al. 2002). Some
fluorimetric determination of beta lactamic antibiotics has also been described mainly
based on the formation of fluorescent degradation products either after alkaline or acid
hydrolysis (Murillo, Lemus et al. 1994; Yang, Zhou et al. 1998).
Pefloxacin (Pef)
Pef, 1-ethyl-6-fluoro-1, 4-dihydro- 7-(4-methylpiperazin-1-yl)-4-oxoquinolone-3-carboxylic
acid, is a fluoroquinolone antibacterial agent. Fluoroquinolones are effective against most
Gram-negative and -positive aerobic bacteria (Swoboda, Oberdorfer et al. 2003; Cao, Sui
et al. 2011), and also they have some activity against mycobacteria, mycoplasmas,
rickettsias and the protozoan Plasmodium falciparum (Sharma, Khosla et al. 1994). It is
used for the treatment for diseases of the skin and various kinds of urinary tract infections
and has been widely applied in clinical medicine (Ooishi and Miyao 1997).
Determination methods for Pef include spectrofluorometry (Veiopoulou, Ioannou et al.
1997; El-Kommos, Saleh et al. 2003), HPLC (Montay, Blain et al. 1983; Santoro, Kassab
et al. 2006), and capillary electrophoresis (Yang, Wang et al. 2008; Deng, Li et al. 2009),
nuclear magnetic resonanc e(Fardella, Barbetti et al. 1995), capillary electrophoresis
tandem mass spectrometry (Lara, García-Campaña et al. 2006), UV-vis (Mostafa, El-
Sadek et al. 2002; Sun, Wu et al. 2013), FT-IR(Xie, Song et al. 2010). However, these
methods require extensive sample preparation as well as highly trained individuals to
operate complicated instruments and understand complex chromatogram or spectral
results. Therefore, these traditional methods, although extremely accurate, are time-
consuming, expensive, and usually not suitable for use in the field. However, these
Part II
68
methods are often time-consuming or require substantial expertise in comparison with
spectrophotometry.
PEF had been used increasingly more in the aquaculture industry to treat and prevent a
variety of infectious diseases, but its overuse runs the risk of increasing drug residues in
seafood and contributing to bacterial resistance (Lu, Zhang et al. 2006). The common
sources of their entry into the environment include the excretion in urine, the wash off of
topical treatments of livestock animals and the pharmaceutical industrial unsafe waste.
Part II
69
2.2 RESULTS AND DISCUSSION
2.2.1Synthesis of Gold Clusters with Flexible and Rigid Diphosphine Ligands and
the Effect of Spacer and Solvent on the Size Selectivity
To study the effect of spacer on size selectivity, we selected few extremely wide range
geometrically ligands: a long chain, flexible alkyl diphosphine PPh2 (CH2)MPPh2 with M
= 6 i.e., 1,6-bis (diphenylphosphino)hexane denoted by L6, a completely rigid electronic
cloud rich xantphos denoted by LAr, another rigid ligand acetylenediphosphines (LAC),
and binap (LBn), and a moderately flexible spacer biphenyl (LBp) (Fig. 2.3).
Figure: 2.3 (A) 1,6-bis(diphenylphosphino)hexane denoted by L6, (B) Xantphos denoted by LAr, (C) Binap. LBn, (D) Biphenyl denoted by LBp, (e) Diphenylphosphinoacetylene
denoted by LAc
Furthermore we examined the effect of solvent on the core sizes of gold nanoclusters and
on the stability of molecular ion complexes by altering the solution from non-polar to
polar solvents.
(Polar) Methanol Ethanol Propanol ButanolCH2Cl2 CHCl3 CCl4 (Non-Polar)
2.2.1.1 Synthesis and Characterization of Bisphosphine Gold Nanoclusters
General procedure used for phosphine stabilized Au nanoclusters is similar to that
previously reported. Precisely, the mixture of one mole equivalent of Au (PPh3) Cl and
bisphosphine ligand is taken in a deaerated solvent of choice in an oven dried schlenk
flask under argon atmosphere. Vigorous stirring is continued and BTBC is added with
five mole equivalents as compared to gold precursor. The reaction mixture turned to
Part II
70
orange within 1-2 hours. Further stirring is continued for four hours to make sure the
completion of reaction. Products obtained were characterized by UV-Visible
spectrophotometry, and MALDI mass spectrometry. The main advantage of this one
phase, one pot synthesis system is its ease in handling and scaling up ability. To study the
effect of solvent polarities on stability and size selectivity of Au clusters, we synthesized
the Au clusters in methanol, ethanol, propanol, butanol, and the results are compared with
those synthesized in chloroform.
Characterization of L6 Au Clusters: The UV-Visible spectra recorded are shown in
Fig.2.4. It shows that in higher alcohols clusters of higher size are obtained and the
formed clusters are polydispersed. While in chloroform and methanol size selectivity was
better achieved. In the mixture of equal volumes of both chloroform and methanol two
bands were observed that describes the effects of solvent polarities on the size selectivity
of L6 Au clusters.
Part II
71
Figure 2.4 UV-Visible Spectrum of L6 Au Clusters in all solvents
MALDI spectrum of L6 Au Clusters revealed very interesting results (Fig.2.5). We
observed [Au8(L6)4]2+ at m/z 1695 while the intense peak at 1893 is designated to
[Au10(L6)4]2+. Other interesting peak appear at higher m/z ratio values at 2740 for clusters
of higher nuclearity like [Au21(L6)9]3+ corresponding to N>20 while another intense peak
is appeared at 3080 and is designated as cluster of highest nuclearity [Au40(L6)3]3+. We
also observed a chloro ligated cluster [Au16(L6)7Cl]2+ at m/z 3204.
Part II
72
Figure: 2.5 MALDI spectra of L6 obtained in all solvents
Characterization of LAr
Au Clusters:A comparative UV-Visible spectra is displayed in
Fig. 2.6. The best results here were obtained with chloroform. Absorption maxima at 418
nm show the formation of smaller clusters predominantly, and in alcohols we observed
clusters of higher nuclearity. Here again in the mixture of chloroform and methanol we
observed two prominent absoption bands which represents the size selectivity as a
function of solvent polarities.
Part II
73
Figure: 2.6 UV-Vis spectrum of LAr Au Clusters in all solvents used
MALDI analysis shown in Fig. 2.7 gives the clear comparison of solvents effect on
selectivity of clusters produced. The formation of [Au9(LAr)6]3+ is observed at 1747 and a
comparatively less intense peak corresponds to the formation of [Au10(LAr)4]2+ which
appeared at 2141. A less intense peak was observed, indicating the formation of high
nuclearity complex [Au29(LAr)3]3+ at m/z 2482 in ethanol and butanol. In most polar
solvent of the study methanol, greater selectivity is observed for smaller clusters. We
observed a peak at m/z 1448 in all the solvents and is assigned to any gold cluster
[AunLArn]n+.
Part II
74
An interesting approach was used to conclude the effect of polarities of solvents on
size selectivity of nanoclusters by taking the MALDI spectrum in 1:1 mixture of
chloroform and methanol. This result led us to a tremendous technique that can be
used to optimize the best suitable solvent to get the core size of desired nuclearity, the
only clusters obtained was Au9 and Au10. When the reaction was carried out in
butanol, [AunLArn]n+ was observed as most abundant peak, however partial ligand
elimination gave an almost equal abundant peak at m/z 1408 and is assigned to
[Au3LArP((C6H3)2OC(CH3)2)]+.
[AuxLAry]z+
[Au3LArP((C6H3)2OC(CH3)2)]+ + PPh3 + [Au2LArPh]+
To further investigate the effect of polar solvents we investigated higher homologues
of alcohols, the results led us to the formation of [AunLArn]n+, and relatively small
peak of Au9 is also obtained, while partial ligand dissociation gave two fragments of
low intensity at m/z 1090 and 1408 and are corresponded as
[AuxLAry]z+
[Au3P((C6H3)2OC(CH3)2)PPh3]+ + [Au3LArP ((C6H3)2OC(CH3)2)]++PPh3
Part II
75
Figure 2.7 MALDI Spectral Ananlysis of LAr Ligated Au Clusters in different solvents
Characterization of LAc
Au Clusters: In the case of most smallest and rigid ligand of the
study we observed that pronounced selectivity is achieved in alcohols while absorption at
higher wavelength in chloroform suggest that non polar medium does not favour the
formation of small cluster. UV-Vis spectrum is given below (Fig. 2.8)
Part II
76
Figure: 2.8 UV-Vis Spectra of LAc Au Clusters in all Solvents
MALDI analysis (Fig. 2.9) shows that clusters of variable size are obtained in this case.
We observed the peak of [Au9L3]3+ at m/z 985 in all the solvents, while signal at m/z 1643
is assigned to [Au13L6]3+. [Au14L6Cl]2+ is obtained in chloroform and in methanol and is
observed at m/z 2560. While Au19 as [Au19L2Cl2]2+ was also observed at m/z 2990 in only
propanol.
Part II
77
Figure: 2.9 MALDI Spectral Ananlysis of LAc Ligated Au Clusters in different solvents
Characterization of LBn
Au Clusters: The most selective solvents for LBn for smaller
clusters for example Au9 and Au10 were found to be methanol and mixture of chloroform
and methanol as compared to other alcohols since they showed a broad absorption band
in the range of higher wavelengths as depicted in Fig. 2.10 below.
Part II
78
Figure: 2.10 UV-Vis Spectra of LBn Au Clusters in all solvents
MALDI spectra of LBn ligated Au Clusters is shown in Fig. 2.11. In methanol it gave the
formation of [Au9L3]3+ at m/z 1213 along with the [Au9L6]3+ at 1835. We also found
[Au10L4]2+ and [Au14L4]2+ as moderately intense peaks at m/z 2229 and 2623 respectively.
The MALDI spectrum of clusters synthesized in chloroform gave largely the Au10, and
when these two results are compared with the one synthesized in mixture of both solvents
we obtained appreciable peaks of Au9, Au10 along with less intense Au13 and Au14 those
were also found in chloroform but not in methanol. The intensity of higher nuclearity
cluster that is [Au14L4]2+ and [Au14L6]2+ were greater in chloroform as compare to
methanol, and were not formed in other alcohols. In propanol we detected [Au13L6]3+ as a
moderate abundant signal while ethanol showed selectivity for Au13 and butanol for Au14.
Part II
79
Figure: 2.11 MALDI Analysis of LBn Au Clusters in all Solvents
Part II
80
Characterization of LBp
Au Clusters: UV-Vis spectra of LBp Ligated Au clusters are
displayed in Fig. 2.12.
It is evident that smaller clusters are selectively formed in chloroform, methanol, ethanol
and the mixture of chloroform and methanol because their absorption maxima lie within
the range of 410 to 450 nm. The best results are obtained with chloroform, it is showing a
narrower band at about 420 nm which suggest the formation of monodisperse small Au
clusters. In other solvents we also observed that another broader absorption band arise in
the higher wavelengths range which describes those chances of polydispersity of larger
clusters in this case.
Part II
81
MALDI spectra of LBp Au clusters (Fig. 2.13) explains that, smaller cluster [Au8L5]2+ is
obtained in chloroform and methanol at m/z 2094 as suggested by UV-Vis spectra, while
we also observed [Au8L5Cl]3+which appeared at m/z 1407 in all solvents except propanol
and butanol, and its absorption intensity is greatest in chloroform and when the spectra is
recorded in mixture of chloroform and methanol it gave a small peak. Au9 is formed in all
solvents and is observed at m/z 1635 as [Au9L6]3+, and the most abundant signal was
observed in mixture of chloroform and methanol as compared to others. [Au10L4]3+ is also
formed in low amounts and is obtained at m/z 2029. A higher nuclearity cluster is formed
in propanol, butanol and in mixture of chloroform and methanol that is [Au14L6]2+ which
appeared at m/z 2945. Although methanol and ethanol are found to be very selective for
the formation of Au9 and Au10.
Figure: 2.13 MALDI Analysis of LBp Au Clusters in all Solvents
Part II
82
2.2.2 Atomic Force Microscopic guided detection of Copper in real samples and T-
lymphocytes with Pyridinium Thioacetate capped Silver Nanoparticles
Pyridinium compounds are known to form ionic liquids (ILs) which have been
extensively used as stabilizing/capping agents for metal nanoparticles (Dupont, Fonseca
et al. 2002; Kim, Demberelnyamba et al. 2003). Reports show that these systems usually
tend to cause aggregation of nanoparticles due to instability and their phase transfer
characteristics (Cassol, Umpierre et al. 2005; Wang and Yang 2005). The uses of
pyridinium ligands are not only limited to ILs but their wide spread occurrence in nature
make them fascinating candidate for biological and photophysical evaluation. Several
pyridinium nanoconjugates and complexes have recently being developed for catalysis
(Tourneux, Gornitzka et al. 2007; Zhu, Jang et al. 2011), drug delivery, biocidal activities
(Patil, Kokate et al. 2011) and molecular recognitions (Saha, Agasti et al. 2012;
Bagherzadeh, Pirmoradian et al. 2014).
2.2.2.1 Synthesis of 4-formylpyridiniumpropylthioacetate Coated Silver
Nanoparticles (4-Py-AgNPs)
1 mL of 0.1 mM solution of 4-Py was added in a shaking solution of 10 mL of 0.1 mM
silver nitrate. After 15 minutes, 0.1 mL of freshly prepared 4 mM NaBH4 solution was
added in the reaction mixture. The colorless reaction mixture turned yellow-brown
instantly after addition of NaBH4 solution, but the solution was stirred further for 2 hours.
The AgNPs colloidal dispersions were centrifuged for 20 min till all nanoparticles
precipitated at 10,000 rpm to separate the potential large aggregates and excess unbound
ligand from the broth followed by redispersion in deionized water. The yellow
supernatant was freeze dried and further used whenever required. The resulting solution
was subjected to UV-visible spectrophotometry and AFM imaging.
2.2.2.2 Characterization of 4-Py-AgNPs
Upon contacting the 0.1 mM solution of 4-Py with 0.1 mM AgNO3 and 4 mM NaBH4
solution, the UV–vis absorption spectra of the Ag nanoparticle showed surface plasmon
band (SPR) at 430 nm (Fig. 2.14) indicating that the reaction is spontaneous. The SPR
Part II
83
band steadily increased in intensity and finally reached to maximum after lapse of 30
minutes. However, the color change of the reaction mixture from colorless to dark brown
was immediately observed. The color change is mainly due to the excitation of surface
plasmon vibrations in Ag nanoparticles.
300 400 500 600 7000.0
0.1
0.2
Ab
sorb
ance
(un
its)
Wavelength(nm)
0.05mM AgNO3
AgNP
0.05mM Ligand
Figure: 2.14 UV-visible spectra of the AgNPs
Thermal stability of the colloidal solution was determined by boiling the nanoparticles at
100 ºC for 2 hours. Fig. 2.15 indicate that there is no change in the absorption band after
heating.
Part II
84
300 400 500 600 7000.0
0.1
0.2
Ab
sorb
an
ce(u
nit
s)
Wavelength(nm)
Boiled
Unboil
Figure: 2.15 UV-visible spectra of the AgNPs solution at room temperature and
boiling point
The effect of electrolytes on the stability of colloidal silver was investigated by mixing
different concentrations of aqueous NaCl solutions (10 uM, 50 uM, 1mM, 5mM, 10 mM,
50 mM, 100 mM, 500 mM and 1 M) with 0.05 mM AgNPs solution and the spectra are
shown in Fig. 2.16. The SPR band of colloidal AgNPs unaffected till mixing of 50 mM
concentration of NaCl but mixing of higher concentration of salt solution led to the
agglomeration of colloids.
Part II
85
300 400 500 6000.0
0.1
0.2
Ab
sorb
ance
(un
its)
Wavelength(nm)
ZAgNp(50uM)
Z+10uM NaCl
Z+50uM NaCl
Z+1mMNaCl
Z+5mMNaCl
Z+10mMNaCl
Z+50mM NaCl
Figure: 2.16 UV-visible spectra of salt addition stability of AgNPs
2.2.2.3 Chemosensing of Cu(I) by 4-Py-AgNPs
The 4-Py-AgNPs possesses a quaternary nitrogen and benzaldehyde. The aldehyde group
of the ligand can interact with metal cations so a number of metal cations (50 µM) such
as, Cu(I), Ca(II), Zn(II), La(III), Sb(III), Fe(III), Co(II), Pd(II), Rb(I), Y(III), Cu(II), and
Na(I)were titrated against AgNPs (50 µM) and the interaction was evaluated with the
help ofUV spectrophotometer (Evolution 300). The UV visible titration profile of
AgNPsbefore andafter mixing of selected metal cations (Fig.2.17A) revealed selective
interaction of AgNPs with Cu(I). A decline in the absorption intensity of AgNPs was
observed at 403 nm after contacting it with Cu(I). Reduction in theabsorbance of
AgNPsis attributed to its selective binding and accessibility for Cu(I) ions via an
intermolecular complexation. The Cu(I)is believed to be engulfed by the chelating
functional groups of the AgNPs.
Part II
86
280 350 420 490 560 6300.00
0.04
0.08
0.12
0.16
0.20
ZAgNPs
ZAgNP+Cu(I)
ZAgNP+Ca(II)
ZAgNP+Co(II)
ZAgNP+Fe(III)
ZAgNP+La(III)
ZAgNP+Na(I)
ZAgNP+Ni(II)
ZAgNP+Pd(II)
ZAgNP+Rb(I)
ZAgNP+Y(III)
ZAgNP+Zn(II)
ZAgNP+Cu(II)
ZAgNP+Sb(III)Ab
sorb
ance
(un
its)
Wavelength(nm)
A
4030.0
0.1
0.2
Ab
sorb
ance
(un
its)
Wavelength(nm)
ZAgNPs
ZAgNPs+Cu(I)
ZAgNPs+Ca(II)
ZAgNPs+Co(II)
ZAgNPs+Fe(III)
ZAgNPs+La(III)
ZAgNPs+Na(I)
ZAgNPs+Ni(II)
ZAgNPs+Pd(II)
ZAgNPs+Rb(I)
ZAgNPs+Y(III)
ZAgNPs+Zn(II)
ZAgNPs+Cu(II)
ZAgNPs+Sb(III)
B
Figure: 2.17 A) UV-visible spectra of metal cations after titrations with nanoparticles. B)
Bar graph representation of metal cations after titrations with nanoparticles at λmax
Part II
87
Functional groups of 4-Py-AgNPs were deduced after evaluation of FTIR spectrum of
AgNPs (Fig. 2.18). The stretching band at 1350-1400 cm-1 confirms the presence of
sulfur. Stretching at 668cm-1 was attributed to C-S bond. As soon as the AgNPs are
treated with Cu(I) a bathochromic shift in the stretching of C-S was observed which also
indicated the tendency of Cu(I) to form metallic bond with Ag.
Figure: 2.18: A) FT-IR spectrum of 4-Py AgNPs
Part II
88
Figure: 2.18 B) FT-IR spectrum of 4-Py AgNPs after treating with Cu(I)
The size and morphology of the nanoparticles were established with atomic force
microscope (AFM). As shown in Fig. 2.19 that the nanoparticles are spherical in shape
and its size is in the range of 5-10 nm. It is evident from Fig. 2.19 that the nanoparticle
size decreases after addition copper`s solution. Reduction in size of AgNPs after mixing
with Cu(I) solution could be attributed to change in aqueous electrical conductivity of the
colloids due to addition of salt solution which leads the particles to further split into
smaller size to overcome the equilibrium disturbance (Jang, Ortega et al. 2012). Another
possible explanation for the size reduction of AgNPs after interactions with Cu(I)
solution may be alloying of metal ions which can link the two metals and hence contracts
the particle size (Valodkar, Modi et al. 2011).
Part II
89
Figure: 2.19 a) Topographical AFM image of 4-Py AgNPs b) Topographical AFM
image of AgNPs after mixing with Cu(I)
Part II
90
The kinetics of interactions between AgNPs and Cu(I) solutions were studied by
recording absorption spectra of the soup bearing AgNPs and Cu(I) at various interval of
time As shown in Fig. 2.20 that immediate change in the SPR band of AgNPs was
observed.
Figure 2.20 Kinetic studies after interaction of Cu(I) with4-Py AgNPs
The synthesized AgNPs was found to be stable in basic media while in acidic media (pH
1-7) the colloids were not stable and prone to aggregation. The results are summarized in
Fig. 2.21. Peak broadening is observed at pH lower than 7 which is in line with literature
reports (Mulvaney 1996). Prathna et al. described that increase in pH of citrate stabilized
AgNPs lead to blue shift due to reduction of particle size (Prathna, Chandrasekaran et al.
2011) at lower pH.
Part II
91
The effect of pH on the interaction of AgNPs with Cu(I) was evaluated. The Fig. 2.21
clearly shows that best interactions between AgNPs and Cu(I) is observed around neutral
pH. The acidic pH can protonate the electron donating groups of the ligand coated around
AgNPs thus decreasing its interaction with Cu(I). Similarly under highly basic conditions
the Cu(I) can precipitate out hence decreasing the concentration of Cu(I) in the media.
Figure: 2.21: Effect of pH on the selective recognition of Cu(I) by AgNPs.
Sensitivity is an important factor that should be evaluated while studying an acceptable
chemosensor. A 50 μM aqueous solution 4-Py AgNPs was titrated against various
concentrations of Cu(I) at pH 8. Absorption spectra (Fig. 2.22) revealed that mixing of
Cu(I) with a 50 μM aqueous solution of 4-Py AgNPs caused a gradual and smooth
decrease in the absorption intensity of 4-Py AgNPs upon gradual increase in the
concentration of Cu(I).
Part II
92
The limit of detection of 4-Py AgNPs as a chemosensor for the detection of Cu(I) was
deduced from the plot of absorption intensity as a function of the concentration of Cu(I).
The lowest limit of detection found to be 1 µM with a regression coefficient of 0.99.
Figure: 2.22 A) Selective recognition of Cu(I) by 4-Py AgNPs at different concentrations B)
Linear regression curve obtained after treating varies concentration of Cu(I) with 4-Py AgNPs.
Part II
93
The affinity of AgNPs toward Cu1+ was studied in the presence of competing metal
cations discussed earlier. The absorption intensity of AgNPs was monitored after titrating
mixture of AgNPs (50 µM) and Cu1+ (50 µM) against 50 µM aqueous solution of each
metal cations such Ca2+, Cu2+, Co2+, Fe2+, Na1+, Ni2+, Sb3+, Y3+, Zn2+, Rb1+ and Pd2+.As
shown in Fig. 2.23 that thecompeting metal cations didn`t interfere in the absorption
behaviour of AgNPs-Cu1+complex. It is established that AgNPsis discriminating Cu1+ in
the presence of other competing metal cations.
280 350 420 490 560 6300.00
0.04
0.08
0.12
0.16
0.20
Abso
rban
ce(u
nits
)
Wavelength(nm)
ZAgNP
ZAgNP+Cu(I)+Water
ZAgNP+Cu(I)+Ca(II)
ZAgNP+Cu(I)+Co(II)
ZAgNP+Cu(I)+Cu(II)
ZAgNP+Cu(I)+Fe(III)
ZAgNP+Cu(I)+La(III)
ZAgNP+Cu(I)+Na(I)
ZAgNP+Cu(I)+Ni(II)
ZAgNP+Cu(I)+Sb(III)
ZAgNP+Cu(I)+Y(III)
ZAgNP+Cu(I)+Zn(II)
ZAgNP+Cu(I)+Rb(I)
ZAgNP+Cu(I)+Pd(II)
A
4040.00
0.02
0.04
0.06
0.08
0.10
0.12 ZAgNP
ZAgNP+Cu(I)+Water
ZAgNP+Cu(I)+Ca(II)
ZAgNP+Cu(I)+Co(II)
ZAgNP+Cu(I)+Cu(II)
ZAgNP+Cu(I)+Fe(III)
ZAgNP+Cu(I)+La(III)
ZAgNP+Cu(I)+Na(I)
ZAgNP+Cu(I)+Ni(II)
ZAgNP+Cu(I)+Sb(III)
ZAgNP+Cu(I)+Y(III)
ZAgNP+Cu(I)+Zn(II)
ZAgNP+Cu(I)+Rb(I)
ZAgNP+Cu(I)+Pd(II)
Abso
rban
ce(u
nits
)
Wavelength(nm)
B
Figure: 2.23 A) Absorption spectra of 4-Py AgNPs+Cu(I) in presence of other
competing metal cations B) bar graph representation Absorption spectra of 4-Py AgNPs+Cu(I) in presence of other competing metal cations
Part II
94
Stochiometry of interaction of AgNPs with Cu(I) was carried out through Job’s plot
experiment. It is shown in Fig. 2.24 that absorbance maxima intersect at the 0.5 mole
fraction on the x-axis (mole fraction). It implies that the complexation ratio between
AgNPs and Cu(I) is 1:1.
Figure: 2.24 Complexation ratio between 4-Py AgNPs and Cu(I) The analytical potential of the AgNPswas evaluated by using it for selective detection and
quantification of Cu1+ in tape water of Karachi University. Tape water samples from
University of Karachi were spiked with50 µM Cu1+. The AgNPs proved to be valuable probe
for detection of Cu1+ with lower limit of detection 2 μM, which is much lower than the
maximum allowed limit (∼20 μM) of Cu1+ in drinking water permitted by the U.S.
Environmental Protection Agency (EPA) (G. Georgopoulos 2001). Nearly similar results
were obtained when tape water was used instead of deionized water for preparation of
copper`s solution and titrated against AgNPs (Fig. 2.25). UV-visible guided titrations of
AgNPs (50µM) with Cu1+ (50 µM) spiked in human blood plasma lead to reduction of
absorption intensity (400 nm) with the increase of Cu1+ concentration (Fig. 2.25). The
obtained decline in absorption intensity is directly proportional to the increase of Cu1+
concentration in a wide range (i.e. 2-50 µM), that is inline with the copperconcentration
range in healthy body plasma samples (Malavolta, Giacconi et al. 2010; Fayet-Moore, Petocz
et al. 2014). This shows that AgNPs can be a valuable tool which shows the plasma sample is
healthy or not.
Part II
95
300 400 500 600 700
0.0
0.1
0.2
0.3
Abs
orba
nce(
unit
s)
Wavelength(nm)
Tap water
CuCl in tap water
AgNPs
AgNPs + CuCl in tap water
A
300 400 500 600 7000.0
0.5
1.0
1.5
Abs
orba
nce(
unit
s)
Wavelength(nm)
AgNPs
Plasma
AgNPs+Cu+Plasma
B
Figure 2.25 UV-visible spectra of 4-Py AgNPs recorded in presence Cu(I) spiked in (A)
Human plasma (B) Tap water
Part II
96
2.2.2.4 Intracellular Cu(I) detection in T-lymphocytes:
Cell incubation
Cytotoxic T lymphocytes (CTLL-2) from (ECACC, ATCC) for AFM based study were
used. For this purpose, the CTLL-2 (ATCC® TIB-214™) cells was cultured in ATCC-
formulated complete RPMI 1640 ((1mM Sodium pyruvate, 2M HEPES and 10% FBS,
10% T-STIM & Con A, 1, 1% L-glutamine and Non-Essential Amino Acids) culturing
media in 75 cm2 flasks, and kept in 5% CO2 incubator at 37 oC. After complete
confluences about 5×104 cell/well (in 96 well flat bottom plate corning) of cell line were
used to conduct the experiments.
Cell Treatment
The cell permeability of the AgNPs on T-cells was evaluated by incubating AgNPs at
different doses with T-cells for 30 min at 37 oC in 5% CO2 incubator. After incubation
the cells were centrifuged at 1300 rpm for 4 min and the cell pellet were washed twice
with 1X, PBS (Phosphate buffer saline) buffer to remove the unbound test sample. Then
the treated cells were incubated for 20 min with multiple concentration of Cu+ solution at
37 oC in 5% CO2 incubator followed by AFM microscopy.
MTT Based Cytotoxicity Assay
Cytotoxicity of test samples were evaluated in 96-well flat-bottomed micro plates by
using the standard MTT (3-[4,5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium
bromide) colorimetric assay. For this purpose, the lymphocytes cells were cultured in
their respective media RPMI 1640 in 75 cm2 flasks, and kept in 5% CO2 incubator at 37 oC. Cell culture with the concentration of 5x104cells/ml was prepared and introduced
(100 µL/well) into 96-well plates. After overnight incubation, medium was removed and
200 µL of fresh medium was added with different concentrations of test sample (30,
50,100 µM). After 48 hrs, 200 µL MTT (0.5 mg/ml) was added to each well and
incubated further for 4 hrs. Subsequently, 100µL of DMSO was added to each well. The
extent of MTT reduction to formazan within cells was calculated by measuring the
absorbance at 540 nm, using a micro plate reader (Spectra Max plus, Molecular Devices,
CA, USA). The percent inhibition was calculated by using the following formula:
Part II
97
Inhibition (%) = [1 - (treated/control)] × 100
Viability (%) = 100 – inhibition (%)
The potential of nanoparticles to selectively detect metal cations in living cells is vitally
important for biological application. Taking into that copper accumulation in the body
leads to toxicity, we primarily conducted investigation using AgNPs on T-cell lines in
which copper is known to be accumulated To determine the cell permeability of AgNPs,
T cells were supplemented with multiple doses of AgNPs in the range of 10-0.5 μM for
10-20 min at 37 °C, and washed with PBS to remove the surplus AgNPs. One can clearly
observe significant accumulation of nanoparticles inside the cells, and the cells are still
intact, indicating permeability of nanoparticles through cell membrane. At 1 μM lower
concentration of AgNPs, the nanoparticles are appeared as small grooves on the cell
surface but at higher concentration, the cells are fully loaded with nanoparticles and they
are still in good health and shape as in the case of 10 μM. While images were taken upon
the addition of CuCl (5, 10, 30, 40 and 50 μM, respectively) at 37 °C for 20 min. In Fig.
2.26, an untreated T-cell image is shown which is used as control throughout the
microscopic studies. We have distributed the host guest intracellular interactions in three
categories. First at lower concentrations of both the guest and host (for example at 0.5
μM nanoparticles and 5 μM), the morphology of cell surface is shrinked by the
interaction between the two. While even on addition of more copper (10 μM) the cells are
not broken (Fig. 2.26). Similar results were obtained when we increased the nanoparticles
and copper concentration to 1 μM and 30 μM respectively (Fig. 2.27). Then at higher
doses (10 μM nanoparticles, we observed a few interesting things. First, the nanoparticles
are completely inside the cell, and they quenched the cytotoxicity at lower concentration
of copper that is 5 μM. Secondly, at higher doses of copper (50 μM), its starts to outweigh
the effect of nanoparticle interaction and a disrupt in cell morphology can be seen in Fig.
2.28 along with a little or no effect on two different cells. This effect of concentration is
completely supported by MTT cytotoxicity assay results given below.
The MTT cytotoxicity assay results suggest that when cells were treated with varying
concentrations of copper, the cell viabilities are found to be 12, 20, and 26 % at 30, 40,
and 50 μM respectively. While AgNPs did not exert any adverse effect on cell viability,
and the viabilities are recovered to 53 % and 47 % at 10 and 1 μM concentrations of
Part II
98
AgNPs respectively. The effect was more pronounced in higher concentration and
showed the recovery of cells viability in a dose-dependent manner.
Figure: 2.26 A) T cell (Control)
Figure: 2.26 B) 0.5 μM 4-Py AgNPs treated T cell
Part II
99
Figure: 2.26 C) 0.5 μM 4-Py AgNPs treated T cell with 5 μMCu (I), cell
surface is shrinked due to interactions.
Part II
100
Figure: 2.26 D) 0.5 micromolar 4-Py AgNPs treated T cell with 10 micromolar Cu (I)
Part II
101
Figure: 2.27 A) 1 micromolar 4-Py AgNPs treated T cell with 5 micromolar Cu (I)
Figure: 2.27 B) 1 micromolar 4-Py AgNPs treated T cell with 30 micromolar Cu (I)
Part II
102
Figure: 2.28 A) 10 micromolar 4-Py AgNPs treated t cell
Figure: 2.28 B) 10 micromolar 4-Py AgNPs treated t cell with 5 micromolar copper
Part II
103
Figure: 2.28 C) 10 micromolar 4-Py AgNPs treated t cell with 50 micromolar copper
Part II
104
2.2.2.5 Chemosensing of 6-AminopenicillanicAcid by 4-Py-AgNPs
6-Aminopenicillanic acid (6-APA) is the core of Beta lactam antibiotic class of compound
penicillins. The penicillins are being used commonly for their antimicrobial activity against
both gram positive and gram negative organisms (Wishart 1984). However, pencillins have
limited stability, and are susceptible to form different degradation products(Tyczkowska,
Voyksner et al. 1992). The presence of degradation and related substances may cause the
reduced activity of the drug, as well as some side effects in humans. These antibacterial
agents can inactivate penicillin-binding proteins (PBP), which are used in the synthesis of
bacterial cell walls through the formation of stable covalent acyl complexes. As an
outcome of this irreversible substrate binding, the cell-wall-synthesizing activity of PBP is
strongly inhibited, which results into cell death.(Matagne, Lamotte-Brasseur et al. 1998;
Fisher, Meroueh et al. 2005). On the other hand, the occurrence of these antibiotics applied
for human or veterinary use in lakes and streams all over the world has created an alarming
situation due to the toxicity of these drugs for aquatic creature (Wilson, Smith et al. 2003;
Sanderson, Johnson et al. 2004). The common sources of their entry into the environment
include the excretion in urine, the wash off of topical treatments of livestock animals and
the pharmaceutical industrial unsafe waste. Hence, during our recent work of chemical
analyses of the drinking water system of Karachi, Pakistan we found a large number of
pharmaceuticals drugs in considerably high concentrations (Scheurell, Franke et al. 2009;
Selke, Scheurell et al. 2010).
Currently the most common analytical procedures which are employed for the detection
and/or determination of beta lactam antibiotics involve spectrometry(Abdalla 1991;
Devani, Patel et al. 1992), voltammetry (Ali and Mohammad 1994; Leszczynska, Koncki et
al. 1996), high-performance liquid-chromatographic (HPLC) (Pinto, Pavon et al. 1995),
(LC-MS) ( a -Cruz, López de Alda et al. 2003; Poole 2003), chemiluminescence method
(Yang, Zhou et al. 1998), and NMR (Shamsipur, Talebpour et al. 2002). Some fluorimetric
determination of beta lactamic antibiotics has also been described mainly based on the
formation of fluorescent degradation products either after alkaline or acid hydrolysis
(Murillo, Lemus et al. 1994; Yang, Zhou et al. 1998). However, all these reported methods
have their own disabilities and limitations, since the fluorescence probes are difficult to
Part II
105
design and involve a tedious protocol of purifying the molecules and comprise highly
skilled synthetic expertise.
Nanoparticles synthesis and their applications in chemosensing have grabbed lots of
interest especially in biosensing of metals and drugs (Jung, Lee et al. 2011; Saha, Agasti et
al. 2012; Liu, Zhou et al. 2014). Nanoparticles probes have tremendous advantages over
other methods, due to their simplicity, cost effectivity and robustness. Recently ateeq etal
reported a natural product capped AuNPs for the chemosensing of piroxicam (Ateeq, Shah
et al. 2014), and in another report yang etal published a tetracycline detection system based
on nanomaterial probe (Yang, Zhu et al. 2014). Inspired by these reports, we put our
attention towards developing a beta lactam antibiotic nanosensor.
Application of 4-PyAgNPs as 6-APA Sensing Probe
The recognition behavior of fully characterized 4-PyAgNPs was evaluated for various
drugs by the 1:1 mixture of 0.05 µM drugs solutions and AgNPs. The UV–vis spectra of
AgNPs were recorded instantly after addition of drug solutions. The effect of tested drugs
such as 6-APA, amoxicillin, aspirin, cefaclor, cefotaxime sodium, ceftriaxone sodium,
cephalexime, cephradine, diclofenac sodium, paracetamol, pefloxacin, and penicillin on
SPR band absorption intensity was investigated and plotted in Fig. 2.29. All drugs
decreased the absorbance intensity of 4-PyAgNPs, but the maximum quenching in SPR
band is observed with 6-APA. The slight decrease in absorbance of AgNPs is associated
with the dilution as in case of all the drugs, but the considerable large difference between
rest of the drugs and 6-APA absorption behavior indicates some chemical or physical
interactions. These results of spectrophotometric drugs screening can be exploited as a
selective assay towards 6-APA detection.
Part II
106
350 400 450 500 5500.00
0.05
0.10
0.15
0.20
Abs
orba
nce(
unit
s)
Wavelength(nm)
0.05mM ZAgNp
ZAgNp+6APA
ZAgNp+Amoxillin
ZAgNp+Aspirin
ZAgNp+Cefaclor
ZAgNp+CefotoximeSodium
ZAgNp+CeftrixonSodium
ZagNp+Cephalexime
ZAgNp+Cephradine
ZAgNp+Diclofinic
ZAgNp+Paracetmol
ZAgNp+Pefloxime
ZAgNp+Penicillin
Figure: 2.29 Drugs screening study
AFM and FT-IR spectral analysis to understand the interactions between 4-PyAgNPs
and 6-APA
The interaction of nanoparticles with other organic species like metal ions and drugs may
cause some morphological change. Some recent reports indicate the decrease in
nanoparticles size upon interaction with other chemical entities (Pingali, Deng et al.
2008; Ohkubo, Seino et al. 2014). We also observed the size of 4-PyAgNPs changed
largely from 40-60 to 0.1-5 nm (Fig. 2.30). This behavior in the size domain of
nanoparticles is explained by comparative FT-IR spectral studies.
The comparative FT-IR spectral analysis is carried out to have a deeper understanding of
the mechanism of nanoparticles formation and their 6-APA recognition. Ligand FT-IR
spectrum shows the C-S stretches at 621 and 1365 1/cm, while thioester carbonyl is
appeared at 1638. As it is very well established fact these days, we also observed that the
thioester peak is diminished upon the formation of AgNPs which is an indication that
Part II
107
stabilization of AgNPs is occurred directly by Sulfur. We also recorded the FT-IR spectra
of 6-APA, which showed all the characteristics peaks of the drug including lactam
carbonyl at 1623, acid carbonyl at 1773 and N-H stretchings at 2904 and 2987 1/cm,
while due to carboxylic hydroxyl group, the whole region around 2900-3300 1/cm
showed a broad signal. Interestingly, when 6-APA was treated with 4-Py-AgNPs, the
acid carbonyl peak which was appeared at 1773 1/cm in the drug was absent here. These
results describes that, in the slightly basic pH of the colloidal suspension the carboxylic
acid group shows hydrogen bonding with free aldehydes on 4-Py AgNPs surface. Due to
hydrogen bonding interactions, acid carbonyl peak is broadened and merged with that of
lactam carbonyl, as well as a broad signal of hydroxyl (H-bonding) is appeared at 3412
1/cm.
Figure: 2.30 Morphological change in the nanoparticles after interaction with 6-APA
Effect of the pH on the Stability of 4-Py AgNPs and 6-APA Recognition
Since the pH of the medium has a great influence on the optical response of the
complexation reaction, an optimum pH is thus essential to develop for the interaction of
4-Py AgNPs-6APA complex. A study of the pH in the range of 2–12 with the optimized
conditions for interaction of nanoparticles with the drug is shown in Fig. 2.31. The
Part II
108
absorbance of SPR band of the AgNPs-6APA mixture shows the maxima at pH 9, and
before and after which it gradually decreases. Moreover, it can be seen with the
comparative pH plots that the interaction of drug and nanoparticles follows the trend of
the buffer stability of the nanoparticles with subsequent quenching in the absorbance of
SPR band. Therefore on the basis of these results it can be safely concluded that the drug
and nanoparticles interaction is independent of the pH change, and our system works
almost equally well at most of the pHs.
Figure: 2.31 Effect of pH on the stability of 4-Py AgNPs and accumulation of
nanoparticles with 6-APA
Analytical Performance of the Nanosensor
The performance of our optimized detection system is demonstrated by evaluation of the
concentration studies. We used various concentrations of 6-APA to measure the sensitive
response of developed analytical system. Fig. 2.32 represents that the data strictly
followed beer’s law and exhibited a good linear correlation to 6-APA concentration in the
range from 1 to 50 μM with the regression constant (R2) equal to 0.992, while the limit of
detection (LOD) is found to be 19.08μM. Moreover, to estimate the binding
stoichiometry between AgNPs and 6-APA Job's plot experiment was performed.
0 0.02 0.04 0.06 0.08
0.1 0.12 0.14 0.16 0.18
0 1 2 3 4 5 6 7 8 9 10 11 12
Ab
sorb
ance
(un
its)
pH(units)
ZAgNp
APA
Part II
109
Absorption spectra were recorded by varying the mole ratio of 6-APA to AgNPs. The
minimum on the plot between absorbance and mole fraction is appeared at 0.5, which
correspond to the 1:1 stoichiometric relation between the stable AgNPs-6APA
complexes.
Part II
110
Figure: 2.32 A) UV-visible spectra of concentration study, B) Calibration
curve and C) Job’s plot
Specificity
The specificity of this nanosensor was tested by evaluating the response of optimized
system towards other interfering drugs. All the drugs were used in 1:1 (v/v) with 6-APA.
Fig. 2.33 shows that the additions of competing drugs such as amoxicillin, aspirin,
cefaclor, cefotaxime sodium, ceftriaxone sodium, cephalexin, cephradine, diclofenac
sodium, paracetamol, pefloxacin, and penicillin respectively to the AgNPs-6APA
complex showed no change in the absorption of SPR band. These data suggested that 6-
APA specifically caused quenching of the SPR band, concluding that this analytical
strategy may serve as a potential spectrophotometric probe for assaying 6-APA in the
mixture of other drugs.
Part II
111
4040.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Ab
sorb
ance
(un
its)
Wavelength(nm)
ZAgNp+Water
ZAgNp+APA+Water
ZAgNp+APA+Amoxillin
ZAgNp+APA+Aspirin
ZAgNp+APA+Cefaclor
ZAgNp+APA+CefotoximeSodium
ZAgNp+APA+CeftriaxonSodium
ZAgNP+APA+Cephalexin
ZAgNP+APA+Cephradine
ZAgNP+APA+Diclofinic
ZAgNp+APA+Paracetmol
ZAgNP+APA+Pefloxacin
ZAgNp+APA+Penicillin
Figure: 2.33 Effect of interfering drugs on 6-APA quenching
Quenching effect of 6-APA in HumanBlood Plasma
The real samples analysis of our detection system was also explored to check the
quenching sensitivity and selectivity of 4-PyAgNPs for 6-APA in human blood plasma.
Human blood plasma was spiked with 0.05 mM 6-APA solution before use, and the
spiked plasma sample was treated with AgNPs. Fig. 2.34 indicates that the ingredients of
the complex and critically important physiological sample of human plasma did not alter
the photophysical properties of AgNPs, and a considerable magnitude of SPR band
quenching is still observed.
Part II
112
Figure: 2.34 6-APA quenching in Human blood plasma sample
2.2.2.6 Antimicrobial Activity of 4-Py-AgNPs
Thiolated organic moieties are known to have a broad spectrum activities against both
gram positive and gram negative microbes and fungi, therefore these are the active part of
pharmacophores in many developed antibiotic drugs for example cephalosporin,
amoxicillin etc.
In this study we used a complex strain of E. coli ATCC 8739; it is a human pathogen that
is considered as a reason of urinary tract infections, gastroenteritis, and neonatal
meningitis (Croxen and Finlay 2010). To study the antibacterial activity of 4-Py-AgNPs,
we utilized AFM along with the conventional Agar well diffusion method. The minimum
inhibitory concentrations (MIC) were calculated by using zone of inhibition method.
The membrane surface of E. coli is not considered as smooth at nanoscale since it is
made up of inhomogeneous packing of lipopolysaccharide (LPS). The packing of the
Part II
113
adjacent patches is tight, which provides an effective permeability barrier for the E. coli
(Coughlin, Caldwell et al. 1981). Metal ions play a key role in upholding the assembly of
the LPSs and membrane proteins within the outer membrane (Coughlin, Tonsager et al.
1983). This technique is usefully exploited to increase the cell permeability and the
liberation of proteins. This phenomenon is being repeatedly reported in the literature as a
method to attain the liberation of the periplasmic substances of Gram-negative bacteria
can be attained by the depletion of metals from the surface of these organisms (Amro,
Kotra et al. 2000).
Cu(I) accumulation dose for E. coli ATCC 8739 is found to be 25µg/ml to 35µg/ml in
which the bacterial cells digested the Cu(I) for their metabolic system regulation. In
excesses amount of Cu(I) like 40µg/ml and above, accumulation was stated toxic for E.
coli cells. Data is represented in table 2.1.
Table 2.1 Zone of inhibition data at multiple dose of Cu(I) accumulation
CuCl Cuprous chloride
CuCl accumulation dose µg/ml Inhibition
1 -
5 -
10 -
25 -
40 9mm±.1
50 10mm±.1
100 12mm±0.3
200 14mm±0.5
300 15mm±0.5
400 17mm±0.4
500 19mm±0.3
Part II
114
Minimum Inhibitory concentration of 4-Py ligand is calculated from 200µg/ml to
300µg/ml, whereas 4- Py-AgNPs of Para-formyl pyridinium propyl thioacetate ligand has
contained 100µg/ml to 200µg/ml. The MIC of 4- Py-AgNPs as compared to ligand is
found to be two times higher, while the bar graph in Fig. 2.35 represents the data.
Figure: 2.35 MIC of ligand in comparison with ligand stabilized 4- Py-AgNPs
in the presence of Cu(I).
The nanoparticles can easily penetrate the bacterial cell membrane by adhering to
proteins in the cell membrane due to the high affinity of 4- Py-AgNPs owards treated
Cu(I) ions. The pathways involved in bacterial responses to 4- Py-AgNPs are highly
dependent on physicochemical properties of the nanoparticles, and in particular the surface
characteristics. These results haveimportant implications for the regulation and testing of
silver nanoparticles (Carlson, Hussain et al. 2008).
0
5
10
15
20
25
30
5 10 25 50 100 200 300 400 500
Zon
e o
f in
hib
itio
n m
m
MIC µg/ml
Zones of inhibition of Ligand(mm)
Zones of inhibition of AgNPs Ligand(mm)
Part II
115
AFM analysis also confirmed the results of the zone of inhibition assay. Since the MIC of
ligand was around 2 times less as compared to its 4- Py-AgNPs, severe morphological
changes are expected in the nanoparticles treated E. coli. While unconjugated 4- Py-
AgNPs exhibited very poor MIC values in our previous report, hence its role as cytotoxic
agent itself is ruled out (Shah, Ali et al. 2014).
Images of untreated E. coli (control) cells revealed a relatively smooth surface with no
ruptures. The surface of E. coli is found to be smooth and shows no indentations in Fig.
2.36A. While Fig. 2.36B represents the Cu(I) treated E. coli, it can be clearly seen that
Cu(I) treated cellslooks healthier than the control cells. Since at 30 µg/mL (below the
MIC), Cu(I) ions are used in the metabolism of the E. coli.
AFM images were acquired at MIC of sample on E. coli. Only relatively intact cells were
imaged, and the images therefore represent only cells with minimal lytic damage at a
particular sample concentration. In general, the roughness of the outer membrane
increased with the increase in sample concentration, with ligand coated 4- Py-AgNPs
having the most pronounced effect on the surface roughness.
The cultures were then treated with formyl pyridinium thioacetate ligand at a dose of 200
µg/mL, and cell morphology was analyzed via AFM as a function of incubation time.
The sample exhibited rough surfaces after 4 h (Fig. 2.37). While the cellular degradation
increased with time, and after 8 h the cells were significantly degraded. Since the MIC
data suggests that 4- Py-AgNPs more effectively inhibit the growth of E. coli as
compared to pure ligand. The AFM analysis presented an interesting feature in
accordance with this data. 4- Py-AgNPs were found to degrade cells faster than pure
compound, Ag conjugates below MIC that is at 100 µg/mL concentration induced cell
rupture by 4 h (Fig. 2.38A). While at higher incubation time that is after eight hours, the
cells were completely melted and destroyed. Significantly, unconjugated Au and Ag NPs
at even higher dosage did not induce any significant morphological changes even after
long incubation time (i.e.48 h).
Part II
116
Figure: 2.36 A) E. coli Control. B) E. coli after treating Cu(I)
Figure: 2.37 Ligand treated E. coli at A) 4 hours. B) 8 hours
Figure: 2.38 4- Py-AgNPs treated E. coli at A) 4 hours. B) 8 hours
Part II
117
2.2.3 Synthesis of Pyrazinium Thioacetate Gold Nanoparticles Pyr-AuNPs and their
Applications
2.2.3.1 Synthesis of Pyr-AuNPs
Pyrwas dissolved in 10% mixture of methanol to deionized water for preparing 0.1 mM
solution, while 0.1 mM solution of tetrachloroauric (III) acid was prepared in deionized
water only. 1 mL (0.1 mM) of Pyr was added into 10 mL (0.1 mM) of tetrachloroauric
(III) acid and the mixture was stirred for 10 minutes. Then 0.1 mL of freshly prepared 4
mM NaBH4 solution was added in the reaction mixture. The color of the reaction mixture
rapidly changed from colorless to wine red. The resulting mixture was stirred for further
1 hour at room temperature, and was used as reference AuNPs solution throughout this
study. The colloidal AuNPs are stable at room temperature and ordinary atmosphere for
more than 1 month. Solid AuNPs were collected by freeze drying.
2.2.3.2 Characterization of Pyr-AuNPs
350 400 450 500 550 600 6500.00
0.05
0.10
0.15
0.20
0.25
0.30
Abso
rban
ce(u
nits
)
Wavelength(nm)
0.1mMAuNPs
Figure: 2.39 UV-visible spectrum of Pyr-AuNPs
In this study, the AuNPs are synthesized by simple reduction of HAuCl4 via NaBH4 in
the presence of the stabilizing agent (one phase method). It is the most widely used
method for the synthesis of AuNPs due to its robustness and rapidity. The one phase
Part II
118
method is more useful as compared to other common methods because it is simple, and
does not require any special treatment like heating, incubation etc. However, using
NaBH4 for the rapid reduction of metal salt may lose the size selectivity for the formation
of relatively small nanoparticles. Along with compromising size selectivity, the toxicity
of borohydrides restricts its use for the synthesis of nanoparticles for medical application.
But for the purpose of chemosensing, this method works fine.
For optimization of the synthetic procedure, various ratios of Pyr and HAuCl4 were
applied, among which 1 ratio 10 was finally chosen, due to stability and prominent SPR
band absorption of formed AuNPs. The SPR maxima were observed at 520 nm, Fig.2.39
displays the typical absorption spectra of colloidal gold stabilized with thiolated ligand.
Notably, the synthesis process only required 15 min, and no further purification or any
treatment was needed. Further characterization of Pyr-AuNPs was achieved by FT-IR
spectroscopy and AFM which will be discussed later.
2.2.3.3 Photophysical Applications of Pyr-AuNPs
In the present study we developed a simple two steps procedure for the synthesis of 1-(3-
(acetylthio) propyl) pyrazin-1-ium capping agent for the stabilization of AuNP. The
obtained AuNPs were used as sensor for detection and quantification of Fe(III) and
Pd(II). The analytical potential of this nanosensor was evaluated by screening tap water
and blood plasma containing Fe(III) and Pd(II). Our schematic design offers key
advantages over several other metal detecting systems as it is stable and robust. Unlike
particular organic fluorophores used recently for detecting heavy metals, the synthesis of
Pyr is quite simple, while AuNPs can also be prepared rapidly without the need of tedious
separation techniques. The high dispersibility of the AuNP probes in aqueous solution
gives this method an edge over recent reported methods for the sensitive detection of
heavy metal ions in water. It provides rapid sensing of heavy metals like Fe (III) and
palladium in very economical way through UV-visible spectrophotometry, while the
sensitivity and specificity of the system is easily reproducible. Finally our system can be
used potentially for the detection and removal of trace heavy metals from drinking water
and physiological fluids.
Part II
119
2.2.3.3.1 Recognition of Fe (III )with Pyr-AuNPs
400 450 500 550 600 650 700 7500.0
0.1
0.2A
bso
rban
ce(u
nit
s)
Wavelength(nm)
AYNP only
AYNP+Ca(II)
AYNP+Cu(I)
AYNP+Cu(I)
AYNP+Cu(II)
AYNP+Cu(I)
AYNP+Fe(II)
AYNP+Fe(III)
AYNP+Fe(II)
AYNP+K(I)
AYNP+Na(I)
AYNP+Ni(II)
AYNP+Rb(I)
AYNP+Cu(I)
AYNP+Sb(III)
Figure: 2.40 UV-visible spectra of metal salts screening
Because heterocyclic ligands are known to form complexes with Fe(III) ions as in heme,
we explored our pyrazinium stabilized AuNPs for the sensing of Fe(III) ions, through a
mechanism based on the complexation of the Fe(III) ions in solution with the Nitrogen
atom of the pyrazine.
The optical sensitivity of heavy metal ions with respect to prepared nanoparticles was
investigated in 1:1 mixture of 0.1 mM metal salt solutions and AuNPs. The UV–vis
spectra (Fig. 2.40) of AuNPs were recorded after 15 minutes of addition of salt solutions.
A significant decrease in absorption of SPR band was observed within 15 min after the
addition of Fe (III) ions to AuNPs, presumably because of aggregation-induced
quenching. The appearance of a quenched SPR band for Fe (III), indicates that the
prepared AuNPs was sensitive and selective towards Fe(III).
Part II
120
Figure: 2.41 Buffer stability of AuNPs with and without Fe(III)
For the stability of AuNPs in aqueous solution, pH of the media needs to be optimized so
that the background agglomeration will be minimal. The effect of pH on the Pyr-AuNPs
is shown in Fig. 2.41. The pH of colloids is found to be 8.4, the solution is slightly basic
due to free pyrazine groups on the surface of gold nanoparticles. The effect of pH on
SPR band was studied by varying pH from 2–12. It is observed that the variation of pH
did not affect the stability of AuNPs, but due to dilution a decrease in absorption of SPR
band was observed.
We further investigated the effect of pH on the optical responses of the AuNPs to Fe(III).
In the presence of Fe(III) ions (0.1 mM), the colloids showed the highest absorbance at
pH=12, while the absorbance was quenched most at pH=2. Since interaction of the
Fe(III) ions with AuNPs is influenced by pH of the solution so investigation of pH is an
important step in chemosensing. The best pH for interaction of Fe(III) ions with AuNPs
was achieved around 2-3. Such a profile indicates, that for pH values lower and higher
than 2-3 in accumulation step, the amount of accumulated Fe(III) ions on AuNPs was
decreased, due to H+ competition for functional groups of the surface, and OH−for Fe(III)
in solution.
0 0.02 0.04 0.06 0.08
0.1 0.12 0.14 0.16 0.18
2,3 4,5 6,7 8,9 10,11 11,12
Ab
sorb
ance
(un
its)
pH(units)
AYNP +FeCl3
AYNP only
Part II
121
2.2.3.3.3 FT-IR spectral analysis
Figure: 2.42 Comparative FT-IR spectra of Pyr-AuNPs before and after mixing of Fe(III)
The FT-IR spectrum of ligand indicates the presence of two peaks of interest as, a weak
band of C-S at 617, and thioester’s carbonyl at 1635 cm-1, while the rest of characteristics
peaks of the chemical structure of Pyr are present in the spectrum. The formation of Pyr-
AuNPs is ascribed to the disappearance of the thioester peak, which implies that the
stabilizing head group of the ligand must be –SH. Fig. 2.42 represents the FT-IR
spectrum of the typical thiolated AuNPs, where strong and broad band around 3500 is
associated with the water in the medium. FT-IR spectra of AuNPs before and after
mixinng with Fe(III) is shown in Fig. 2.42, and on the basis of the observation that no
change is occurred between the two spectra, it can be safely concluded that the
mechanism of SPR quenching is not related to any kind of chemical transformation or
detachment of the ligand from AuNPs.
0
10
20
30
40
50
350 850 1350 1850 2350 2850 3350 3850
% T
ran
smit
tan
ce
Wave number(1/cm)
AuNPs+Fe
AuNPs
Part II
122
2.2.3.3.4 Morphology of AuNPs
Figure: 2.43 AFM images of AuNPs alone and of AuNPs treated with Fe(III)
Part II
123
The morphological information of the AuNPs is obtained by AFM, and is displayed in
Fig. 2.43. It showed the presence of polydispersed and spherical particles with in the size
range of 15-100 nm. Hence the applied reaction conditions do not give rise to size
selectivity for the synthesis of AuNPs. The AFM images reveals that the AuNPs were
initially small, and tended to aggregate in the presence of Fe(III) ions, resulting in the
formation of huge complexes of size in the range of 100-1000 nm. However, the obtained
behavior of AuNPs is very attractive for its application in optical sensors, since the shape
of the nanoparticles is related to its optical properties and it justifies the instant decrease
in SPR band of AuNPs was occurred due to aggregation.
2.2.3.3.5 Quantitative relationship between Nanosensor and Fe(III)
Figure: 2.44 Concentration study with the calibration curve
On the basis of FT-IR spectral analysis, it can be safely assumed that, when ferric
chloride is added to the AuNPs, Fe(III) ions interact with the Nitrogen of pyrazine which
are free at the surface of the AuNPs. So, to demonstrate the sensitivity of our nanosensor
for the quantitation of Fe(III) ions by the mechanism mentioned above, UV-visible
Part II
124
spectra of AuNPs in the presence of various concentrations of Fe(III) was recorded (Fig.
2.44). It shows that there is a decrease in absorbance on increasing the concentration of
Fe(III) ions. The data exhibited a good linear correlation to Fe(III) concentration in the
range from 1 to 100 μM. The regression constant (R2) is 0.9813, while the limit of
detection (LOD) and the limit of quantification (LOQ) are found to be 4.3 μM and 13.19
μM respectively. Since, The LOD of our detection system is lower than the maximum
level (5.4 μM) of Fe(III) ions permitted in drinking water by the US Environmental
Protection Agency, we hope that this method can turn out as a cheap alternative for the
detection of Fe(III) ions in drinking water.
2.2.3.3.6 Selective interaction of Pyr-AuNPs with Fe(III) in the presence of competing
metal ions
5380.00
0.02
0.04
0.06
0.08
0.10
Ab
sorb
ance
(un
its)
Wavelength(nm)
AYNP
AYNP+Fe(III)
AYNP+Fe(III)+K(I)
AYNP+Fe(III)+Na(I)
AYNP+Fe(III)+Ni(II)
AYNP+Fe(III)+Cu(II)
AYNP+Fe(III)+Cu(I)
AYNP+Fe(III)+Cu(I)
AYNP+Fe(III)+Na(I)
AYNP+Fe(III)+Sb(III)
Figure: 2.45 Effect of interfering metal salts on the Fe(II) sensing
Since a practical detection system must possess a high selectivity and rapid response time
to differentiate and recognize the target among potentially competing entities. Therefore,
to test the specificity of the developed optical probe for Fe(III) ions, the nanosensor
discriminates Fe(III) in the presence of other competing metal ions. The concentration of
Part II
125
the interfering salts was kept same as that of Fe(III) (0.1 mM). As shown in Fig.2.45, in
the presence of Fe(III) ions, no response is observed toward K(I), Na(I), Ni(II), Cu(II),
Cu(I), and Sb(III). Small shift in the plasmon band energy to longer wavelength is
observed in the presence of K(I) and Cu(I), however, the responses are negligible
compared with that of Fe(III). Hence Fig. 2.45 establishes that the chemosensor has
relatively desirable selectivity towards Fe(III).
Part II
126
400 500 600 700 800
0.0
0.1
0.2
Ab
sorb
ance
(un
its)
Wavelength(nm)
Fe(III)+tap water
Fe(III)+tap water+AYNPs
AYNPs
Tap water
A
400 500 600 700 8000.0
0.1
0.2
Ab
sorb
ance
(un
its)
Wavelength(nm)
AYNPs
Plasma+Fe(III)+AYNPs
Plasma+Fe(III)
Plasma
B
Figure: 2.46 Fe(III) sensing in drinking water and human blood plasma
Part II
127
The analytical potential of the nanosensor was exploited through analysis of Fe(III) in
real samples. For this purpose, we selected human blood plasma taken from healthy
human volunteer, which was used after standard pretreatment protocol, and tap water
taken from Karachi University. Both of these samples were spiked with the 0.1 mM
FeCl3 solution before use. We observed the Fe(III) dependent quenching in the SPR band
of AuNPs is not affected by the complex analyte samples and the nanosensor response is
distinctive towards Fe(III) (Fig. 2.46).
2.2.3.3.8 UV-visible Spectrophotometric Detection of Pd(II)via Pyr-AuNPs
A series of eighteen salts of 0.1mM concentration were mixed with Pyr-AuNPs in 1:1
ratio. Interestingly we observed the disappearance of color of AuNPs with Pd(II) (Fig.
2.47A). All other salt solutions showed no visible changes at all on AuNPs color such as
Ag(I), Ba(II), Ca(II), Cd(II), Co(I), Cr(III), Cu(I) , Cu(II), Sb(III), Fe(II), Hg(II), K(I),
Mg(II), Na(I), Ni(II), Pb(II), and Rb(I). The significant color change can be attributed to
the destabilization of AuNPs induced by Pd(II). Further spectral analysis results of
mixture of metal ions and Pyr-AuNPs screening (Fig. 2.47) demonstrates that Pd(II)
induced aggregation of AuNPs, hence the surface plasmon resonane (SPR) band is
completely quenched. These results also reveals that Pyr-AuNPs can serve as a selective
“naked-eye” off–on probe for Pd(II) recognition.
Part II
128
400 500 600 7000.0
0.1
0.2Ab
sorb
ance
(uni
ts)
Wavelength(nm)
AYNP+Ag(I)
AYNP+Ba(II)
AYNP+Ca(II)
AYNP+Cd(II)
AYNP+Co(I)
AYNP+Cr(III)
AYNP+Cu(I)
AYNP+Cu(II)
AYNP+Sb(III)
AYNP+Fe(II)
AYNP+Hg(II)
AYNP+K(I)
AYNP+Mg(II)
AYNP+Na(I)
AYNP+Ni(II)
AYNP+Pb(II)
AYNP+Rb(I)
AYNP+Sb(III)
AYNP+Pd(II)
AYNP only
B
Figure: 2.47A) Pictorial display of colorimetric sensing of Pd(II), B) UV-visible
spectrum of salt screening
2.2.3.3.9 Effect of pH on AuNPs Stability and Pd(II) Sensing
For the stability of AuNPs in aqueous solution, pH of the media needs to be optimized so
that the background agglomeration become minimal. The bar graph diagram (Fig. 2.48)
shows the effect of pH on the Pyr-AuNPs. The pH of colloids is found to be 8.4, the
solution is slightly basic due to free pyrazine groups on the surface of AuNPs. The effect
of pH on SPR band was studied by varying pH from 2–12. It is observed that the
variation of pH did not affect the stability of AuNPs, but due to dilution a little decrease
in absorption of SPR band was observed.
The pH of the medium has great influence on the optical response of the complexation
reaction, thus an optimum pH level is essential to develop maximum interaction between
AuNPs and Pd(II) complex. The comparison in intensity of SPR band of the AuNPs-
Pd(II) mixture proves a unique feature of this detection system that the quenching is
independent of the pH change (Fig. 2.48). Since pH variation yields no significant change
on the absorption behaviour of Pyr-AuNPs so our system can be used at any pH.
Part II
129
Figure: 2.48 pH stability of AuNPs with and without Pd(II)
2.2.3.3.10 Morphological investigation of Pyr-AuNPs and its adduct with Pd(II)
The morphology of the AuNPs is investigated by AFM images, the results and analysis
are displayed in Fig.2.49. Fig.2.49a shows the presence of polydispersed and spherical
particles with in the size range of 20-90 nm. Since the adopted reaction conditions do not
give rise to size selectivity for the synthesis of AuNPs, so nanoparticles in wide range of
size were obtained as revealed by AFM analysis. The AFM images in Fig. 2.49b reveals
that the AuNPs were initially small, and were prone to aggregation in the presence of
Pd(II) ions, resulting in the formation of huge complexes of upto 1 µm scale. These
results are in excellent agreement with the UV-visible spectrophotometric data, and
thebehaviour of AuNPs is turned out to be attractive for its application in optical sensing.
Since the size and shape of the metal nanoparticles are related to their optical properties,
it justifies the instant decrease in SPR band of AuNPs which occurred due to aggregation.
Part II
130
Figure: 2.49 A) AFM image of AuNPs, B) AFM image of AuNPs after mixing
with Pd(II)
Part II
131
2.2.3.3.11 FT-IR Spectral Analysis
Since Pd(II) species are known to form complexes with nitrogenated ligands, FT-IR
spectra of AuNPs before and after treatment with Pd(II) suggest the interaction of Pd(II)
with the pyrazine moiety as a result of which C-N band is shifted from 1115 to 1250 cm-
1a region of much higher energy.
2.2.3.3.12 Kinetic evaluation of supramolecular interaction between Pyr-AuNPs and
Pd(II)
Kinetics associated to supramolecular interaction of Pyr-AuNPs and Pd(II) was
investigated by varying the concentration of Pd(II) while keeping constant the
concentration of nanoparticles during mixing. Fig. 2.50a represents the spectral results,
while inset shows the calibration curve which indicated that the data strictly followed
beer’s law and exhibited a good linear correlation to Pd(II) concentration in the range
from 1 to 80 μM with the regression constant (R2) equal to 0.9402. The limit of detection
(LOD), and limit of quantification (LOQ) are found to be 4.23 μM and 12.83 μM.
Moreover, to estimate the binding stoichiometry between AuNPs and Pd(II) Job's plot
experiment was performed. Absorption spectra were recorded by varying the mole ratio
of Pd(II) to AuNPs. Fig. 2.50b displays that the stable AuNPs-Pd(II) is formed at 0.4, 0.6
mole ratios of Pd(II) and AuNPs respectively.
400 450 500 550 600 650 700 750 800
0.00
0.05
0.10
0.15
0.20 y = 0.0073x - 0.0204R² = 0.9402
0
0.02
0.04
0.06
0.08
0.1
0.12
Abso
rban
ce(u
nits
)
Wavelength(nm)
.01uM
.05uM
.1uM
.5uM
1uM
5uM
10uM
20uM
30uM
40uM
50uM
60uM
70uM
80uM
90uM
100uM
A
Part II
132
Figure: 2.50 A) UV-visible spectral profile of the concentration study, inset represents
calibration curve, B) Job’s plot
2.2.3.3.13 Selective interaction of Pyr-AuNPs with Pd(II)in presence ofCompeting
Metals Ions
Since a practical metal chemosensor must possess a high selectivity and rapid response
time to differentiate and recognize the target metal ion among potentially competing
entities, the Pd(II) specificity was assessed by competitive experiments. The absorbance
changes of AuNPs were measured by contacting 1 equivalent of Pd(II) in the presence of
1 equivalent of other interfering metal ions including Cu(I) , Cu(II), Na(I), K(I), Rb(I),
Ni(II), and Sb (III). As shown in Fig. 2.51, the tested metal ions experienced no
interference between the interaction of Pd(II) and Pyr-AuNPs. Hence this specific nano
sensor can be used to recognize Pd(II) ions in a mixture of several other metal ions
without interfering from competing metal ions.
Part II
133
5400.00
0.02
0.04
0.06
0.08
0.10A
bso
rban
ce(u
nit
s)
Wavelength(nm)
AYNP
AYNP+Pd(II)
AYNP+Pd(II)+Cu(II)
AYNP+Pd(II)+Cu(I)
AYNP+Pd(II)+Na(I)
AYNP+Pd(II)+K(I)
AYNP+Pd(II)+Rb(I)
AYNP+Pd(II)+Ni(II)
AYNP+Pd(II)+Sb(III)
Figure: 2.51 Effect of interfering metal ions on Pd(III) sensing
2.2.3.3.14 Detection of Pd(II) in Tap Water and Human Blood Plasma
The analytical application of Pyr-AuNPs to detect Pd(II) in real samples like tap water
and Human blood plasma was also explored. For this purpose, we used human blood
plasma obtained from healthy human volunteer, which was used after standard
pretreatment protocol, and tap water taken from Karachi University. Fig. 2.52a indicates
that a significant magnitude of SPR band quenching is still observed in AuNPs with 0.1
mM Pd(II) spiked tap water. While in case of blood plasma a slight blue shift is observed
with less quenching of SPR band absorbance than 0.1 mM Pd(II) aqueous solution (Fig.
2.52B). These results suggest that our system can be effectively used to detect Pd(II) in
water and blood testing. It is also concluded from the study that the ingredients of tap
water and Human blood plasma did not interfere in the detection process.
Part II
134
Figure: 2.52 UV-visible spectra of Pyr-AuNPs after mixing with A) tap water, B)
Human blood plasma spiked with Pd(II)
Part II
135
2.2.4 A New Pefloxacin Chemosensor Based on Thioacetate Functionalized DMAP
coated Gold Nanoparticles
We developed a synthetic route for the facile synthesis of dimethyl aminopyridine based
thiolated capping agent. The capping agent was used to coat AuNPs which effectively
recognized Pefloxacine (Pef) in aqueous solution. Importantly this protocol provides an
easy, relatively rapid, economical and sensitive method for detection of Pef in water and
in human blood plasma samples. We hope that this method can be utilized for the
recognition and control of this antibiotic in drinking water as well as in important
physiological fluids.
2.2.4.1 Synthesis of Dimethylaminopyridinium Propylthioacetate (D2) Stabilized Gold
Nanoparticles (D2-AuNPs)
1 mL of 0.1 mM solution of D2 was added in a shaking solution of 10 mL of 0.1
Chloroauric acid trihydrate mM . After 15 minutes, 0.1 mL of freshly prepared 4 mM
NaBH4 solution was added in the reaction mixture. The colorless reaction mixture turned
into clear wine red instantly after mixing of NaBH4 solution, and the reaction mixture
was kept on shaking for 2 more hours. The resulting solution was subjected to UV-visible
spectrophotometry and AFM imaging.
D2-AuNPs were synthesized via one phase method by NaBH4 reduction. Various ratios
of Gold salt and D2 were used, and 10:1 with respect to Gold salt and D2 was selected
based on the prominent peak of AuNPs surface plasmon band at 520 nm. UV-visible
spectrum of the colloids was recorded after 2 and 4 hours. Since there was no difference
in absorbance intensity at longer time, the formation of nanoparticles was achieved at 2
hours shaking. However an increase in color intensity of nanoparticles solution was
observed after 3 days, and the UV-visible spectrum indicates the formation of larger
aggregates at prolonged time (Fig 2.52A). Fig. 2.52b reveals the morphology of the
nanoparticles, they were found to be spherical and poly dispersed in size. While the
particles size lie in the range of 15-45 nm (Fig. 2.52C).
Part II
136
Part II
137
Figure: 2.53 A) UV-Visible spectrum of the AuNPs stabilized with D2, B) AFM image
of the D2 capped AuNPs, C) Size distribution histogram of D2-AuNPs
2.2.4.2 Stability of D2-AuNPs
Thermal stability of AuNPs was tested at 100 oC for 1 hour. Only a slight increase in
intensity of SPR was observed, but no change was appeared in the dispersety of the
solution, even on heating for longer time (Fig.2.53A). This effect in the SPR band
maxima is described by electron dephasing mechanism in the literature (Link and El-
Sayed 1999). Furthermore, chloride promoted aggregation of the nanoparticles was also
studied. Fig. 2.53b shows that the D2-AuNPswere stable over the wide range of 0.01 mM
to 10 mM concentration of sodium chloride (NaCl) with the decrease in intensity of SPR
band (Bae, Nam et al. 2002). At NaCl concentration of 50 mM or above only, the SPR
band broadening and visible aggregation of the AgNPs was observed.
Part II
138
Figure: 2.54 A) Effect of heating on SPB, B) Aggregation of D2-AuNPs induced by
various concentration of NaCl
Part II
139
2.2.4.3 Exploration of Chemosensing Potential of D2-AuNPs
The recognition behavior of fully characterized D2-AuNPswas assessed for various drugs
by mixing 1:1 v/v ratio of 0.1 mM drugs solutions and AuNPs. The UV–vis spectra of
AuNPs were recorded instantly after addition of drug solutions. The effect of tested drugs
such as Pefloxacin mesylate, Paracetamol, Penicillin, Amoxicillin, Cefaclor, Cefotaxime
sodium, Ceftriaxone sodium, Cephalexime, and Cephradine on absorption intensity was
investigated and plotted in Fig. 2.55. All drugs induced the enhancement in the
absorbance intensity of D2-AuNPs, but Pefloxacin mesylate resulted in the hyperchromic
shift, while a new peak around 700 nm is appeared. The red shifted band was presumably
observed due to the induced aggregation of AuNPs. The substantially large difference
between rest of the drugs and Pefloxacin mesylate added absorption behaviour indicates
some chemical or physical interactions between the D2-AuNPsand this drug. Hence,
these results of spectrophotometric drugs screening suggest that this nano probe can be
exploited as a selective sensor for the detection of Pef.
Figure: 2.55 UV-Visible spectral profile generated after mixing drugs with D2-AuNPs
Part II
140
2.2.4.4 Effect of the pH on the Stability of D2-AuNPs and Pef Response
The pH of colloids is found to be in the range of 4-5, the solution is slightly acidic in
nature due to cationic protonated form. The synthesized D2-AuNPs was found to be
stable in both basic media, as well as acidic media. While at neutral pH, the colloids were
prone to aggregation and resulted in the peak broadening of the SPR band.
Since the pH of the medium has a great impact on the optical response of the
complexation reaction, an optimum pH is thus essential to develop for the interaction of
AuNPs-Pef complex. While a practical sensor needs to be smart enough to remain
effective in a wide range of pH. A study of the pH in the range of 2–12 with the
optimized conditions for interaction of nanoparticles with the drug is shown in Fig. 2.56.
The absorbance of SPR band of the D2-AuNPs-Pef mixture shows the maxima at pH 10-
11, and before and after which it gradually decreases. On the basis of these results it can
be safely concluded that the drug and nanoparticles interaction is independent of the pH
change, and our system does not rely on pH of media.
Figure: 2.56 Effect of pH on the interaction of D2-AuNPs with Pef
Part II
141
2.2.4.5 FT-IR Spectral Analysis
To have a deeper understanding of the mechanism of nanoparticles formation, a
comparison between FT-IR spectrum of D2, and D2-AuNPsis studied. Results suggest
that thioester functionality is cleaved and Gold nanoparticle surface is stabilized with
thiol group since the thioester peak at 1716 1/cm in D2 spectrum is diminished after the
formation of AuNPs, while the rest of the characteristics peak for DMAP were all
present. We also recorded the FT-IR spectra of Pef, which showed O-H stretch at 3427,
N-CH3 at 2646, carboxyl carbonyl at 1716, C-O at 1627, C-F at 1091 1/cm. While
sulfoxide asymmetric and symmetric stretches were observed at 1197 and 1051 1/cm
(Dorofeev 2004). Interestingly, in a mixture of D2-AuNPsand Pef, the acid carbonyl
peak which appeared at 1716 1/cm in the drug was absent, and a new peak at 1583 1/cm
appeared. This peak at 1583 1/cm is attributed to the carboxylate moiety, which is
responsible for the accumulation of Pef with AuNPs via hydrogen bonding.
2.2.4.6 AFM investigation of the interaction between D2-AuNPs and Pef
AFM image of the 1:1 mixture of D2-AuNPs and Pef revealed that the nanoparticles are
prone to agglomeration upon interaction with the drug. Fig.2.57 depicts the presence of
larger aggregates ranging in 50-150 nm size. These results are in agreement with that of
UV-visible and FT-IR spectra, and it can be deduced that the drug-nanoparticle
interaction is based on aggregation induced mechanism.
Part II
142
Figure: 2.57 AFM image of the D2-AuNPs -Pef 1:1 mixture
2.2.4.7 Analytical Performance of the Pef Nanosensor
The analytical performance of Pef chemosensor is demonstrated with the help of
calibration curve. The absorption titration of Pef to measure the sensitive optical response
of D2-AuNPsat 700 nm (Fig.2.58) strictly followed beer’s law and exhibited a good
linear correlation in the range from 0.1 to 90 μM with the regression constant (R2) equal
to 0.945, and the limit of detection (LOD) 10.9 μM. Moreover, to estimate the binding
stoichiometry between stable D2-AuNPs-Pef complex, Job's plot experiment was
performed. Absorption spectra were recorded by varying the mole ratio of Pef to D2-
AuNPs. The minimum on the plot between absorbance and mole fraction appeared at 0.5,
which correspond to the 1:1 stoichiometric relation between the stable D2-AuNPs-Pef
complexes.
Part II
143
Part II
144
Figure: 2.58 A) Concentration study of Pef with D2-AuNPs, B) Calibration
curve at 700 nm C) Job’s plot for binding ratio
2.2.4.8 Interference Study
Since it is essential for a practical detection system that it can specifically target the
binding specie in an environment where other competing entities are present. Therefore
to test the specificity of this nanosensor, we evaluated the response of optimized Pef
recognition strategy in the presence of other interfering drugs. All the drugs were used in
1:1 (v/v) against Pef, and the UV-Visible spectra of the mixtures were recorded which are
presented in Fig. 2.59. It shows that the addition of competing drugs such as 6-amino
penicillanic acid, amoxicillin, aspirin, cefaclor, diclofenac sodium, flurbiprofen,
paracetamol, and penicillin respectively to the D2-AuNPs-pef mixture showed no
significant change in the absorption intensity of 700 nm band. These results conclude that
this analytical strategy may serve as a potential spectrophotometric probe for assaying
Pef in the mixture of other drugs.
Part II
145
Figure: 2.59 Effect of competing drugs on Pef response towards nanosensor
2.2.4.9 Analysis of Pef in Real Samples
The utility of the optimized Pef detection system was explored to check the sensitivity
and selectivity of optical response in real and complex samples. We tested the efficiency
of this Pef chemosensor in the analysis of tap water and human blood plasma. Human
blood plasma was collected from healthy human volunteer, which was used after standard
pretreatment protocol, while tap water was taken from Karachi University. Both samples
were spiked with 0.1 mM concentration of pef before analysis. Fig. 2.60A indicates that
the distinctive pef recognition signal is still observed in D2-AuNPs with 0.1 mM Pef
spiked tap water. While in case of blood plasma only a hyperchromic shift without the
700 nm band is observed (Fig. 2.60B). These results suggest that our system can be
effectively used to detect Pef in drinking water treatment and blood testing.
Part II
146
Figure: 2.60 UV-visible spectra of Pef recognition in (A) tap water (B)
Human blood plasma
Part II
147
2.3 EXPERIMENTAL
2.3.1 Materials
All the chemicals (Solvents and reagents) used in the synthesis of ligand were of
analytical grade and used as provided. Oven dried glassware were used, washed with
aqua regia and rinsed by deionized water. Freshly prepared solutions were used
throughout synthesis and analysis. 4-Pyridinecarboxaldehyde, Pyrazine, and Dimethyl
aminopyridine were purchased from Tokyo chemicals industry (TCI, China). Dibromo
propane and potassium thioacetate were purchased from Alfa-Aesar (MA, USA).
Chloroauric acid trihydrate, Triphenylphosphine gold (I) chloride, Borane tert-butylamine
complex (BTBC), and all the bisphosphines were purchased from Sigma-Aldrich (St.
Louis, USA). Other chemicals were purchased from local chemical suppliers.
2.3.2 Instruments
UV-Visible spectrophotometer used for characterization of nanoparticles was Thermo
Scientific Evolution 300 spectrophotometer, operated at 2 nm resolution with a quartz
cuvette of 1cm path length. The spectra were reordered in range 200-800 nm. The FT-IR
spectra were recorded with Bruker-Vector-22, in diffuse reflectance modewith2 cm-1
resolution. Sample preparation was consist of mixing test sample separately with KBr in
1:1 ratio to form the KBr disk. The spectra were recorded between 400 and 4000 cm-1.
Oakton, Eutech model-510 pH-meter having glass-electrode and Ag/AgCl electrode was
used as a reference in pH measurements. Morphological analysis of synthesized nano
particles was carried out by Atomic Force Microscope (AFM) Agilent-5500 operated in
tapping-mode. Sample preparation for AFM consist of sorption of nanoparticles on
freshly cleaved mica wafer surface from a drop for 30 minutes and was then air dried
under controlled environment. AFM was operated in tapping-mode having triangular
shape silicon-nitride cantilever made by Veeco model-MLCT-AUHW with 0.01-0.01N/m
nominal value of spring-constant.
Part II
148
2.3.3 Synthesis of Bisphosphines Coated Gold Nanoclusters
In a typical synthesis, AuPPh3Cl was dissolved in solvent of choice to reach a
concentration of 10-3 M. The desired bisphosphine ligand was then added in equal
molarity and solution was stirred until completely dissolved. Lastly, BTBC is added at a
concentration of 510-3 M and stirring was continued for about 30 min, under Ar gas.
The solution was turned orange and displayed absorption maxima at around 400 nm.
2.3.4 Synthesis of 1-(3-(acetylthio)propyl)-4-formylpyridin-1-ium (4-Py) Coated
Silver Nanoparticles (4-Py-AgNPs)
1 mL of 0.1 mM solution of 4-Py was added in a shaking solution of 10 mL of 0.1 mM
silver nitrate. After 15 minutes, 0.1 mL of freshly prepared 4 mM NaBH4 solution was
added in the reaction mixture. The colorless reaction mixture turned yellow-brown
instantly after addition of NaBH4 solution, but the solution was stirred further for 2 hours.
The AgNPs colloidal dispersions were centrifuged for 20 min till all nanoparticles
precipitated at 10,000 rpm to separate the potential large aggregates and excess unbound
ligand from the broth followed by redispersion in deionized water.
2.3.5 Synthesis of 1-(3-(acetylthio)propyl)pyrazin-1-ium (Pyr) Coated Gold
Nanoparticles (Pyr-AuNPs)
Pyr was dissolved in 10% mixture of methanol and deionized water to prepare 0.1 mM
solution, while 0.1 mM solution of tetrachloroauric (III) acid was prepared in deionized
water only. 1 mL (0.1 mM) of Pyr solution was added into 10 mL (0.1 mM) of
tetrachloroauric (III) acid and the mixture was stirred. After 10 minutes 0.1 mL of freshly
prepared 4 mM NaBH4 solution was added in the reaction mixture. The colour of the
reaction mixture rapidly changed from colourless to wine red, stirring was continued for
2 hours. The colloidal AuNPs are found stable at room temperature and ordinary
atmosphere for more than 1 month. Solid AuNPs were collected by freeze drying.
Part II
149
2.3.6 Synthesis of 1-(3-(acetylthio)propyl)-4-(dimethylamino)pyridin-1-ium (D2)
Coated Gold Nanoparticles (D2-AuNPs)
1 mL of 0.1 mM solution of D2 was added in a shaking solution of 0.1 Chloroauric acid
trihydrate mM (10 mL). After 15 minutes, 0.1 mL of freshly prepared NaBH4 solution (4
mM) was added in the reaction mixture. The colourless reaction mixture was turned into
clear wine red solution instantly after addition of NaBH4 solution, and the reaction
mixture was kept on shaking for 2 more hours. The resulting solution was subjected to
UV-visible spectrophotometry and AFM analysis.
2.3.7 Synthesis of Chlorhexidine (CHX) Coated Gold Nanoparticles (CHX-AuNPs)
10 mL, of AuCl4 (0.5 mM) solution is added into the 1 mL CHX solution (0.5 mM) and
the mixture was allowed to stir for 10 minutes. After 10 minutes 30 microlitres, sodium
borohydride (4mM) was added as reducing agent. The intense deep red color
characteristics of colloidal gold were obtained instantly on addition of borohydride. The
solution was stirred for two more hours. This solution was subjected to UV-Visible
spectrophotometry and Atomic Force Microscopy in order to confirm the successful
formation of gold nanoparticles.
2.3.8 Synthesis of Neomycin (Neo) Coated Silver Nanoparticles (Neo-AgNPs)
10 mL, Silver nitrate aqueous solution (0.1mM) is added into the 2 mL, Neo aqueous
solution (0.1 mM) and the mixture was allowed to stir for 10 minutes. Then 0.5 mL,
sodium hydroxide solution (30mM) was added as mild reducing agent. Shaking was
continued for six hours, after which the solution was subjected to characterization as
mentioned above.
2.3.9 Synthesis of Guanidinobutyric Acid (GBA) Coated Silver Nanoparticles (Neo-
AgNPs)
1 mL, Silver nitrate aqueous solution (0.1mM) is added into the 1 mL, GBA solution (0.1
mM) and the mixture was allowed to stirr. After 10 minutes 1.5 mL, sodium hydroxide
Part II
150
solution (30mM) was added as mild reducing agent, and the reaction was further stirred
for 6 hours.
2.3.10 Cell Incubation
Cytotoxic T lymphocyte (CTLL-2) from (ECACC, ATCC) for AFM based study were
used. For this purpose, the CTLL-2 (ATCC® TIB-214™) cells was cultured in ATCC-
formulated complete RPMI 1640 ((1mM Sodium pyrovate, 2M HEPES and 10% FBS,
10% T-STIM & Con A, 1, 1% L-glutamine and Non-Essential Amino Acids) culturing
media in 75 cm2 flasks, and kept in 5% CO2 incubator at 37oC. After complete
confluences about 5×104 cell/well (in 96 well flat bottom plate corning) of cell line were
used to conduct the experiments.
2.3.11 Cell Treatment
The cell permeability of the 4Py-AgNPs on T-cells was evaluated by incubating 4Py-
AgNPs at different doses with T-cells for 30 min at 37 oC in 5% CO2 incubator. After
incubation the cells were centrifuged at 1300 rpm for 4 min and the cell pellet were
washed twice with 1X, PBS (Phosphate buffer saline) buffer to remove the unbound test
sample. Then the treated cells were incubated for 20 min with multiple concentration of
Cu+ solution at 37 oC in 5% CO2 incubator followed by AFM microscopy.
2.3.12 MTT Based Cytotoxicity Assay
Cytotoxicity of test samples were evaluated in 96-well flat-bottomed micro plates by
using the standard MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium
bromide) colorimetric assay. For this purpose, the lymphocytes cells were cultured in
their respective media RPMI 1640 in 75 cm2 flasks, and kept in 5% CO2 incubator at 37 oC. Cell culture with the concentration of 5x104cells/ml was prepared and introduced
(100 µL/well) into 96-well plates. After overnight incubation, medium was removed and
200 µL of fresh medium was added with different concentrations of test sample (30,
50,100 µM). After 48 hrs, 200 µL MTT (0.5 mg/ml) was added to each well and
incubated further for 4 hrs. Subsequently, 100µL of DMSO was added to each well. The
extent of MTT reduction to formazan within cells was calculated by measuring the
Part II
151
absorbance at 540 nm, using a micro plate reader (Spectra Max plus, Molecular Devices,
CA, USA). The percent inhibition was calculated by using the following formula:
Inhibition (%) = [1 - (treated/control)] × 100
Viability (%) = 100 – inhibition (%)
2.3.13 Minimum Inhibitory Concentration by Agar Well Diffusion Method
To calculate minimum inhibitory concentration (MIC), the agar well diffusion method
was employed. Estimation of Minimum inhibitory concentration for 4-py ligand was
measured with or without Ag nanoparticle. In brief, Mueller Hinton agar was used as
medium to grow a lawn of E.coli ATCC 8739at the concentration of 106 cells in one ml.
Separately, Mueller Hinton agar plates carried Cu(I) was made for 4-Py-AgNPs.
Duplicate dilutions were used to calculate minimum inhibition zones. The 60mm well
was made by using a borer. The 100 µg ml¯1 stock solution of 4-Py was used to avoid
nonspecific merged zones of inhibition. In each well different amounts of various
concentrations ranging from 80-100µg ml¯1were added. The plates were incubated at
room temperature for 2 hours to allow the diffusion process to take place before it was
incubated for 24-48 hours at 37 ºC ±1. The zones of inhibition were measured by using a
millimeter scale.
2.3.14 Atomic Force Microscopy Imaging
E. coliATCC 8739 was developed on tryptic soy agar (Oxoid UK) at 37 oC for 24 h in
static conditions, which were marked as stock E. coli culture. A 10 µL droplet of each
test sample was applied onto a poly-L-lysine (PLL) coated glass slide and allowed to
stand at 25 °C for drying, in the meanwhile, a freshly incubated culture of E. coli on
tryptic soy agar (Oxoid UK) was diluted in distilled water to make 106 cfu of E. coli and
10 µL droplets of this solution were transferred onto a freshly cleaved mica surface and
left to dry. Control samples were not treated with the ligand. After deposition, the
samples were rinsed several times with Milli-Q water, and air-dried at 25 °C. The dried
sample was characterized by atomic force microscopy to check its size and morphology.
The test samples were added into vials of distilled water containing 106 cfu of E. coli
Part II
152
bacteria and incubated for 2 h at 37 oC. All experiments were performed with duplicate
cultures for each sample.
The same procedure was applied for 4, and 8 h to check the degradation of E. coli cells.
4-Py-AgNPs dose of 100 µg were treated with 106 cfu of E. coli for 4 and 8 h and were
characterized by atomic force microscopy to check the cell changes and note the effects
of these AgNPs. Atomic force microscope was used (AFM, Agilent Technologies 5500,
USA) in the ACAFM mode. We used a high frequency Si cantilever of 125 µm length,
force constant 42 N m-1 and resonance frequency 330 kHz. All the E. coli samples were
prepared and analyzed under same condition.
Part II
153
REFERENCES
Abdalla, M. A. (1991). "Selective Spectrophotometric Determination of Some
Cephalosporins in Pharmaceutical Formulations." Analytical Letters 24(1): 55-67.
Ackerson, C. J., P. D. Jadzinsky, et al. (2005). "Thiolate Ligands for Synthesis of Water-
Soluble Gold Clusters." Journal of the American Chemical Society 127(18):
6550-6551.
Ajlec, R. and J. Štupar (1989). "Determination of Fe(III) species in wine by ion-exchange
chromatography-flame atomic absorption spectrometry." Analyst 114(2): 137-
142.
Ali, M. and A. Mohammad (1994). "Polarogaphic determination of cephradine in
aqueous and biological media." Bioelectrochemistry and Bioenergetics 33(2):
201-204.
Amro, N. A., L. P. Kotra, et al. (2000). "High-resolution atomic force microscopy studies
of the Escherichia coli outer membrane: structural basis for permeability."
Langmuir 16(6): 2789-2796.
Andrews, N. C. (2000). "Fe(III) metabolism: Fe(III) deficiency and Fe(III) overload."
Annual Review of Genomics and Human Genetics 1(1): 75-98.
Annie Ho, J.-a., H.-C. Chang, et al. (2012). "DOPA-mediated reduction allows the facile
synthesis of fluorescent gold nanoclusters for use as sensing probes for ferric
ions." Analytical Chemistry 84(7): 3246-3253.
Ateeq, M., M. R. Shah, et al. (2014). "Green synthesis and molecular recognition ability
of patuletin coated gold nanoparticles." Biosensors and Bioelectronics.
Badawy, A. M. E., T. P. Luxton, et al. (2010). "Impact of environmental conditions (pH,
ionic strength, and electrolyte type) on the surface charge and aggregation of
silver nanoparticles suspensions." Environmental Science & Technology 44(4):
1260-1266.
Part II
154
Bae, C. H., S. H. Nam, et al. (2002). "Formation of silver nanoparticles by laser ablation
of a silver target in NaCl solution." Applied Surface Science 197–198(0): 628-
634.
Bagherzadeh, M., M. Pirmoradian, et al. (2014). "Electrochemical Detection of Pb and
Cu by Using DTPA Functionalized Magnetic Nanoparticles." Electrochimica
Acta 115(0): 573-580.
Bao, H., E. Wang, et al. (2006). "One-Pot Synthesis of CdTe Nanocrystals and Shape
Control of Luminescent CdTe–Cystine Nanocomposites." Small2(4): 476-480.
Bergeron, D. E., O. Coskuner, et al. (2008). "Ligand exchange reactions in the formation
of diphosphine-protected gold clusters." The Journal of Physical Chemistry C
112(33): 12808-12814.
Bertino, M. F., Z.-M. Sun, et al. (2006). "Facile syntheses of monodisperse ultrasmall Au
clusters." The Journal of Physical Chemistry B 110(43): 21416-21418.
Bindhu, M. and M. Umadevi "Green Synthesized Gold Nanoparticles as a Probe for the
Detection of Fe3+ Ions in Water." Journal of Cluster Science: 1-10.
Biver, T., A. Corti, et al. (2012). "Analysis of 4-dimethylaminopyridine (DMAP)-gold
nanoparticles behaviour in solution and of their interaction with calf thymus DNA
and living cells." Journal of Nanoparticle Research 14(2): 1-12.
Blonder, R. (2010). "The story of nanomaterials in modern technology: An advanced
course for chemistry teachers." Journal of Chemical Education 88(1): 49-52.
Bogart, L. K., G. Pourroy, et al. (2014). "Nanoparticles for Imaging, Sensing, and
Therapeutic Intervention." ACS nano 8(4): 3107-3122.
Boisselier, E. and D. Astruc (2009). "Gold nanoparticles in nanomedicine: preparations,
imaging, diagnostics, therapies and toxicity." Chemical Society Reviews 38(6):
1759-1782.
Part II
155
Bolshakova, A., O. Kiselyova, et al. (2001). "Comparative studies of bacteria with an
atomic force microscopy operating in different modes." Ultramicroscopy 86(1):
121-128.
Bouzigues, C., T. Gacoin, et al. (2011). "Biological Applications of Rare-Earth Based
Nanoparticles." ACS Nano 5(11): 8488-8505.
Bruchez, M., M. Moronne, et al. (1998). "Semiconductor nanocrystals as fluorescent
biological labels." Science 281(5385): 2013-2016.
Brust, M., M. Walker, et al. (1994). "Synthesis of thiol-derivatised gold nanoparticles in a
two-phase Liquid-Liquid system." Journal of the Chemical Society, Chemical
Communications(7): 801-802.
Cao, L., J. Sui, et al. (2011). "Generic immunoassay of quinolones: production and
characterization of anti-pefloxacin antibodies as broad selective receptors." Food
Analytical Methods 4(4): 517-524.
Carey, J. S., D. Laffan, et al. (2006). "Analysis of the reactions used for the preparation of
drug candidate molecules." Organic &Biomolecular Chemistry 4(12): 2337-2347.
Carlson, C., S. M. Hussain, et al. (2008). "Unique cellular interaction of silver
nanoparticles: size-dependent generation of reactive oxygen species." The Journal
of Physical Chemistry B 112(43): 13608-13619.
Cassol, C. C., A. P. Umpierre, et al. (2005). "The Role of Pd Nanoparticles in Ionic
Liquid in the Heck Reaction." Journal of the American Chemical Society 127(10):
3298-3299.
Chah, S., M. R. Hammond, et al. (2005). "Gold Nanoparticles as a Colorimetric Sensor
for Protein Conformational Changes." Chemistry & Biology 12(3): 323-328.
Chen, X., Y.-F. Huang, et al. (2008). "Using aptamer–nanoparticle conjugates for cancer
cells detection." Journal of Biomedical Nanotechnology 4(4): 400-409.
Part II
156
Chen, X., Y.-F. Huang, et al. (2008). "Using AptamerNanoparticle Conjugates for Cancer
Cells Detection." Journal of Biomedical Nanotechnology 4(4): 400-409.
Chen, Y. S., Y. C. Hung, et al. (2009). "Assessment of the In Vivo Toxicity of Gold
Nanoparticles." Nanoscale Research Letters 4(8): 858-864.
Cho, J. and F. Caruso (2005). "Investigation of the interactions between ligand-stabilized
gold nanoparticles and polyelectrolyte multilayer films." Chemistry of Materials
17(17): 4547-4553.
Colman, B. P., C. L. Arnaout, et al. (2013). "Low concentrations of silver nanoparticles
in biosolids cause adverse ecosystem responses under realistic field scenario."
PLoS One8(2): e57189.
Cookson, J. (2012). "The preparation of palladium nanoparticles." Platinum Metals
Review 56(2): 83-98.
Corthey, G. n., A. A. Rubert, et al. (2012). "New insights into the chemistry of thiolate-
protected palladium nanoparticles." The Journal of Physical Chemistry C 116(17):
9830-9837.
Coughlin, R. T., C. R. Caldwell, et al. (1981). "A cationic electron spin resonance probe
used to analyze cation interactions with lipopolysaccharide." Biochemical and
Biophysical Research Communications 100(3): 1137-1142.
Coughlin, R. T., S. Tonsager, et al. (1983). "Quantitation of metal cations bound to
membranes and extracted lipopolysaccharide of Escherichia coli." Biochemistry
22(8): 2002-2007.
Craig F. Bohren, D. R. H. (1983). Absorption and scattering of light by small particles.
New York, Wiley: 530.
Croxen, M. A. and B. B. Finlay (2010). "Molecular mechanisms of Escherichia coli
pathogenicity." Nature Reviews Microbiology 8(1): 26-38.
Part II
157
Daniel, M.-C. and D. Astruc (2003). "Gold Nanoparticles: Assembly, Supramolecular
Chemistry, Quantum-Size-Related Properties, and Applications toward Biology,
Catalysis, and Nanotechnology." Chemical Reviews 104(1): 293-346.
Daniel, M. C. and D. Astruc (2004). "Gold Nanoparticles: Assembly, Supramolecular
Chemistry, Quantum-Size-Related Properties, and Applications Toward Biology,
Catalysis, and Nanotechnology." Chemical Reviews 104(1): 293-346.
Dass, A., K. Holt, et al. (2008). "Mass spectrometrically detected statistical aspects of
ligand populations in mixed monolayer Au25L18 nanoparticles." The Journal of
Physical Chemistry C 112(51): 20276-20283.
Davis, R. E., E. Bromels, et al. (1962). "Boron hydrides. III. Hydrolysis of sodium
borohydride in aqueous solution." Journal of the American Chemical Society
84(6): 885-892.
Deng, B., L. Li, et al. (2009). "Pharmacokinetics of pefloxacin mesylate in human urine
using capillary electrophoresis electrochemiluminescence detection." Journal of
Chromatography B 877(24): 2585-2588.
Deng, H.-H., G.-W. Li, et al. (2014). "Thermally treated bare gold nanoparticles for
colorimetric sensing of copper ions." Microchimica Acta 181(9-10): 911-916.
Deng, H.-H., G.-W. Li, et al. (2014). "Thermally treated bare gold nanoparticles for
colorimetric sensing of copper ions." Microchimica Acta: 1-6.
Devani, M., I. T. Patel, et al. (1992). "Spectrophotometric determination of ampicillin
and its dosage forms." Talanta 39(10): 1391-1394.
a -Cruz, M. S., M. a. J. López de Alda, et al. (2003). "Environmental behavior and
analysis of veterinary and human drugs in soils, sediments and sludge." TrAC
Trends in Analytical Chemistry 22(6): 340-351.
Dorofeev, V. (2004). "Infrared spectra and the structure of drugs of the fluoroquinolone
group." Pharmaceutical Chemistry Journal 38(12): 693-697.
Part II
158
Drbohlavova, J., V. Adam, et al. (2009). "Quantum Dots — Characterization, Preparation
and Usage in Biological Systems." International Journal of Molecular Sciences
10(2): 656-673.
Duan, L., Y. Xu, et al. (2008). "Highly sensitive and selective Pd 2+ sensor of
naphthalimide derivative based on complexation with alkynes and thio-
heterocycle." Chemical Communications(47): 6339-6341.
Dupont, J., G. S. Fonseca, et al. (2002). "Transition-metal nanoparticles in imidazolium
ionic liquids: recycable catalysts for biphasic hydrogenation reactions." Journal of
the American Chemical Society 124(16): 4228-4229.
Dupont, J., G. S. Fonseca, et al. (2002). "Transition-Metal Nanoparticles in Imidazolium
Ionic Liquids: Recycable Catalysts for Biphasic Hydrogenation Reactions."
Journal of the American Chemical Society 124(16): 4228-4229.
El-Kommos, M. E., G. A. Saleh, et al. (2003). "Spectrofluorometric determination of certain
quinolone antibacterials using metal cHeLation." Talanta 60(5): 1033-1050.
El-Safty, S. A. and M. Shenashen (2013). "Optical mesosensor for capturing of Fe (III)
and Hg (II) ions from water and physiological fluids." Sensors and Actuators B:
Chemical 183: 58-70.
El Badawy, A. M., R. G. Silva, et al. (2010). "Surface charge-dependent toxicity of silver
nanoparticles." Environmental Science & Technology 45(1): 283-287.
Fardella, G., P. Barbetti, et al. (1995). "Quantitative analysis of fluoroquinolones by 1 H-and
19 F-NMR spectroscopy." International Journal of Pharmaceutics 121(1): 123-127.
Fayet-Moore, F., P. Petocz, et al. (2014). "Micronutrient Status in Female University
Students: Fe(III), Zinc, Copper, Selenium, Vitamin B12 and Folate." Nutrients
6(11): 5103-5116.
Part II
159
Fisher, J. F., S. O. Meroueh, et al. (2005). "Bacterial Resistance to β-Lactam
Antibiotics:Compelling Opportunism, Compelling Opportunity†." Chemical
Reviews 105(2): 395-424.
Frens, G. (1973). "An experiment concerning the dispersion forces between very small
metal spheres." Physics Letters A 44(3): 208-210.
G. Georgopoulos, A. R., MJ Yonone-Lioy, RE Opiekun, PJ Lioy, P (2001).
"Environmental copper: its dynamics and human exposure issues." Journal of
Toxicology and Environmental Health Part B: Critical Reviews 4(4): 341-394.
Gandubert, V. J. and R. B. Lennox (2005). "Assessment of 4-(dimethylamino) pyridine
as a capping agent for gold nanoparticles." Langmuir 21(14): 6532-6539.
Gao, S., N. Koshizaki, et al. (2009). "Innovative platform for transmission localized
surface plasmon transducers and its application in detecting heavy metal Pd (II)."
Analytical Chemistry 81(18): 7703-7712.
Gao, Y., N. Shao, et al. (2011). "Catalytic activities of subnanometer gold clusters (Au16–
Au18, Au20, and Au27–Au35) for CO oxidation." ACS nano 5(10): 7818-7829.
Garrett, C. E. and K. Prasad (2004). "The art of meeting palladium specifications in
active pharmaceutical ingredients produced by Pd‐catalyzed reactions." Advanced
Synthesis & Catalysis 346(8): 889-900.
Gittins, D. I. and F. Caruso (2001). "Spontaneous Phase Transfer of Nanoparticulate
Metals from Organic to Aqueous Media." Angewandte Chemie International
Edition 40(16): 3001-3004.
Gnanaprakash, G., S. Mahadevan, et al. (2007). "Effect of initial pH and temperature of
Fe(III) salt solutions on formation of magnetite nanoparticles." Materials
Chemistry and Physics 103(1): 168-175.
Part II
160
Grace, A. N. and K. Pandian (2007). "Antibacterial efficacy of aminoglycosidic
antibiotics protected gold nanoparticles—A brief study." Colloids and Surfaces A:
Physicochemical and Engineering Aspects 297(1): 63-70.
Gu, H., P. Ho, et al. (2003). "Presenting vancomycin on nanoparticles to enhance
antimicrobial activities." Nano letters 3(9): 1261-1263.
Hattori, Y., S. Mukasa, et al. (2011). "Synthesis of zinc and zinc oxide nanoparticles from
zinc electrode using plasma in liquid." Materials Letters 65(2): 188-190.
Hong, S., G. Shafai, et al. (2011). "Toward an understanding of ligand selectivity in
nanocluster synthesis." The Journal of Physical Chemistry C 115(30): 14478-
14487.
Hong, Y., Y. M. Huh, et al. (2012). "Nanobiosensors Based on Localized Surface
Plasmon Resonance for Biomarker Detection." Journal of Nanomaterials.
Hua, M., S. J. Zhang, et al. (2012). "Heavy metal removal from water/wastewater by
nanosized metal oxides: A review." Journal of Hazardous Materials 211: 317-331.
Huang, H., K. Wang, et al. (2004). "Design of a Modular‐Based Fluorescent Conjugated
Polymer for Selective Sensing." Angewandte Chemie International Edition
43(42): 5635-5638.
Huang, L.-S. and K.-C. Lin (2001). "Detection of Fe(III) species using inductively
coupled plasma mass spectrometry under cold plasma temperature conditions."
Spectrochimica Acta Part B: Atomic Spectroscopy 56(1): 123-128.
Hudgens, J. W., J. M. Pettibone, et al. (2011). "Reaction mechanism governing formation
of 1, 3-bis (diphenylphosphino) propane-protected gold nanoclusters." Inorganic
Chemistry 50(20): 10178-10189.
Hutchings, G. J., M. Brust, et al. (2008). "Gold-an introductory perspective." Chemical
Society Reviews 37(9): 1759-1765.
Part II
161
Ivask, A., A. ElBadawy, et al. (2013). "Toxicity mechanisms in Escherichia coli vary for
silver nanoparticles and differ from ionic silver." ACS Nano8(1): 374-386.
Iwasawa, T., M. Tokunaga, et al. (2004). "Homogeneous palladium catalyst suppressing
Pd black formation in air oxidation of alcohols." Journal of the American
Chemical Society 126(21): 6554-6555.
Jadzinsky, P. D., G. Calero, et al. (2007). "Structure of a Thiol Monolayer-Protected Gold
Nanoparticle at 1.1 Å Resolution." Science 318(5849): 430-433.
Jamieson, T., R. Bakhshi, et al. (2007). "Biological applications of quantum dots."
Biomaterials 28(31): 4717-4732.
Jang, K.-D., A. Ortega, et al. (2012). "Effect of Lithium Ions on Copper Nanoparticle
Size, Shape, and Distribution." Journal of Nanotechnology 2012.
Jiang, J., H. Jiang, et al. (2011). "A colorimetric and ratiometric fluorescent probe for
palladium." Organic Letters 13(18): 4922-4925.
Ju, J. and W. Chen (2014). "Synthesis of highly fluorescent nitrogen-doped graphene
quantum dots for sensitive, label-free detection of Fe (III) in aqueous media."
Biosensors and Bioelectronics 58: 219-225.
Jung, J. H., J. H. Lee, et al. (2011). "Functionalized magnetic nanoparticles as
chemosensors and adsorbents for toxic metal ions in environmental and biological
fields." Chemical Society Reviews 40(9): 4464-4474.
Kabir, N., H. Ali, et al. (2014). "Silymarin coated gold nanoparticles ameliorates CCl 4-
induced hepatic injury and cirrhosis through down regulation of hepatic stellate
cells and attenuation of Kupffer cells." RSC Advances 4(18): 9012-9020.
Kaplan, O., N. C. Yildirim, et al. (2011). "Assessment of some heavy metals in drinking
water samples of Tunceli, Turkey." Journal of Chemistry 8(1): 276-280.
Part II
162
Kielhorn, J., C. Melber, et al. (2002). "Palladium–a review of exposure and effects to
human health." International Journal of Hygiene and Environmental Health
205(6): 417-432.
Kim, K.-S., D. Demberelnyamba, et al. (2003). "Size-Selective Synthesis of Gold and
Platinum Nanoparticles Using Novel Thiol-Functionalized Ionic Liquids."
Langmuir 20(3): 556-560.
Kim, K. S., S. B. Suh, et al. (2002). "Assembling phenomena of calix [4] hydroquinone
nanotube bundles by one-dimensional short hydrogen bonding and displaced π-π
stacking." Journal of the American Chemical Society 124(47): 14268-14279.
Kim, Y., R. C. Johnson, et al. (2001). "Gold nanoparticle-based sensing of
“spectroscopically silent” heavy metal ions." Nano Letters1(4): 165-167.
Kirubaharan, C. J., D. Kalpana, et al. (2012). "Biomediated Silver Nanoparticles for the
Highly Selective Copper(II) Ion Sensor Applications." Industrial & Engineering
Chemistry Research 51(21): 7441-7446.
Knoppe, S., J. Boudon, et al. (2011). "Size exclusion chromatography for semipreparative
scale separation of Au38 (SR) 24 and Au40 (SR) 24 and larger clusters."
Analytical Chemistry 83(13): 5056-5061.
Lara, F. J., A. M. García-Campaña, et al. (2006). "Multiresidue method for the determination
of quinolone antibiotics in bovine raw milk by capillary electrophoresis-tandem mass
spectrometry." Analytical Cchemistry 78(22): 7665-7673.
Larguinho, M. and P. V. Baptista (2012). "Gold and silver nanoparticles for clinical diagnostics
- From genomics to proteomics." Journal of Proteomics 75(10): 2811-2823.
Lee, H.-C., T.-H. Chen, et al. (2012). "Novel core etching technique on synthesized gold
nanoparticles for colorimetric detection of dopamine biosample". Nano/Micro
Engineered and Molecular Systems (NEMS), 2012 7th IEEE International
Conference on, IEEE.
Part II
163
Leszczynska, E., R. Koncki, et al. (1996). "Biosensors with immobilized penicillin
aminohydrolase and penicillinase for determination of -lactam antibiotics."
Chemia Analityczna 41: 839-844.
Li, J., X. Li, et al. (2003). "Au20: A tetrahedral cluster." Science 299(5608): 864-867.
Link, S. and M. A. El-Sayed (1999). "Spectral properties and relaxation dynamics of
surface plasmon electronic oscillations in gold and silver nanodots and nanorods."
The Journal of Physical Chemistry B 103(40): 8410-8426.
Liu, J.-F., L.-G. Zhang, et al. (1998). "Fabrication of colloidal gold micro-patterns using
photolithographed self-assembled monolayers as templates." Thin Solid Films
327–329(0): 176-179.
Liu, Q., Q. Zhou, et al. (2014). "Nanomaterials for analysis and monitoring of emerging
chemical pollutants." TrAC Trends in Analytical Chemistry 58: 10-22.
Liu, T. Z., S. D. Lee, et al. (1979). "Toxicity of palladium." Toxicology Letters 4(6): 469-
473.
Liu, X., J. G. Worden, et al. (2006). "Kinetic study of gold nanoparticle growth in
solution by Brust-Schiffrin reaction." Journal of Nanoscience and
Nanotechnology 6(4): 1054-1059.
Lo, C.-t. F., K. Karan, et al. (2007). "Kinetic studies of reaction between sodium
borohydride and methanol, water, and their mixtures." Industrial & Engineering
Chemistry Research 46(17): 5478-5484.
Locatelli, C. (2006). "Possible interference in the sequential voltammetric determination
at trace and ultratrace concentration level of platinum group metals (PGMs) and
lead: Application to environmental matrices." Electrochimica Acta 52(2): 614-
622.
Locatelli, C. (2007). "Voltammetric Analysis of Trace Levels of Platinum Group Metals–
Principles and Applications." Electroanalysis 19(21): 2167-2175.
Part II
164
Lu, L. T., L. D. Tung, et al. (2008). "Size and shape control for water-soluble magnetic
cobalt nanoparticles using polymer ligands." Journal of Materials Chemistry
18(21): 2453-2458.
Lu, S., Y. Zhang, et al. (2006). "Preparation of anti-pefloxacin antibody and development
of an indirect competitive enzyme-linked immunosorbent assay for detection of
pefloxacin residue in chicken liver." Journal of Agricultural and Food Chemistry
54(19): 6995-7000.
Magano, J. and J. R. Dunetz (2011). "Large-scale applications of transition metal-
catalyzed couplings for the synthesis of pharmaceuticals." Chemical Reviews
111(3): 2177-2250.
Mah C, Z. I., Fraites TJ, Dobson J, Batich C, Byrne BJ (2000). "Microsphere-mediated
delivery of recombinant AAV vectors in vitro and in vivo." Molecular Therapy
1(5): S239.
Mahtab, R., J. P. Rogers, et al. (1995). "Protein-Sized Quantum-Dot Luminescence Can
Distinguish between Straight, Bent, and Kinked Oligonucleotides." Journal of the
American Chemical Society 117(35): 9099-9100.
Malavolta, M., R. Giacconi, et al. (2010). "Plasma copper/zinc ratio: an
inflammatory/nutritional biomarker as predictor of all-cause mortality in elderly
population." Biogerontology 11(3): 309-319.
Matagne, A., J. Lamotte-Brasseur, et al. (1998). "Catalytic properties of class A beta-
lactamases: efficiency and diversity." Journal of Biochemistry 330(2): 581-598.
Matsukawa, K., S. Watase, et al. (2006). Preparation of metal nano-particles containing
organic-inorganic hybrid thin films and their application.
Mehta, V. N., S. K. Kailasa, et al. (2014). "Sensitive and selective colorimetric sensing of
Fe 3+ ion by using p-amino salicylic acid dithiocarbamate functionalized gold
nanoparticles." New Journal of Chemistry 38(4): 1503-1511.
Part II
165
Meincken, M., D. Holroyd, et al. (2005). "Atomic force microscopy study of the effect of
antimicrobial peptides on the cell envelope of Escherichia coli." Antimicrobial
Agents and Chemotherapy 49(10): 4085-4092.
Miles, D. T. and R. W. Murray (2003). "Temperature-dependent quantized double layer
charging of monolayer-protected gold clusters." Analytical Chemistry 75(6):
1251-1257.
Montay, G., Y. Blain, et al. (1983). "High-performance liquid chromatography of
pefloxacin and its main active metabolites in biological fluids." Journal of
Chromatography B: Biomedical Sciences and Applications 272: 359-365.
Morones, J. R., J. L. Elechiguerra, et al. (2005). "The bactericidal effect of silver
nanoparticles." Nanotechnology 16(10): 2346.
Mostafa, S., M. El-Sadek, et al. (2002). "Spectrophotometric determination of
ciprofloxacin, enrofloxacin and pefloxacin through charge transfer complex
formation." Journal of Pharmaceutical and Biomedical Analysis 27(1): 133-142.
Mulvaney, P. (1996). "Surface Plasmon Spectroscopy of Nanosized Metal Particles."
Langmuir 12(3): 788-800.
Murillo, J. A., J. M. Lemus, et al. (1994). "Spectrofluorimetric analysis of cefoxitin in
pharmaceutical dosage." Talanta 41(4): 557-563.
Murray, C. B., D. J. Norris, et al. (1993). "Synthesis and characterization of nearly
monodisperse CdE (E = S, Se, Te) semiconductor nanocrystallites." Journal of the
American Chemical Society 115(19): 8706-8715.
Nandakumar, R., C. E. Santo, et al. (2011). "Quantitative proteomic profiling of the
Escherichia coli response to metallic copper surfaces." Biometals 24(3): 429-444.
Neely, A., C. Perry, et al. (2009). "Ultrasensitive and highly selective detection of
Al heimer’s disease biomarker using two-photon Rayleigh scattering properties
of gold nanoparticle." ACS Nano 3(9): 2834-2840.
Part II
166
Nikiyan, H., A. Vasilchenko, et al. (2010). "AFM investigations of various disturbing
factors on bacterial cells." Microscopy: Science, Technology, Applications and
Education. Badajoz, Spain: Formatex Research Center: 523-529.
O'Farrell, N., A. Houlton, et al. (2006). "Silicon nanoparticles: Applications in cell
biology and medicine." International Journal of Nanomedicine 1(4): 451-472.
Ohkubo, Y., S. Seino, et al. (2014). "Effect of decrease in the size of Pt nanoparticles
using sodium phosphinate on electrochemically active surface area." Journal of
Nanoparticle Research 16(2): 1-9.
Ohyama, J., Y. Hitomi, et al. (2009). "Size Controlled Synthesis of Gold Nanoparticles
by Porphyrin with Four Sulfur Atoms." Topics in Catalysis 52(6-7): 852-859.
Ooishi, M. and M. Miyao (1997). "Antibiotic sensitivity of recent clinical isolates from
patients with ocular infections." Ophthalmologica 211(Suppl. 1): 15-24.
Patil, R., M. Kokate, et al. (2011). "A novel one step synthesis of silver nanoparticles
using room temperature ionic liquid and their biocidal activity." Comptes Rendus
Chimie 14(12): 1122-1127.
Pérez-López, B. and A. Merkoçi (2011). "Nanomaterials based biosensors for food
analysis applications." Trends in Food Science and Technology 22(11): 625-639.
Perry, C. C., M. Weatherly, et al. (2009). "Atomic force microscopy study of the
antimicrobial activity of aqueous garlic versus ampicillin against Escherichia coli
and Staphylococcus aureus." Journal of the Science of Food and Agriculture
89(6): 958-964.
Pettibone, J. M. and J. W. Hudgens (2010). "Synthetic approach for tunable, size-
selective formation of monodisperse, diphosphine-protected gold nanoclusters."
The Journal of Physical Chemistry Letters 1(17): 2536-2540.
Part II
167
Pettibone, J. M. and J. W. Hudgens (2011). "Gold cluster formation with phosphine
ligands: etching as a size-selective synthetic pathway for small clusters?" ACS
Nano 5(4): 2989-3002.
Pettibone, J. M. and J. W. Hudgens (2012). "Predictive Gold Nanocluster Formation
Controlled by Metal‐Ligand Complexes." Small 8(5): 715-725.
Pettibone, J. M. and J. W. Hudgens (2012). "Reaction network governing diphosphine-
protected gold nanocluster formation from nascent cationic platforms." Physical
Chemistry Chemical Physics 14(12): 4142-4154.
Pettibone, J. M. and N. R. Reardon (2012). "Nucleation products of ligated nanoclusters
unaffected by temperature and reducing agent." Nanoscale 4(18): 5593-5596.
Piao, J., J. Lv, et al. (2014). "A dansyl–rhodamine chemosensor for Fe (III) based on off–
on FRET." Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy 128: 475-480.
Pingali, K. C., S. Deng, et al. (2008). "Effect of Ammonium Nitrate on Nanoparticle Size
Reduction." Research Letters in Nanotechnology 2008.
Pinto, C. G., J. L. P. Pavon, et al. (1995). "Micellar liquid chromatography of zwitterions:
retention mechanism of cephalosporins." Analyst 120(1): 53-62.
Podsiadlo, P., S. Paternel, et al. (2005). "Layer-by-Layer Assembly of Nacre-like
Nanostructured Composites with Antimicrobial Properties." Langmuir 21(25):
11915-11921.
Poole, C. F. (2003). "New trends in solid-phase extraction." TrAC Trends in Analytical
Chemistry 22(6): 362-373.
Prathna, T., N. Chandrasekaran, et al. (2011). "Studies on aggregation behaviour of silver
nanoparticles in aqueous matrices: effect of surface functionalization and matrix
composition." Colloids and Surfaces A: Physicochemical and Engineering
Aspects 390(1): 216-224.
Part II
168
Prichard, H. M. and P. C. Fisher (2012). "Identification of platinum and palladium
particles emitted from vehicles and dispersed into the surface environment."
Environmental Science & Technology 46(6): 3149-3154.
Rademacher, C. and B. Masepohl (2012). "Copper-responsive gene regulation in
bacteria." Microbiology 158(Pt 10): 2451-2464.
Rasouli, S., S. Davaran, et al. (2014). "Positively charged functionalized silica
nanoparticles as nontoxic carriers for triggered anticancer drug release." Designed
Monomers and Polymers 17(3): 227-237.
Ren, W. X., T. Pradhan, et al. (2012). "Rapid responsive palladium sensor under mild
condition." Sensors and Actuators B: Chemical 171: 1277-1282.
Rogach, A. L. (2000). "Nanocrystalline CdTe and CdTe(S) particles: Wet chemical
preparation, size-dependent optical properties and perspectives of optoelectronic
applications." Materials Science and Engineering B: Solid-State Materials for
Advanced Technology 69: 435-440.
Rogach, A. L., A. Eychmüller, et al. (2007). "Infrared-Emitting Colloidal Nanocrystals:
Synthesis, Assembly, Spectroscopy, and Applications." Small 3(4): 536-557.
Rosen, J. E., L. Chan, et al. (2012). "Fe(III) oxide nanoparticles for targeted cancer
imaging and diagnostics." Nanomedicine: Nanotechnology, Biology and
Medicine 8(3): 275-290.
Saha, K., S. S. Agasti, et al. (2012). "Gold nanoparticles in chemical and biological
sensing." Chemical Reviews 112(5): 2739-2779.
Sanderson, H., D. J. Johnson, et al. (2004). "Ranking and prioritization of environmental
risks of pharmaceuticals in surface waters." Regulatory Toxicology and
Pharmacology 39(2): 158-183.
Santoro, M. I. R., N. M. Kassab, et al. (2006). "Quantitative determination of
gatifloxacin, levofloxacin, lomefloxacin and pefloxacin fluoroquinolonic
Part II
169
antibiotics in pharmaceutical preparations by high-performance liquid
chromatography." Journal of Pharmaceutical and Biomedical Analysis 40(1): 179-
184.
Scheinberg, I. H. and I. Sternlieb (1996). "Wilson disease and idiopathic copper
toxicosis." The American Journal of Clinical Nutrition 63(5): 842S-845S.
Scheurell, M., S. Franke, et al. (2009). "Occurrence of diclofenac and its metabolites in
surface water and effluent samples from Karachi, Pakistan." Chemosphere 77(6):
870-876.
Schwarze, T., H. Müller, et al. (2007). "Luminescence Detection of Open‐Shell
Transition‐Metal Ions by Photoinduced Electron Transfer Controlled by Internal
Charge Transfer of a Receptor." Angewandte Chemie International Edition
46(10): 1671-1674.
Selke, S., M. Scheurell, et al. (2010). "Identification and enantioselective gas
chromatographic mass-spectrometric separation of desmethylnaproxen, the main
metabolite of the drug naproxen, as a new environmental contaminant." Journal of
Chromatography A 1217(3): 419-423.
Semetey, V., C. Didierjean, et al. (2002). "Self‐Assembling Organic Nanotubes from
Enantiopure Cyclo‐N, N′‐Linked Oligourease: Design, Synthesis, and Crystal
Structure." Angewandte Chemie 114(11): 1975-1978.
Shah, M. R., S. Ali, et al. (2014). "Morphological analysis of the antimicrobial action of
silver and gold nanoparticles stabilized with ceftriaxone on Escherichia coli using
atomic force microscopy." New Journal of Chemistr y38(11): 5633-5640.
Shamsipur, M., Z. Talebpour, et al. (2002). "Monitoring of ampicillin and its related
substances by NMR." Journal of Pharmaceutical and Biomedical Analysis 30(4):
1075-1085.
Part II
170
Sharma, A., R. Khosla, et al. (1994). "Fluoroquinolones: antimicrobial agents of the
90’s." Indian Journal of Pharmacology 26(4): 249.
Sharma, V. K., R. A. Yngard, et al. (2009). "Silver nanoparticles: green synthesis and
their antimicrobial activities." Advances in Colloid and Interface Science 145(1):
83-96.
Shinkai, M., M. Yanase, et al. (1999). "Intracellular hyperthermia for cancer using
magnetite cationic liposomes." Journal of Magnetism and Magnetic Materials
194(1-3): 176-184.
Shiraishi, Y., M. Ikeda, et al. (2011). "One-pot synthesis of imines from alcohols and
amines with TiO2 loading Pt nanoparticles under UV irradiation." Chemical
Communications 47(16): 4811-4813.
Siripinyanond, A., S. Worapanyanond, et al. (2005). "Field-flow fractionation-inductively
coupled plasma mass spectrometry: an alternative approach to investigate metal-
humic substances interaction." Environmental Science & Technology 39(9):
3295-3301.
Sondi, I. and B. Salopek-Sondi (2004). "Silver nanoparticles as antimicrobial agent: a
case study on E. coli as a model for Gram-negative bacteria." Journal of Colloid
and Interface Science 275(1): 177-182.
Song, F., A. L. Garner, et al. (2007). "A highly sensitive fluorescent sensor for palladium
based on the allylic oxidative insertion mechanism." Journal of the American
Chemical Society 129(41): 12354-12355.
Sun, S., C. B. Murray, et al. (2000). "Monodisperse FePt Nanoparticles and
Ferromagnetic FePt Nanocrystal Superlattices." Science 287(5460): 1989-1992.
Sun, S. and H. Zeng (2002). "Size-controlled synthesis of magnetite nanoparticles."
Journal of the American Chemical Society 124(28): 8204-8205.
Part II
171
Sun, X., L. Wu, et al. (2013). "Longitudinal surface plasmon resonance assay enhanced
by magnetosomes for simultaneous detection of Pefloxacin and Microcystin-LR
in seafoods." Biosensors and Bioelectronics 47: 318-323.
Sun, Y. F., S. B. Liu, et al. (2012). "Metal Oxide Nanostructures and Their Gas Sensing
Properties: A Review." Sensors 12(3): 2610-2631.
Swoboda, S., K. Oberdorfer, et al. (2003). "Tissue and serum concentrations of
levofloxacin 500 mg administered intravenously or orally for antibiotic
prophylaxis in biliary surgery." Journal of Antimicrobial Chemotherapy 51(2):
459-462.
Taglietti, A., Y. A. Diaz Fernandez, et al. (2012). "Antibacterial activity of glutathione-
coated silver nanoparticles against gram positive and gram negative bacteria."
Langmuir 28(21): 8140-8148.
Tang, B., H. Zhang, et al. (2004). "Determination of trace palladium with salicyladehyde
furfuralhydrazone (SAFH) by catalytic kinetic spectrofluorimetry." Analytical
Letters 37(6): 1219-1231.
Thomson, A. M., J. T. Rogers, et al. (1999). "Fe(III)-regulatory proteins, Fe(III)-
responsive elements and ferritin mRNA translation." The International Journal of
Biochemistry & Cell Biology 31(10): 1139-1152.
Toikkanen, O., S. Carlsson, et al. (2009). "Solvent-Dependent Stability of Monolayer-
Protected Au38 Clusters." The Journal of Physical Chemistry Letters 1(1): 32-37.
Tourneux, E., H. Gornitzka, et al. (2007). "Palladate Salts from Chiral Pyridinium Ionic
Liquids: Synthesis and Crystal Structures." Molecules 12(8): 1940-1949.
Tripathy, S. K., J. Y. Woo, et al. (2013). "Colorimetric detection of Fe (III) ions using
label-free gold nanoparticles and acidic thiourea mixture." Sensors and Actuators
B: Chemical 181: 114-118.
Part II
172
Tshikhudo, T. R., D. Demuru, et al. (2005). "Molecular recognition by calix[4]arene-
modified gold nanoparticles in aqueous solution." Angewandte Chemie -
International Edition 44(19): 2913-2916.
Turkevich, J., P. C. Stevenson, et al. (1951). "A study of the nucleation and growth
processes in the synthesis of colloidal gold." Discussions of the Faraday Society
11(0): 55-75.
Tyczkowska, K. L., R. D. Voyksner, et al. (1992). "Solvent degradation of cloxacillin in
vitro: Tentative identification of degradation products using thermospray liquid
chromatography-mass spectrometry." Journal of Chromatography A594(1–2):
195-201.
Uauy, R., M. Olivares, et al. (1998). "Essentiality of copper in humans." The American
Journal of Clinical Nutrition 67(5): 952S-959S.
Umbreit, J. (2005). "Fe(III) deficiency: a concise review." American Journal of
Hematology 78(3): 225-231.
Ung, D., L. D. Tung, et al. (2009). "Variant shape growth of nanoparticles of metallic Fe-
Pt, Fe-Pd and Fe-Pt-Pd alloys." CrystEngComm 11(7): 1309-1316.
Valodkar, M., S. Modi, et al. (2011). "Synthesis and anti-bacterial activity of Cu, Ag and
Cu–Ag alloy nanoparticles: A green approach." Materials Research Bulletin
46(3): 384-389.
Veiopoulou, C., P. Ioannou, et al. (1997). "Application of terbium sensitized fluorescence
for the determination of fluoroquinolone antibiotics pefloxacin, ciprofloxacin and
norfloxacin in serum." Journal of Pharmaceutical and Biomedical Analysis15(12):
1839-1844.
Verma, P. K., A. Giri, et al. (2010). "Superparamagnetic fluorescent nickel-enzyme
nanobioconjugates: synthesis and characterization of a novel multifunctional
biological probe." Journal of Materials Chemistry 20(18): 3722-3728.
Part II
173
Vertelov, G., Y. A. Krutyakov, et al. (2008). "A versatile synthesis of highly bactericidal
Myramistin® stabilized silver nanoparticles." Nanotechnology 19(35): 355707.
Vigderman, L. and E. R. Zubarev (2013). "Therapeutic platforms based on gold
nanoparticles and their covalent conjugates with drug molecules." Advanced Drug
Delivery Reviews 65(5): 663-676.
Wang, B., J. Hai, et al. (2010). "Selective Detection of Fe(III) (III) by
Rhodamine‐Modified Fe3O4 Nanoparticles." Angewandte Chemie International
Edition 49(27): 4576-4579.
Wang, J. and K. Varughese (1987). "Determination of traces of palladium by adsorptive
stripping voltammetry of the dimethylglyoxime complex." Analytica Chimica
Acta 199: 185-189.
Wang, R., F. Yu, et al. (2012). "A turn-on fluorescent probe based on hydroxylamine
oxidation for detecting ferric ion selectively in living cells." Chemical
Communications 48(43): 5310-5312.
Wang, S., S. Sato, et al. (2003). "Preparation of hexagonal-close-packed colloidal crystals
of hydrophilic monodisperse gold nanoparticles in bulk aqueous solution."
Chemistry of Materials 15(12): 2445-2448.
Wang, Y. and H. Yang (2005). "Synthesis of CoPt Nanorods in Ionic Liquids." Journal of
the American Chemical Society 127(15): 5316-5317.
Weissleder, R., G. Elizondo, et al. (1990). "Ultrasmall superparamagnetic Fe(III) oxide:
Characterization of a new class of contrast agents for MR imaging." Radiology
175(2): 489-493.
Wilson, B. A., V. H. Smith, et al. (2003). "Effects of three pharmaceutical and personal
care products on natural freshwater algal assemblages." Environmental Science &
Technology 37(9): 1713-1719.
Part II
174
Wishart, D. (1984). "Recent advances in antimicrobial drugs: the penicillins." Journal of
the American Veterinary Medical Association 185(10): 1106-1108.
Wu, P., Y. Li, et al. (2009). "CdTe Quantum Dots (QDs) Based Kinetic Discrimination of
Fe2+ and Fe3+, and CdTe QDs-Fenton Hybrid System for Sensitive
Photoluminescent Detection of Fe2+." Analytical Chemistry 81(15): 6252-6257.
Wu, S.-P., Y.-P. Chen, et al. (2011). "Colorimetric detection of Fe 3+ ions using
pyrophosphate functionalized gold nanoparticles." Analyst 136(9): 1887-1891.
Xiang, G., Y. Zhang, et al. (2010). "Determination of trace copper in food samples by
flame atomic absorption spectrometry after solid phase extraction on modified
soybean hull." Journal of Hazardous Materials 179(1): 521-525.
Xiao, Q., H. Gao, et al. (2012). "Gold nanoparticle-based optical probes for sensing
aminothiols." TrAC - Trends in Analytical Chemistry 40: 64-76.
Xie, Y., Y. Song, et al. (2010). "Near-infrared spectroscopy quantitative determination of
Pefloxacin mesylate concentration in pharmaceuticals by using partial least
squares and principal component regression multivariate calibration."
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy75(5):
1535-1539.
Yang, J., G. Zhou, et al. (1998). "Study on the fluorescence characteristics of alkaline
degradation of cefadroxil, cephradine, cefotaximum sodium and amoxicillini."
Analytical Letters 31(6): 1047-1060.
Yang, X., S. Zhu, et al. (2014). "Novel and remarkable enhanced-fluorescence system
based on gold nanoclusters for detection of tetracycline." Talanta 122: 36-42.
Yang, Z., X. Wang, et al. (2008). "Capillary electrophoresis–chemiluminescence
determination of norfloxacin and prulifloxacin." Analytica Chimica Acta 623(2):
231-237.
Part II
175
Yantasee, W., K. Hongsirikarn, et al. (2008). "Direct detection of Pb in urine and Cd, Pb,
Cu, and Ag in natural waters using electrochemical sensors immobilized with
DMSA functionalized magnetic nanoparticles." Analyst 133(3): 348-355.
Zhang, H., X. Wang, et al. (2000). "Functional crystals: Search criteria and design
principles." Journal of Solid State Chemistry 152(1): 191-198.
Zhang, Y. L., R. W. Cattrall, et al. (2011). "Gold, an alternative to platinum group metals
in automobile catalytic converters." Gold Bulletin 44(3): 145-153.
Zhao, Z. and Z. Gao (1988). "Single sweep polarography of palladium-dimethylglyoxime
complex." Journal of Electroanalytical Chemistry and Interfacial Electrochemistry
256(1): 65-75.
Zhou, Y., J. Zhang, et al. (2012). "A highly sensitive and selective “off–on” chemosensor
for the visual detection of Pd 2+ in aqueous media." Sensors and Actuators B:
Chemical 171: 508-514.
Zhu, Y., S. H. A. Jang, et al. (2011). "An Efficient and Recyclable Catalytic System
Comprising Nano-Iridium(0) and a Pyridinium Salt of nido-Carboranyldiph
osphine for the Synthesis of One-Dimensional Boronate Esters via Hydroboration
Reaction." Organometallics31 (7): 2589-2596.
Conclusions
176
CONCLUSIONS
We have shown that the size selectivity of diphosphine capped gold clusters is dependent
on size and flexibility of spacer groups. Results revealed that the smallest spacer
(bis(diphenylphosphino)propane/L3) exhibits the greatest size selectivity towards cluster
formation of smaller core sizes in all solvents used as compared to other flexible and
rigid spacer. The molecular recognition of 3 synthesized metal nanoparticles was also
developed.We have successfully established a quick and sensitive optical sensor for the
selective detection of Cu(I), based on Para-formylpyridinium propylthioacetate ligand
stabilized silver nanoparticles. The applied protocol is feasible for detection of copper in
both the T-lymphocytes and human blood plasma. This nanosensor offers fast, simple and
cost efficient method for selective detection of Cu(I) as well as 6-APA. Furthermore, a
robust method for the synthesis of 1-(3-(acetylthio)propyl)pyrazin-1-ium protected stable
AuNPs was developed. The novel AuNPs were applied for selectively sensing of Fe(III)
on the basis of aggregation-induced absorbance quenching, and colorimetric detection of
Pd(II). Finally, weoptimized a facile and rapid nanosensor based on 1-(3-
(acetylthio)propyl)-4-(dimethylamino)pyridin-1-ium capped AuNPs for the detection of
Pefloxacin mesylate. The optical response of Pefloxacin mesylate is distinctive, and
follows linear correlation in a wide range of the drug with comparable LOD as compared
to other reported methods. Importantly, these detection systems worked precisely with
good selectivity towards analytes in the presence of many interfering species, as well as
in tap water for drinking, and in physiologically important human blood plasma samples.
A series of novel dihydropyridine derivatives was synthesized, and it was screened by
cytotoxicity, antioxidant and urease inhibition assays. Two compounds were found highly
potent as antioxidant, while one significantly cytotoxic. Moreover, to specifically target
Acanthamoeba, we achieved the synthesis of photosensitising compound porphyrin and
conjugated it with mannose and tested its photochemotherapeutic efficacy in vitro. The
results revealed that mannose-conjugated porphyrin produced potent trophicidal effects
and blocked excystation of the microbe. These data suggest that mannose-conjugated
porphyrin has application for the targeted photodynamic therapy of Acanthamoeba
infections. The finding of this study will help in the development of new chemosensors
based on nanoparticles and also new antibacterial drug development.
List of Publications
177
LIST OF PUBLICATIONS
1) Aqeel, Y., R. Siddiqui, A. Anwar, et al. (2015). "Photochemotherapeutic strategy
against Acanthamoeba infections." Antimicrobial Agents and Chemotherapy:
AAC. 05126-05114.
2) Anwar, A., A. Hameed, et al. (2014). "1, 1-Diphenyl-2-picrylhydrazyl radical
scavenging activity of novel dihydropyridine derivatives." European Journal of
Chemistry 5 (1): 189-191.
3) Hameed, A., A. Anwar, et al. (2013). "Urease inhibition and anticancer activity of
novel polyfunctional 5, 6-dihydropyridine derivatives and their structure-activity
relationship." European Journal of Chemistry 4 (1): 49-52.
4) Hameed, A., A. Anwar, et al. (2012). "Tetra-n-butylammonium fluoride-mediated
dimerization of (α-methylbenzylidene) malononitriles to form polyfunctional 5, 6-
dihydropyridines derivatives under solvent-free conditions." European Journal of
Chemistry 3 (2): 179-185.
5) Anwar, A. M. Shah, et al. (2015). “Optical sensing of Cu(I) in real samples and T-
lymphocytes with Thia-pyridinium capped silver nanoparticles” (Submitted)
6) Anwar, A. M. Shah, et al. (2015). “Atomic Force Microscope based intra-
microbial sensing of Cu(I) by Silver nanoparticles with enhanced antimicrobial
activity” (Submitted)
7) Aqeel, Y., R. Siddiqui, A. Anwar, et al. (2015).” Gold nanoparticle conjugation
enhanced anti-Acanthamoebic effects of chlorhexidine” (Submitted)
