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CHAPTER-2
SiO2 BASED NANOBIOSENSOR
MONITORING MITOXANTRONE-DNA
INTERACTION
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 33
2.1 ABSTRACT
The present study involves the development of nanobiosensor to determine toxicological
behavior of Mitoxantrone (MTX). Mitoxantrone intercalates with DNA and produce
MTX-DNA adduct, resulting in blockade of protein synthesis and excessive production
of free radicals in the myocardium eventually leads to cardiac toxicity. Potentiometry was
applied to develop an electroanalytical procedure for the determination of MTX and its
interaction with DNA immobilized on the electrode surface modified with Silicon
dioxide (SiO2) nanoparticles. The nanobiosensor immersed in MTX solution to monitor
MTX-DNA interaction with respect to time and alters the resistance of the
nanobiosensor. It was observed that MTX-DNA interaction is fast initially and as time
elapses, the change in interaction gets slow due to formation of MTX-DNA adduct. The
MTX-DNA adduct was validated by UV-visible and FT-IR spectroscopy. Determination
limit of the nanobiosensor is up to10 ng/ml. This study suggests that the nanobiosensor
allows non-invasive, real-time monitoring of the drug-DNA interaction changes by
measuring the potential at DNA/sensor interface which can prove to be an important tool
in drug discovery pipelines and molecular toxicology.
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 34
2.2 INTRODUCTION
Nanobiosensors and nanobiochips are gaining importance in the field of life science
because of its faster, direct, more accurate, more selective detection at very low
concentrations. Enormous research has been carried out for the development of
nanobiosensor which can be useful in life science field like clinical diagnosis, genomics,
proteomics and toxicology. But, until now nanobiosensor has never been developed
which can monitor or detect toxicity at nano gram level. Nanoparticles play a key role in
adsorption of biomolecules due to their large specific surface area and high surface free
energy. The combination of nanomaterials and biomolecules is of considerable interest in
the field of nanobiotechnology. Recently, many kinds of nanometer materials such as
gold1-3
, platinum4 and silicon dioxide
5-6 nanoparticles are widely applied for
Electrochemical based nanobiosensor due to its conducting and semiconducting
properties. Also, these nanoparticles have been used to catalyze biochemical reactions,
improving coverage and binding ability of the functional components and this capability
can be usefully employed in biosensor design7.
SiO2 nanoparticles have been used to construct biosensor due to its biocompatibility as
well as good electron transfer properties. In the work of Xi-Liang Luo and co-workers,
SiO2 nanoparticles were introduced in the construction of field-effect transistors
(ENFETs) biosensor which can provide a biocompatible environment and improve the
enzyme activity8. Chen and his team reported the effect of SiO2 nanoparticles on the
adsorbability and enzymatic activity of glucose oxidase9. The oligonucleotide-modified
silica nanoparticles were prepared by Lisa R. Hilliard and her coworkers which provide
an efficient substrate for hybridization and used in the development of DNA biosensors
and biochips10
. Ningning Zhu and his group developed electrochemical DNA
nanobiosensor which consists of platinum nanoparticles combined with Nafion-
solubilized Mutli-walled carbon nanotubes.4 M. Yousef Elahi and his team developed
polypyrrole (PPy) nanofiber modified electrode to monitor DNA-salicylic acid/Aspirin
interaction. A Platinum electrode was electrochemically modified by the polymerization
of pyrrole to obtain a nanofiber PPy film using a pulse potential method. The reaction
rate of Aspirin with DNA was lower than that of Salicylic Acid with DNA, potentially
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 35
due to the steric hindrance of the acetyl group when binding to the minor groove11
. Many
research papers have been reported on silicon dioxide nanowire based Nanosensor to
study DNA interaction and DNA hybridization studies12,13
. Overall, these studies suggest
that silicon dioxide shows good biocompatibility as well as good electron transfer
properties. Hence, it can be useful for construction of biosensor to improve its
functionality.
The electromotive force (EMF) is the maximum potential difference or charge between
two electrodes. This causes electrons to move so that there is an excess of electrons at
one point and a deficiency of electrons at a second point14
. The electrochemical signals
are usually generated by redox reactions and changes in ionic composition.
Potentiometric sensors measure the potential of an electrode at equilibrium (i.e. in the
absence of the appreciable currents) by measuring the electrochemical cell potential vs. a
reference electrode potential15
. MTX allow extensive stabilization of the intercalated
adduct by hydrogen-bonding interactions with DNA16
. Thus, this nanobiosensor has been
designed to monitor interaction of MTX with DNA.
In this report, we developed a real-time nanobiosensor by modifying the electrode with
SiO2 nanoparticle and DNA. This nanobiosensor was immersed in the solution containing
MTX to monitor MTX-DNA interaction (Figure 2.1). Mitoxantrone, an anti-cancer agent
has a planar heterocyclic ring structure and the basic side groups are critical for
intercalation into DNA. Binding of MTX to DNA inhibits both DNA replication and
RNA transcription. Mitoxantrone inhibits DNA and leads excessive production of free
radicals in myocardium responsible for producing cardiotoxicity17,18
. Therefore, the
change in MTX-DNA interaction was observed by measuring changes in EMF (mV). All
experiments were carried out at neutral pH and at room temperature since double – strand
of DNA breaks at neutral pH and single - strand breaks at high pH19
.
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 36
Figure 2.1: Schematic representation of MTX-DNA interaction development of
nanobiosensor and monitoring of MTX-DNA interaction
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 37
2.3 EXPERIMENTAL
2.3.1 Chemicals
Highly polymerized calf thymus DNA (MP Biomedicals, US) was used in this study.
DNA dilutions were prepared in Phosphate buffer pH 7. Phosphate buffer was prepared
by dissolving 0.1M disodium hydrogen phosphate in water and adjusting the pH by
adding 0.1M HCl. Tetraethylorthosilicate (TEOS), ammonium hydroxide (NH4OH) and
ethanol were used to prepare SiO2 nanoparticles. All chemicals were purchased from E-
Merck (India, Mumbai) and were all of analytical reagent grade. Mitoxantrone was
obtained from Cipla Ltd (India, Mumbai) and used without purification. All aqueous
solutions were prepared in Milli-Q water from a Millipore purification system and all
experiments were done at room temperature.
2.3.2 Apparatus
The potential measurements were carried out at 25.0±0.1 ◦C with a digital pH meter
(Model LI120, ELICO, India). An Saturated calomel electrode (SCE) was used. UV and
FT-IR spectra were obtained on a JASCO V-630 spectrophotometer and a JASCO FT-IR
4100, respectively. Particle size of SiO2 was carried out by Malvern Zeta-sizer (Model-
The Zetasizer Nano ZS, UK).
2.3.3 Preparation Of SiO2 Nanoparticles
SiO2 nanoparticles were prepared according to the literature 20-22
. To 20 ml of ethanol,
2ml of TEOS was added followed by 4ml of concentrated NH4OH. After that it was
stirred for 12-14 hours at 200-300 rpm. Then the mixture obtained was centrifuged at
3000 rpm for 30-50 min. Finally a white color powder was formed which was named as
silica nanoparticle.
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 38
2.3.4 Fabrication of Electrode By SiO2 Nanoparticles
The surface of working calomel electrode was modified with SiO2 nanopaticles. An
amount of 2.0 mg of SiO2 were dispersed in a 10 ml of ethanol solution. After about 10
min of sonication, uniformly dispersed SiO2 nanoparticles were formed. Before
modifying the electrode with SiO2, the electrode was cleaned by washing it with distilled
water and was allowed to dry. Then the dry electrode was immersed in solution
containing SiO2 nanoparticles for 30 min with stirring at room temperature. The electrode
was removed and was left for drying for about 15 min. Potentiometric measurement was
performed at working calomel electrode versus reference calomel electrode.
2.3.5 Immobilization Of DNA On SiO2 Modified Electrode
10 ppm DNA solution was prepared in phosphate buffer pH 7. The electrode was
immobilized by drop casting technique. A 10 µL (100 ng) drop of DNA was delivered on
the modified SiO2 surface of electrode by micropipette and allowed to dry in air. After
drying, this nanobiosensor was used for monitoring toxicological behavior of MTX.
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 39
2.4 RESULTS AND DISCUSSION
2.4.1 Characterization of SiO2
The morphology of SiO2 were performed by SEM. Figure 2.2 illustrates that the particles
are predominantly spherical in shape with diameter ranging from 20 to 25 nm. Larger and
uneven shaped particles with diameter 35–70 nm were also obtained.
Figure 2.2: SEM image of SiO2
Particle sizes of SiO2 were determined by Malvern zeta-sizer which was found as an
average 20 nm (Figure 2.3). Particles were ranging from 46.98 nm (81.3%), 0.6549 nm
(7.3%), and 2210 nm (5.8%).
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 40
Figure 2.3: Particle size distribution of SiO2
2.4.2 MTX-DNA Interaction In Solution
MTX solution of varying concentration (100, 75, 50, 25, and 10 ng/ml) was prepared in
distilled water. The MTX-DNA interaction in solution was carried out at room
temperature. 1 ml of 100 ng/ml of MTX and 10 µL of 10 µg/ml of DNA was taken to
perform interaction study by potentiometry. As shown in Figure 2.4, the electrode
potential shifted to negative direction steadily. But, at one point, the change in EMF was
not increased. This indicates the formation of MTX-DNA adduct. Initially the change in
EMF was very fast, but as time elapse, the change in EMF gets slow. In all MTX
concentration series, potential shifted steadily but at one stage it get stopped due to the
formation of MTX-DNA adduct. This MTX-DNA complex was further validated by UV
and FT-IR spectroscopy.
MTX interacts preferentially with DNA binding with guanine, cytosine base pairs. 17
The
sensor measures the two-electron oxidation process of 5,8- hydroxyl substituents on
MTX while interacting with DNA. No more hydrogen from guanine and cytosine base
can be liberated and oxidized which could lead to the stopped EMF change. The change
in EMF with respect to time indicates the interacting behavior of MTX with DNA.
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 41
Table 2.1: Time dependent changes of MTX-DNA interaction determined in solution
(without Nanobiosensor)
Time EMF
100 ng/ml 75 ng/ml 50 ng/ml 25 ng/ml
5 135 ± 3 124.7 ± 1.5 123.3 ± 1.5 114 ± 1
10 133.7 ± 3.1 122.7 ± 1.5 121.7 ± 1.5 113 ± 1
20 132.7 ± 3.1 121.3 ± 1.5 120.3 ± 0.6 112.3 ± 0.6
30 132.3 ± 1.5 120 ± 1 119 ± 1 111.3 ± 1.5
60 131 ± 1 119.3 ± 0.6 117.3 ± 0.6 111 ± 2
120 129.3 ± 2.1 118.7 ± 0.6 116 ± 1 110 ± 2
240 127.6 ± 1.5 118 ± 1 115.7 ± 0.6 109 ± 2
300 127 ± 1 117.7 ± 0.6 115.3 ± 0.6 109 ± 2
360 126 ± 2 117 ± 1 115.3 ± 0.6 109 ± 2
480 125.3 ± 1.5 117 ± 1 - -
600 125.3 ± 1.5 117 ± 1 - -
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 42
In case of 100 ng/ml of MTX, the interaction of MTX with DNA showed more potential
difference (Table 2.1, Figure 2.4) as compare to 75, 50 and 25 ng/ml of MTX due to
higher amount of MTX was available to interact with DNA. Thus, this study suggests that
concentration of drug is directly proportional to the sensitivity of sensor and shows
significant EMF changes. It was also observed, that no measurable change was found in
EMF at 10 ng/ml of MTX concentration. Thus, this study suggests that concentration of
drug is directly proportional to the sensitivity of sensor and at very low concentration no
EMF changes are observed.
Figure 2.4: MTX-DNA interaction in solution at various concentration of MTX
2.4.3 Nanobiosensor Monitoring MTX-DNA Interaction
MTX (100, 75, 50, 25 and 10 ng/ml) and DNA interaction was performed by developed
Nanobiosensor. The results obtained from developed Nanobiosensor were significant
from without modified sensor (Figure 2.5, Table 2.2). In all MTX concentration series,
the MTX-DNA interaction shows more change in electrode potential. The electrode
potential decreases steadily until all the amount of MTX gets interacted with DNA. The
biocompatibility of SiO2 nanoparticles provides a suitable environment for DNA to keep
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 43
its bioactivity and prevent DNA leakage. Moreover, signals from sensor improve much
better due to semiconducting properties of SiO2, which provide a faster pathway for
electrons to be transferred between the active sites of the DNA and the surface of the
SiO2. Thus, Nanobiosensor reveals high sensitivity.
Figure 2.5: Nanobiosensor monitoring MTX-DNA interaction at various concentration of
MTX
The linearity and reproducibility of the nanobiosensor was investigated by performing
three experiments using the same working calomel electrode. It has been observed that in
all MTX concentration series, a significant change in EMF was reported with
nanobiosensor. Also, the change in EMF was remarkable at lower concentration i.e., 10
ng/ml of MTX determined by nanobiosensor, while without nanobiosensor didn’t show
any change in EMF. So, sensitivity was also improved at lower concentration. Thus, the
developed nanobiosensor allows real-time monitoring of MTX-DNA interaction, which
can play a pivotal role in screening of drugs while developing series of new drugs.
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 44
Table 2.2: Time dependent changes of MTX-DNA interaction determined by
nanobiosensor
Time EMF
100 ng/ml 75 ng/ml 50 ng/ml 25 ng/ml 10 ng/ml
5 185 ± 1 171 ± 1 161.3 ± 3.1 157.3 ± 2 153.3 ± 1.5
10 184 ± 1 169 ± 1 160.3 ± 2.5 156 ± 1.7 153 ± 1
20 182.3 ± 1 167.3 ± 0.6 158.7 ± 2.5 155.3 ± 2.5 151.7 ± 0.6
30 180.7 ± 1.5 165.3 ± 1.5 157 ± 1.7 154.3 ± 1.5 150.3 ± 0.6
60 180 ± 0.6 163.3 ± 2.1 155.7 ± 1.5 152.7 ± 2.0 150.7 ± 0.6
120 179.3 ± 0.6 161.7 ± 2.5 155 ± 3 151.3 ± 0.5 148.7 ± 0.6
180 176.3 ± 0.6 160.3 ± 2.5 155 ± 1.7 151.3 ± 1.2 147 ± 1
240 173.7 ± 1.5 158.7 ± 2.5 153.3 ± 1.5 149.7 ± 2 145.7 ± 0.6
360 171 ± 1 157 ± 1.7 151.7 ± 1.2 148 ± 2.0 145.3 ± 1.5
480 170 ± 1 155.7 ± 1.5 150.3 ± 0.6 146.7 ± 2.1 144.3 ± 0.6
600 168 ± 2 155 ± 3 150 ± 2 145.7 ± 1.7 143.3 ± 0.6
720 165.7 ± 3 155 ± 1.7 148.3 ± 1.5 145 ± 2.1 142.7 ± 0.6
840 164 ± 2.6 153.3 ± 1.5 147.3 ± 1.5 144.7 ± 2.5 141.7 ± 1.5
960 162 ± 3 151.7 ± 1.1 146.3 ± 1.5 144.3 ± 2.5 141.7 ±1.5
1080 161 ± 3 150.3 ± 0.6 145.3 ± 1.5 144.3 ± 2.5 141.7 ± 1.5
1100 158.7 ± 3.1 150 ± 2 145.3 ± 1.5 144.3 ± 2.5 141.7 ± 1.5
1220 157.7 ± 3.1 148.3 ± 1.5 145.3 ± 1.5 144.3 ± 141.7 ± 1.5
1440 156.7 ± 2.1 148.3 ± 1.5 - - -
1560 156.7 ± 2.1 148.3 ± 1.5 - - -
1680 156.7 ± 2.1 - - - -
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 45
2.4.4 Sensitivity and selectivity of Nanobiosensor
To evaluate the performance of the Nanobiosensor, potential shift ∆E was calculated, i.e.
the potential change from equilibrium time to the end of the experiment. From 2.6, it is
clearly seen that, the potential shift is directly proportional to MTX concentration. If, the
concentration of MTX is higher, more potential shift was observed. Nanobiosensor
showed more ∆E than without modification of sensor at all MTX concentration series. At
10 ng/ml of MTX concentration series, Nanobiosensor showed remarkable change in
potential shift ∆E, while without modified sensor doesn’t showed any change in potential
shift ∆E. This confirms the improvements of electrical signals and thus, Nanobiosensor
reveals high sensitivity.
Figure 2.6: Potential difference between nanobiosensor and without nanobiosensor at
various MTX concentration series
On addition of incremental concentrations of MTX to DNA, potential difference
increases in all concentration series. It is evident from the Table 2.3 that interacting
behavior of DNA with the stock concentrations of MTX are as follows:
100 ng/ml > 75 ng/ml > 50 ng/ml > 25 ng/ml > 10 ng/ml.
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 46
Table 2.3: Potential difference at various concentration series
MTX
concentration
ng/ml
Nanobiosensor
∆E
SD Without
Nanobiosensor
∆E
SD
100 28.67 2.3 9.67 2.3
75 22.66 0.58 8.33 0.58
50 17.66 2.08 8.33 0.58
25 13.67 0.58 5 1.73
10 9 1.73 0 0
2.4.5 Analytical performance of Nanobiosensor
The linearity and reproducibility of the nanobiosensor was investigated by performing
three different experiments using the same working electrode. Figure 2.7 depicts the
calibration curves of MTX concentration ranging from 100 to 10 ng/ml versus the ∆E
values. It was observed that the nanobiosensor showed good reproducibility for all three
measurements. The working electrode was water-washed to take away the DNA residuals
from the surface of the electrode after each measurement. The stability of the
nanobiosensor was tested by performing the experiments daily for a period of 15 days
while storing in a suitable environment when not in use. Almost 90% of the initial
sensitivity was retained at the end of the period and the biosensor half-life is estimated to
almost 1 month.
Table 2.4: Comparison of the analytical parameters for nanobiosensor and without
nanobiosensor
Method Shelf-
life
Range Reproducibility
Nanobiosensor 15 days 10-100 ng/ml >10 times
Without nanobiosensor - 25-100 ng/ml -
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 47
Figure 2.7: Calibration curves of MTX concentration versus the ∆E values.
The comparison of analytical performances for determining MTX-DNA interaction by
nanobiosensor and without nanobiosensor is given in Table 2.5. For nanobiosensor, the
values of correlation coefficient (R2), slope and intercept were found as 0.994, 32.83and
4.83 respectively. Limit of detection (LOD) and limit of quantification (LOQ) were found
as 1.42 (µg/mL) and 4.36 (µg/mL) respectively. For Without nanobiosensor, the values of
correlation coefficient (R2), slope and intercept were found as 0.842, 13.067and 2.267
respectively. LOD and LOQ were found as 2.51 (µg/mL) and 7.63 (µg/mL) respectively.
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 48
Table 2.5: Comparison of the analytical performance for nanobiosensor and without
nanobiosensor
aLOD =3.3ₓSD/slope
bLOQ = 10ₓSD/slope
2.4.6 Comparative study of MTX-DNA interaction by nanobiosensor
In order to compare the results and hence detect systematic errors between the two
methods, a student t-test was employed to check whether the standard deviations for the
same sample differ significantly (Table 2.6). Since the experimental value of t-test is
higher than the critical, it is concluded that the proposed nanobiosensor technique is more
precise than without nanobiosensor. Table 2.6 shows the statistical comparison between
two methods at various concentration of MTX. Thus, the results obtained from both the
methods were not in agreement, indicating significant difference between two.
Parameters Nanobiosensor Without nanobiosensor
Regression equation (Y)
Slope (b)
Intercept (c)
4.83
2.26
32.82 13.06
Correlation coefficient (r) 0.994 0.842
Limit of Detectiona 1.42 ( µg/mL) 2.51 ( µg/mL)
Limit of Quantitationb 4.36 ( µg/mL) 7.63 (µg/mL)
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 49
Table 2.6: Statistical Comparison between two methods
MTX
concentration
(ng/ml)
Nanobiosensor Without
nanobiosensor
t-test
∆E SD ∆E SD tExperimental tCritical
100 28.67 2.3 9.67 2.3 10.10 2.132
75 22.66 0.58 8.33 0.58 30.48 2.132
50 17.66 2.08 8.33 0.58 7.46 2.132
25 13.67 0.58 5 1.73 8.25 2.132
2.4.7 Confirmation Of MTX-DNA Adduct By UV-Spectroscopy
The MTX-DNA adduct obtained from potentiometric measurement was validated by UV
spectroscopy. In Figure 2.8, the pure DNA shows absorbance at 202 and 259.5 nm, while
MTX shows peak at 222.5 and 288 nm. The MTX-DNA adduct shows peak at 272.5 nm.
The major shifting of peak from 288 to 272.5 nm indicates that the formation of MTX-
DNA adduct. Similar results were obtained in all MTX concentration series.
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 50
Figure 2.8: Validation of MTX-DNA adduct by UV-spectroscopy
2.4.8 Confirmation Of MTX-DNA Adduct By FT-IR Spectroscopy
The MTX-DNA adduct was also validated by FT-IR spectroscopy. The resulting solution
obtained from potentiometric measurement was determined by FT-IR spectroscopy
which confirms the binding of MTX to DNA. Only the spectral interval range between
1800 and 850 cm-1
was examined. The IR peaks of DNA, MTX and MTX-DNA
interaction are shown in Table 2.7 (Figure 2.9).
The major spectral changes (intensity or shifting) of several prominent DNA in-plane
vibrations related to the G-C, A-T bases and the backbone phosphate modes at 1710
(mainly G), 1662 (mainly T), 1610 (A), 1492 (mainly C), and 1226 (asymmetric PO2
vibration) and 1088 cm-1
(PO2 symmetric vibration) 22-24
. However, some perturbations of
DNA vibrations were observed in this study. The guanine band at 1710 cm-1 shifted
toward a lower frequency at 1710, whereas the adenine and cytosine shows very weak
band at 1610 and 1492 cm-1 respectively (Figure 2.9).
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 51
Figure 2.9: Confirmation of MTX-DNA adduct by FT-IR spectroscopy (A: pure DNA,
B: MTX, C: MTX-DNA)
Also, PO2 vibrations shifted towards lower frequency at 1076 cm-1
. The peaks at 991 and
955 cm-1
were observed in case of P-O and Asymmetric O-P-O stretching, respectively.
DNA backbone peaks at 941, 928, 902, 893 and 854 cm-1
were observed and are well in
agreement with results reported by Yan Mao and his team [23].
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 52
Table 2.7: Wavenumbers (in cm-1
) of DNA, MTX and MTX-DNA interaction
DNA
(cm-1)
MTX
(cm-1)
MTX-DNA
adduct
(cm-1)
Assignments
1700 - - C=O of guanine
1662 - 1644 C=C, C=N in the base plane
1583 - 1553 C=C, C=N in the base plane
1492 - - Cytosine
1076 1055 1059 Symmetric PO2- stretching
991 992 993 P-O or C-O backbone stretch
955 962 956 Asymmetric O-P-O
941 948 947 DNA backbone
928 936 933 DNA backbone
902 927 921 DNA backbone
The MTX-DNA adduct was confirmed for all resulting solution obtained from
potentiometric measurement. Disappearance of peak indicate the breakage of DNA, while
shifting of peaks indicates the formation of MTX-DNA adduct. From Figure 2.9 and
Table 2.7, it is clear that, the MTX breaks DNA and forms complex with DNA. In all
case studies, similar results were obtained.
2.4.9 Confirmation of MTX-DNA adduct by Zeta-potential measurement
Zeta potential measurements confirm the formation of MTX-DNA adduct. Figure 2.10
shows the mean zeta-potential of pure DNA which was found to be -31.1 mV. MTX
showed positive potential 0.0226 mV. The MTX-DNA adduct showed potential at -17.6
mV. Thus, this suggests that the formation of drug-DNA adduct.
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 53
Figure 2.10: Zeta-potential of DNA, MTX, and MTX-DNA adduct
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 54
2.5 CONCLUSION
This work has shown experimental evidence of interaction of MTX with DNA and may
contribute to the understanding of the mechanism of action of this drug with DNA. It was
observed that drug-DNA interaction occurring with time which suggests that MTX
intercalates with DNA and slowly interacts with it causing some breaking of the
hydrogen bonds. It is interesting to note that the nanobiosensor experiments suggest
preferential interaction of MTX with DNA at very low concentration. Without surface
modified electrode does not seem to be suitable for the monitoring MTX-DNA
interaction at low concentrations of MTX. The sensor revealed high sensitivity and
selectivity. Formation of MTX-DNA adduct was validated by spectroscopic analysis and
are well in agreement with reported studies. Overall, we developed nanobiosensor which
allows non-invasive, real-time monitoring of the Drug-DNA interaction changes by
measuring potential at sensor interface which can be crucial biosensor in molecular
toxicology and drug discovery pipelines.
Chapter 2
Institute of R & D, GFSU, Gandhinagar, Gujarat 55
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