A Report of Minor Research Project

Triangulenium cation–silica microparticle conjugates as an efficient

tool for the photosensitised disinfection of water contaminated by

bacterial pathogens

Ms. SEENA SEBASTIAN,

(1933-MRP/14-15/KLMG034/UGC-SWRO)

Assistant Professor, Department of Chemistry

Assumption Autonomous College

Changanacherry, Kottayam, Kerala, 686101

Submitted to

University Grants Commission, New Delhi

March 2017

Abstract

Photosensitized processes have been shown to offer favourable perspectives for both the

treatment of several kinds of infectious diseases and addressing some environmental problems of

high scientific and societal impact. The photosensitized production of singlet oxygen can be used

for the disinfection of waste water contaminated with bacterial pathogens. A suitable choice of

the photosensitizer leads to the inactivation of a broad spectrum of pathogens. An extensive drop

in microbial cell survival can be often achieved by irradiation with intrinsically non-damaging

visible light in the presence of photosensitiser doses markedly lower than those found to be

phototoxic for host cells or tissues, as well as for non-target organisms. The trioxatriangulenium

carbocation is a member of the triphenylmethane dye family. Triangulenium cations are

remarkably stable carbenium ion in crystalline form as well as in solution such as in water,

acetonitrile, dichloromethane etc. The aqueous solutions are stable in the pH range of 1-9 so it is

less susceptible to nucleophilic attack. The most interesting property is that its absorption bands

is in the visible spectral region and has very good excited state properties with high fluorescence

and phosphorescence quantum yields. Changing the bridge atom in the trioxatriangulenium

cation from oxygen to nitrogen leads to the formation of triazatriangulenium cation with

significantly increased cation stability. The triazatriangulenium cations have moderate singlet

oxygen quantum yield. In this project triazatriangulenium cation and its derivatives are used as

photosensitizers for water disinfection. The synthesis of heterosensitizers gain attention in recent

times and found application in many fields including photosensitised disinfection of waste water

as they have many advantages over the homogenious photosensitizers. Among the hetero

sensitizers silica gel supported organic compounds received great attention. Immobilization of

organic functional group on silica surfaces produces modified silica.

The main objective of this work is to synthesize a hetrosensitizer in which

triazatriangulenium supported on silica. The silica is functionalised with amino groups using the

aminopropyl trimethoxy silane and methoy trimethyl silanes. The starting material for the

synthesis of triazatriangulenium cation is tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate

.The tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate is cyclised with amino functionalized

silica resulting in the loading of triazatriangulenium on the silica surface. The singlet oxygen

production capacity of the triazatriangulenium-silica microparticle conjugates can be monitored

using the molecular probe disodium 9,10 anthracene dipropionic acid. The disinfection studies

are also conducted inorder to determine the effectiveness of the synthesiszed

triazatriangulenium-silica microparticle conjugates for the waste water treatment. All the four

types of synthesiszed triazatriangulenium-silica microparticle conjugates produced the singlet

oxygen as it is evident from the photobleaching of molecular probe disodium 9, 10 anthracene

dipropionic acid. By the use of four types of synthesiszed triazatriangulenium-silica

microparticle conjugates there is large decrease in the survival of bacterial pathogens as it is

evident from the disinfection studies.

INTRODUCTION

The availability of drinking water is a critical problem for an important part

of the world population, mainly located in third-world countries where over one billion people

suffer diseases related to waterborne microorganisms. Classical water disinfection techniques

such as chlorination, and less-common alternatives based on other oxidizing reagents (ozone,

chlorine dioxide, etc.) or physical treatments (membrane filtration or UV-C illumination), are

typically used in urban or industrial areas. However, they are difficult to apply in isolated

regions, especially those in poorer countries, due to the lack of infrastructures.

Photodynamic microbial inactivation in the presence of cationic photosensitising

agents, aimed at the protection of the environment and improvement of its quality, can find a

particularly useful application for the disinfection of microbiologically polluted waters. The

aqueous milieu can facilitate a real-time electrostatic interaction between the positively charged

groups of the photosensitiser molecule and the array of negatively charged moieties at the

surface of many types of microbial cells, so that the photoinactivation of such cells can be

performed by irradiation after incubation times of the order of minutes, when no appreciable

accumulation of the photosensitiser in other cell types has occurred.

In this project triazatriangulenium cation and its derivatives are used as photosensitizers

for water disinfection. The trioxatriangulenium carbocation is a member of the triphenylmethane

dye family. Its trivial name is trioxatriangulenium and has the acronym TOTA+. Triangulenium

cations are remarkably stable carbenium ion in crystalline form as well as in solution such as in

water, acetonitrile, dichloromethane etc. The aqueous solutions are stable in the pH range of 1-9

so it is less susceptible to nucleophilic attack. The most interesting property is that its absorption

bands is in the visible spectral region (λmax, MeCN = 450 nm).and has very good excited state

properties with high fluorescence and phosphorescence quantum yields. Changing the bridge

atom in the trioxatriangulenium cation from oxygen to nitrogen significantly increases the cation

stability.

Trioxatriangulenium (TOTA) cation exhibits a triplet energy of 228 kJM-1 and a

quantum yield of intersystem crossing of 0.57 in water. The triplet state may therefore

transfer efficiently its energy to molecular oxygen to produce singlet oxygen.

Azatriangulenium cations (TATA) have similar photophysical properties and exhibit a robust

photochemical and thermal stability compared to trioxatriangulenium (TOTA) cation.

Triangulenium cations are efficient sensitizers for singlet oxygen generation.

The photodynamic inactivation of bacterial cell comprises the action of three

components: a photosensitizing agent (PS), a light source of an appropriate wavelength (artificial

light or sunlight) and oxygen. Two oxidative mechanisms of photoinactivation (PI) are

considered to be implicated in the inactivation of the target cells. The type I pathway involves

electron/hydrogen atoms-transfer reactions from the PS triplet state with the participation of a

substrate to produce radical ions while the type II pathway involves energy transfer from that

triplet state to molecular oxygen to produce singlet oxygen (1O2). Both processes lead to highly

toxic reactive oxygen species (ROS) such as 1O2 and free radicals, able to irreversibly alter vital

components of cells resulting in oxidative lethal damage. The main advantages of photodynamic

inactivation are the non-target specificity, the few side effects, the prevention of the regrowth of

the micro organisms after treatment and the lack of development of resistance mechanisms due

to the mode of action and type of biochemical targets.

MATERIALS AND METHODS

Solvents used were of reagent grade and used without further purification. Reagents were

purchased from Sigma-Aldrich and used as received. Absorption spectra were recorded using

Evolution 201 UV-visible spectrophotometer. Emission spectra were recorded using Fluoromax-

3 spectrophotometer. IR spectra were recorded on JASCO 4100 model, FTIR spectrometer. The

1H NMR spectra were recorded on 400 MHz on Bruker FT-NMR spectrometer with

tetramethylsilane(TMS) as internal standard. Chemical shifts were reported in parts per million

(ppm) downfield to tetramethylsilane.Molecular mass was determined by Waters 3100 mass

detector with an Electro-Spray-Ionization unit.

Synthesis of Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate

3.1.1 Preparation of 2, 6, 2’, 6’, 2’’, 6’’-hexamethoxytriphenylcarbinol.

Scheme 1

A solution of phenyllithium was prepared by addition of

bromobenzene (4.56 g, 33.28 mmol) in dry ether (15 mL) to lithium wire (0.52 g, 72.04 mmol).

1,3- dimethoxybenzene (6 g, 35.71 mmol) in dry benzene (25 mL) was added followed by

N,N,N’,N.’tetramethylenediamine (0.0298 g, 0.0256 mmol) and the mixture was stirred at 0oC

for 2hr under nitrogen to give a white suspension of 2,6– dimethoxyphenyllithium. To this

suspension was added diethyl carbonate (1.21 g, 10.95 mmol) dissolved in benzene (35 mL). The

mixture was heated at 80 oC for 12 hr in an inert atmosphere to give a brown solution. The

cooled reaction mixture was poured into 100 mL of water. The phases were separated and the

water phase was extracted with dichloromethane. The organic layer is concentrated and the

compound was precipitated by the addition of diethyl ether (100 mL).

Synthesis of Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate

Scheme 2

Aqueous HBF4 solution(50%,1.25ml,20mmol) was added to a solution of

tris(2,6-dimethoxyphenyl carbinol(2.9g,6.6mmol) in absolute ethanol (50ml).Diethyl ether(50ml)

was then added, followed by petroleum ether. The dark-blue precipitate formed was collected by

filtration and thoroughly washed with diethyl ether, yielding 3.6g of greenish black crystals.

Functionalisation of silica surface

Silica surface was functionalised with (3-aminopropyl)-trimethoxysilane

and with a mixture of (3-aminopropyl)-trimethoxysilane and methyl trimethoxy silane based on

the reported procedure Prior to functionalization the silica material was refluxed in water (25 ml

per g of support) for 1 h. After the water treatment the material was collected by filtration and

washed with toluene (20 ml/g).The wet material was suspended in toluene (100 ml/g) and the

majority of the remaining water was removed during 2 h of azeotropic distillation (2.5 ml/g).

After cooling to ambient temperature pure APTMS (3.6 ml/g),MTMS (2.8 ml/g) or both silanes

were added to the slurry. In the latter case MTMS was added before APTMS. The total amount

of silane was kept constant at 19 mmol of reactive silane per g of support. Mixtures of APTMS

and MTMS are identified by the molar ratio of the silanes in the reaction mixture. For example, 1

g of support with APTMS/MTMS 1:5 was reacted with 0.7 ml (3.2 mmol) APTMS and 2.2 ml

(15.8 mmol) MTMS. The mixture was vigorously stirred for 14 h at room temperature. Then the

solid was filtered off, redispersed in fresh toluene (100 ml/g) and refluxed for 1 h. The solid was

collected by filtration and washed with isopropanol (20 ml/g).In one experiment a Soxhlet

extraction was performed at this stage. The functionalized material was placed in a Soxhlet filter

and treated with a 2:1 diethyl ether: acetonitrile mixture for 24 h. The functionalized material

was dried at 373 K in an evacuated oven.

Synthesis of triazatriangulenium silica microparticle conjugates

The triazatriangulenium silica microparticle conjugates are synthesized by

adding activated amino surface functionalized silica to a solution of tris (2, 6 dimethoxyphenyl)

carbenium tetrafuroborate in NMP under inert condition. The functionalized silicas were

characterized by the elemental analysis of CHN, Infrared spectroscopy was performed using KBr

pellets and a spectral range of 4000 to 400 cm, elemental analysis, SEM, TEM images.

Photophysical studies

The production of singlet oxygen by the triazatriangulenium-silica

particles was determined using the molecular probe disodium 9, 10-anthracene dipropionic acid

(ADPA). Typically, 50 mg of the triazatriangulenium– silica particle conjugate was suspended in

10 ml water. Then, 250 μl of this suspension was diluted to 1 ml with water and ADPA (23 μl,

5.5 mM) was added. The particles were exposed to LED light for 150 min with UV-visible

absorption spectra recorded every 30 min. Control experiments were performed using blank

silica micro particles. The same experiment was performed with sunlight also.

Photosensitization studies of microbial cell cultures with the triazatriangulenium-silica

particles

Irradiated and unirradiated samples of E. coli (10 ml final volume) were

prepared by adding a suitable volume of the triazatriangulenium- silica microparticle conjugates

to the cell suspensions, in order to achieve a final photosensitiser concentration of 10 μM. The

samples thus obtained were incubated at 37 °C in the dark for 5 min and then irradiated for 30

min with sun light. Unirradiated and irradiated bacterial cells were serially 10-fold diluted in

growth medium and the number of colonies found after 18–24 h incubation at 37 °C was

counted.

RESULTS AND DISCUSSION

Functionalisation of silica surface

FT-IR spectroscopy

FT-IR spectra were collected on FT-IR Spectrometer using KBr pellets in

the frequency range of 4000–400 cm-1. It was performed to study the structures and identify the

functional groups of raw silica, silica functionalized with APTMS and with the mixture of

APTMS and MTMS. All the FT-IR spectra present similar features. The intense and broad band

appearing at 1000–1235 cm–1 is corresponding to the asymmetric stretching vibrations of Si–O–

Si and the one at 794 cm–1 is due to the symmetric stretching vibration of Si–O–Si. The

characteristic band at 3300–3600cm–1 is responsible for the broadening Si–OH stretching

vibration and the isolated hydroxyl groups (water or alcohol) stretching by hydrogen bonding.

The characteristic bands located at 3300–3400 cm–1and 1479–1640 cm–1

were assigned to the stretching and bending vibrations of aliphatic amine (N–H) groups,

respectively, for the amino functionalized silica particles, was absent in silica indicates the

successful grafting of aminosilane into the silica surface.

By comparing the FTIR spectra of raw silica and silica functionalized with

APTMS and with the mixture of APTMS and MTMS new bands at 2941cm–1 and 2853 cm–1 are

observed for amine functionalized particles. These are the characteristic peaks of aminosilanes.

The presence of asymmetric and symmetric stretching vibrations of –CH2 on functionalized

silica particles indicates the grafting of aminopropyl groups of APTMS on the surface of raw

silica.

Elemental Analysis

Silica gels were functionalized with amino groups using (3-aminopropyl)-

trimethoxysilane and with a mixture of (3-aminopropyl)-trimethoxysilane and methyl trimethoxy

silane. Results of elemental analysis of the functionalized silica gel samples are summarized in

table 1

Sample C: (mmol/g) N: (mmol/g) H : (mmol/g)

Silica functionalized

with APTMS

5.46

1.26

18.77

Silica functionalized

wit mixture of

APTMS+MTMS

4.07

0.71

18.1

Table 1. Elemental analysis amino functionalized silica gel

Scanning electron microscopy

The SEM images of silica functionalized with APTMS and with the

mixture of APTMS and MTMS are shown in figure3.

Figure1 .SEM images of (a) silica functionalized with APTMS (b) silica functionalized with

APTMS and MTMS.

Synthesis of triazatriangulenium-silica particles.

Four types of triazatriangulenium silica particles were prepared.(1)Silica

surface modified with aptms alone and using this Tris(2,6-dimethoxyphenyl)carbenium

tetrafluroborate was cyclised.(2) Silica surface modified with aptms and monoaza derivative was

prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are

cyclised with ethanolamine.(3) silica surface modified with aptms and mtms using this Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was cyclised.(4) Silica surface modified with aptms

and mtms using this monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with ethanolamine.

FT-IR spectroscopy

(a) Silica surface modified with aptms alone and using this Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was cyclised.

The characteristic stretching and bending vibrations of aliphatic amine

groups observed in the region 1479cm-1 to 1640cm-1 was absent in the FT-IR spectrum of

synthesized triazatriangulenium silica particle insted the band at 1649cm-1 indicates the presence

of triazatriangulenium on the silica surface.

(b) Silica surface modified with aptms and monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with

ethanolamine.

The characteristic stretching and bending vibrations of aliphatic amine groups observed

in the region 1479cm-1 to 1640cm-1 was absent in the FT-IR spectrum of synthesized

triazatriangulenium silica particle insted the band at 1644cm-1 indicates the presence of

triazatriangulenium on the silica surface

(c) silica surface modified with aptms and mtms using this Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was cyclised.

The characteristic stretching and bending vibrations of aliphatic amine groups observed in the

region 1473cm-1 to 1634cm-1 was absent in the FT-IR spectrum of synthesized

triazatriangulenium silica particle insted the band at 1649cm-1 indicates the presence of

triazatriangulenium on the silica surface.

(d)Silica surface modified with aptms and mtms using this monoaza derivative was

prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are

cyclised with ethanolamine.

The characteristic stretching and bending vibrations of aliphatic amine groups observed in the

region 1473cm-1 to 1634cm-1 was absent in the FT-IR spectrum of synthesized

triazatriangulenium silica particle insted the band at 1648cm-1 indicates the presence of

triazatriangulenium on the silica surface.

Elemental Analysis

Silica gels were functionalized with amino groups using (3-aminopropyl)-

trimethoxysilane and with a mixture of (3-aminopropyl)-trimethoxysilane and methyl trimethoxy

silane.This functionalized silica were used for cyclising Tris(2,6-dimethoxyphenyl)carbenium

tetrafluroborate.Thus triazatriangulenium compounds were loaded on the silica surface.

Sample C: (mmol/g) N: (mmol/g) H : (mmol/g)

S1

7.71

1.95

30.7

S2

5.91

2.32

20.8

S3

6.57

0.96

23.1

S4

7.03

2.14

27.7

Table 2. Elemental analysis of triazatriangulenium loaded amino functionalized silica.

S1- Silica surface modified with aptms alone and using this Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was cyclised.

S2- Silica surface modified with aptms and monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with ethanolamine.

S3- silica surface modified with aptms and mtms using this Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was cyclised

S4- Silica surface modified with aptms and mtms using this monoaza derivative was prepared

with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with

ethanolamine

Scanning electron microscopy

Figure2. SEM images of (a) Silica surface modified with aptms (b) Silica surface modified with

aptms alone and using this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised(c)

Silica surface modified with aptms and monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with ethanolamine.

Figure3. SEM images of (a) Silica surface modified with aptms and mtms (b) Silica surface

modified with aptms and mtms and using this Tris(2,6-dimethoxyphenyl)carbenium

tetrafluroborate was cyclised(c) Silica surface modified with aptms and mtms monoaza

derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two

sides are cyclised with ethanolamine.

TEM IMAGES

Figure4. TEM images of (a) Silica surface modified with aptms alone and using this Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was cyclised(b) Silica surface modified with aptms

and monoaza derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate

further two sides are cyclised with ethanolamine(c) Silica surface modified with aptms and mtms

monoaza derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate

further two sides are cyclised with ethanolamine.

Photochemical characterization of the photosensitizing materials

The absorption maximum in the visible region of the of the triazatriangulenium was found to be

525nm in acetonitrile solution but when supported on the silica surface modified with aptms

changed to 485nm. The strong hypsochromic shift is a consequence of the less polar

environment of the silica surface and, probably some contribution of rigidochromism. Its

emission maximum is 557 nm in acetonitrile solution and 562 nm when this sensitizer is

immobilized in the silica surface modified with aptms.

Figure 5.Absorption spectra of (a) Silica surface modified with aptms alone and using this

Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised(b) Silica surface modified

with aptms and monoaza derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium

tetrafluroborate further two sides are cyclised with ethanolamine. (c) Silica surface modified

with aptms and mtms and using this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was

cyclised(d) Silica surface modified with aptms and mtms monoaza derivative was prepared with

Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with

ethanolamine.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

300 400 500 600 700

Ab

so

rba

nc

e

Wavelength,nm

a

b

c

d

Figure6.Emission spectra of (a) Silica surface modified with aptms alone and using this

Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised(b) Silica surface modified

with aptms and monoaza derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium

tetrafluroborate further two sides are cyclised with ethanolamine. (c) Silica surface modified

with aptms and mtms and using this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was

cyclised(d) Silica surface modified with aptms and mtms monoaza derivative was prepared with

Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with

ethanolamine.

Singlet oxygen production

The singlet oxygen production of the triazatriangulenium-silica particle during exposure

to LED was monitored by the molecular probe ADPA. In the presence of singlet oxygen, the

ADPA is photobleached, through the conversion to the endoperoxide, leading to a reduction in

the absorbance bands of the ADPA probe. Fig. 7a shows the change in absorbance intensity of

the ADPA bands upon irradiation with the LED light. Over a period of 60 min, the bands

0

100

200

300

400

500

600

700

800

900

1000

500 550 600 650 700

Inte

ns

ity

Wavelength,nm

a

b

c

d

decrease, suggesting that singlet oxygen is generated. The results obtained when blank silica

particles were left in the light of the LED in the presence of ADPA are shown in Fig. 7b. In this

instance the absorbance bands of the ADPA do not decrease significantly, indicating that singlet

oxygen is not produced by the blank silica particles. The control experiment shows that the

triazatriangulenium is essential for the generation of singlet oxygen. Experiments using the free

triazatriangulenium (Fig. 7c) confirm that the triazatriangulenium is an effective photosensitiser

as the ADPA is completely converted to the endoperoxide by singlet oxygen in the 60 min

period. A comparison of the data shown in Fig. 7a–b is shown in Fig. 7d. Over the 60 min

irradiation period, singlet oxygen is generated by both the triazatriangulenium silica particle and

the free triazatriangulenium as evidenced by the decrease in the absorption band of the ADPA at

400 nm. While the rate of singlet oxygen production is greater for the, triazatriangulenium

clearly shows that singlet oxygen is produced by the triazatriangulenium silica particles. The

singlet oxygen production of all the triangulenium supported samples were monitored using

ADPA.In all the four cases the the photobleaching of ADPA was observed.

Figure7. Production of singlet oxygen in the presence of LED light by (a) Triazatriangulenium

silica particle conjugate (b) blank silica particles; (c) free triazatriangulenium and (d) comparison

of the change in absorbance at A400nm for (■) silica microparticle–triazatriangulenium

conjugate and 8(■) blank silica microparticles.

Comparison of the production of singlet oxygen production by the four synthesized

triazatriangulenium –silica microparticle conjugates in the presence of green LED light.

Inorder to compare the production of singlet oxygen production by the four synthesized

triazatriangulenium-silica microparticle conjugate it was irradiated in the presence of disodium 9,

10-anthracene dipropionic acid (ADPA) using green LED light. The decrease in the absorbance

at 400nm is an indication of the production of singlet oxygen because in the presence of singlet

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

300 400 500 600

Ab

sorb

an

ce

wavelength

adpa

15 Mts

30 Mts

45 Mts

60 Mts

a

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

300 400 500 600

Ab

sorb

ance

wavelength

adpa

15Mts

30Mts

45Mts

60Mts

b

0

0.5

1

1.5

2

2.5

3

300 400 500 600

Ab

sorb

ance

wavelength

0

15 Mts

30Mts

45 Mts

60 Mts

C

1.05

1.1

1.15

1.2

1.25

1.3

0 50 100

Ab

sorb

ance

at

40

0n

m

Lightexposure/min

silicatataconjugate

Blank silica

d

oxygen the ADPA is converted to endoperoxide which has no absorption in the 400nm.The

result was expressed in the following figure14.In x-axis the decrease in absorbance at 400nm of

each triazatriangulenium-silica microparticle conjugate is represented.

Figure8.Comparison of singlet oxygemn production rate of the synthesized triazatriangulenium –

silica microparticle conjugates in the presence of green LED light.

Silica surface modified with aptms alone and using this Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was cyclised,S1 shows maximum production of

singlet oxygen and the least was observed in the case of Silica surface modified with aptms and

monoaza derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate

further two sides are cyclised with ethanolamine,S2.The reason for the maximum production of

singlet oxygen is due to the higher amount of triazatriangulenium loaded on the silica surface as

it is clear from the elemental analysis. The least singlet oxygen production observed in the case

of S2, may be due to the quenching of the produced singlet oxygen by the unreacted amino

groups present on the silica surface. It is clear from the elemental analysis that the amount of

nitrogen content was very high in the case of S2 but the proportionate loading is not as expected.

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0 50 100 150

Ab

sorb

an

ce a

t 4

00

nm

Time in Minutes

s3

s2

s4

s1

In the case of S3 and S4 ie silica surface modified with aptms and mtms using this Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was cyclised and Silica surface modified with

aptms and mtms using this monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with ethanolamine

moderate production of singlet oxygen is observed. In the case of S3 and S4 ie silica surface

modified with aptms and mtms using this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate

was cyclised and Silica surface modified with aptms and mtms using this monoaza derivative

was prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are

cyclised with ethanolamine moderate production of singlet oxygen is observed.

Comparison of the production of singlet oxygen production by the four synthesized

triazatriangulenium –silica microparticle conjugates in the presence of sunlight.

Inorder to compare the production of singlet oxygen production in the presence of

sunlight by the four synthesized triazatriangulenium-silica microparticle conjugates were

irradiated in the presence of disodium 9, 10-anthracene dipropionic acid (ADPA) using sun light.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 20 40 60 80 100 120 140

Ab

sorb

an

ce a

t 4

00

nm

Time in minutes

s1

s2

s3

s4

Figure9.Comparison of singlet oxygemn production rate of the synthesized triazatriangulenium –

silica microparticle conjugates in the presence of sunlight.

The same trend is observed in the production of singlet oxygen while

irradiating these synthesiszed triazatriangulenium microparticle conjugates in the presence of

sunlight as obtained in the case of irradiating these synthesiszed triazatriangulenium

microparticle conjugates in the presence of green LED light. Silica surface modified with aptms

alone and using this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised,S1

shows maximum production of singlet oxygen and the least was observed in the case of Silica

surface modified with aptms and monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with

ethanolamine,S2. In the case of S3 and S4 ie silica surface modified with aptms and mtms using

this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised and Silica surface

modified with aptms and mtms using this monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with ethanolamine

moderate production of singlet oxygen is observed On comparing the irradiation in the presence

of green LED light and sunlight the maximum production of singlet oxygen was observed in the

case were the four synthesized triazatriangulenium-silica microparticle conjugates irradiated in

the presence of green LED light.

Disinfection assays

Irradiated and unirradiated samples of E. coli (10 ml final volume) were prepared by

adding a suitable volume of the triazatriangulenium- silica microparticle conjugates to the cell

suspensions, in order to achieve a final photosensitiser concentration of 10 μM. The samples thus

obtained were incubated at 37 °C in the dark for 5 min and then irradiated for 30 min with sun

light. Unirradiated and irradiated bacterial cells were serially 10-fold diluted in growth medium

an the number of colonies found after 18–24 h incubation at 37 °C was counted.

Figure 10.The E.coli treated with .(1)Silica surface modified with aptms alone and using

this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised,S1.(2) Silica surface

modified with aptms and monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with

ethanolamine,S2.(3) silica surface modified with aptms and mtms using this Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was cyclised,S3.(4) Silica surface modified with

aptms and mtms using this monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with

ethanolamine,S4.(5)Silica alone.

The results shown in Fig.11 clearly demonstrate that the silica surface modified with

aptms alone and using this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised

particles successfully inactivated Gram-negative bacteria through a photosensitization process.

The Gram-negative bacteria underwent a 4 log decrease in survival upon exposure to full

spectrum visible light for 30 min. The triazatriangulenium – silica particle conjugate exerted a

little toxic effect on bacterial cells when incubated in the dark for 30 min.

Figure11.Effect of photosensitized irradiation on E.Coli survival after 30 minutes irradiation in

the presence of sunlight and aptms modified silica surface cyclised with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate.

0

200000000

400000000

600000000

800000000

1E+09

1.2E+09

1.4E+09

control tata-silica

composit

Cel

l S

urv

iva

l

Type of sample

unirradiated

irradiated

Figure12.Effect of photosensitized irradiation on E.Coli survival after 30 minutes irradiation in

the presence of sunlight and silica modified with aptms and monoaza derivative was prepared

with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with

ethanolamine

The results shown in Fig.12 clearly demonstrate that the silica modified with aptms and

monoaza derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate

further two sides are cyclised with ethanolamine shows only 0.09 log decrease in the inactivation

of gram negative bacteria. This is due to the fact that the amount of triazatriangulenium on the

surface was low compare to the previous case and hence the generation of singlet oxygen is also

very low hence this triangulenium silica microparticle conjugate shows decreased efficiency of

bacterial inactivation.

0

500000000

1E+09

1.5E+09

control tata-silicacomposit

unirradiated

irradiated

Figure13.Effect of photosensitized irradiation on E.Coli survival after 30 minutes

irradiation in the presence of sunlight and silica surface modified with aptms and mtms using

this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised

The results shown in Fig.13 clearly demonstrate that the silica modified with aptms and

mtms using this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised shows only

0.38 log decrease in the inactivation of gram negative bacteria. The loading of

triazatriangulenium on the silica surface modified with aptms and mtms was low compared to the

loading of triazatriangulenium on the silica surface modified with aptms alone. This effect is

reflucted in the generation of singlet oxygen and is the reason for the low rate of bacterial

inactivation.

0

200000000

400000000

600000000

800000000

1E+09

1.2E+09

1.4E+09

control tata-silicacomposit

unirradiated

irradiated

Figure14.Effect of photosensitized irradiation on E.Coli survival after 30 minutes

irradiation in the presence of sunlight and silica surface modified with aptms and mtms using

this monoaza derivative was prepared with Tris(2,6-dimethoxyphenyl)carbenium

tetrafluroborate further two sides are cyclised with ethanolamine.

The results shown in Fig.14 clearly demonstrate that silica surface modified with aptms

and mtms using this monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with ethanolamine

shows only 1.5 log decrease in the inactivation of gram negative bacteria. Among the two

triangulenium silica surface modified with aptms and mtms this shows higher efficiency in the

bacterial inactivation.

The disinfection studies were conducted with all the four synthesized

triazatriangulenium-silica microparticle conjugates and the results are discussed above. It shows

that the inactivation of microparticle was taken place in all the four conjugates but pronounced

effect was shown in the case of silica surface modified with aptms alone and using this Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was cyclised particles.

0

200000000

400000000

600000000

800000000

1E+09

1.2E+09

1.4E+09

control tata-silicacomposit

unirradiated

irradiated

Discussion

The need for the clear water is a worldwide problem. Countries from various parts of the world

face difficulties for the pure water since the water sources are heavily polluted with hazardous

chemicals and several kinds of pathogens such as bacteria, fungi, viruses and parasitic protozoa.

Therefore, there is an urgent need for the definition of efficacious, reliable and environmentally

safe techniques, which yield satisfactory levels of water purification and disinfection overcoming

the limitations often exhibited by most of the currently adopted physical and chemical

approaches.

Undoubtedly, photosensitised processes offer favourable perspectives in

this field. The possibility to reduce the population of helminth eggs and faecal coliforms by the

combined action of a meso-substituted tetracationic porphyrin and visible light has been

demonstrated previously. Such encouraging results prompted intensive investigations aimed at

optimizing the treatment protocols in order to tailor them to the specific situations and properties

of a given aqueous system. In particular, the attention of several investigators is being focused on

the use of photosensitising agents associated with matrices which undergo a ready swelling when

introduced into water. This possibility is especially interesting when singlet oxygen-generating

photosensitisers are used: this hyper-reactive oxygen species has a long natural lifetime, so that

the killing of bacteria physically separated for distances of up to 1 mm from the immobilized

photosensitiser has been reported. The triazatriangulenium utilized in this investigation is

characterized by a singlet oxygen quantum yield of 0.35, and has been shown to display an

efficient photocidal activity against Gram-negative bacterium in both the free state and when

associated within silica particles.

The extensive inactivation is probably a consequence of the tight

interaction of the cationic photosensitiser with the array of negatively charged moieties,

including carboxylate groups from teichoic and lipoteichoic acids, which are present on the outer

surface of bacterial cells. The close proximity between the photoexcited photosensitiser and the

photosensitive targets which stabilize the tight three-dimensional architecture of the cell outer

wall causes an increase in the permeability of such structure and facilitates the penetration of the

photosensitiser to the inner cellular compartments, whose integrity is critical for cell survival.

There are important advantages in using the microparticle conjugated

triazatriangulenium as exemplified by the present experiments: the irradiation of water for its

disinfection can be performed prior to its introduction into (for example) fishfarming ponds, thus

avoiding any direct contact between the photoreactive intermediates and the fish or other types of

nontarget organisms, adding to the safety of the process. There is no release of the

photosensitiser during the incubation in water or the irradiation by visible light hence there is no

diffusion into the environment avoiding its pollution by chemical contaminants. Finally, it should

be considered that the efficacy of our protocol which involves generally mild conditions for

experimental parameters.

CONCLUSION

The study of singlet molecular oxygen production and reactivity has

emerged as a rich and diverse area with implications in fields ranging from polymer science to

cancer therapy. Photosensitized production of singlet molecular oxygen is common in the

treatment of waste water disinfection. In the present work we have synthesized four types of

triazatriangulenium-silica microparticle conjugates. The starting material for the

triazatriangulenium cation is tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate. Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was synthesized and characterized using mass,c-13

and NMR spectroscopy. To synthesize silica supported heterosensitizer silica was functionalized

with amino groups using 3-amino propyl trimethoxy silane and methoxy trimethyl silane. The

synthesized amino functionalized silica was characterized using FT-IR spectroscopy, elemental

analysis,SEM images. Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised using

the amino functionalized silica so that triazatriangulenium cation was loaded on the silica

surface. The triazatriangulenium loaded silica was characterized using FT-IR spectroscopy,

elemental analysis, SEM,and TEM images. There are four types of triazatriangulenium-silica

microparticle conjugates were synthesized. Silica surface modified with aptms alone and using

this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate was cyclised (S1), Silica surface

modified with aptms and monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with

ethanolamine(S2), silica surface modified with aptms and mtms using this Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate was cyclised(S3), Silica surface modified with

aptms and mtms using this monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with

ethanolamine(S4) were the four types of synthesized triazatriangulenium-silica microparticle

conjugates.The photochemical characterization of all the four synthesized triazatriangulenium-

silica microparticle were done.

The singlet oxygen production of the triazatriangulenium-silica particle

during exposure to LED was monitored by the molecular probe ADPA. In the presence of singlet

oxygen, the ADPA is photobleached, through the conversion to the endoperoxide, leading to a

reduction in the absorbance bands of the ADPA probe. In all the four types of synthesized

triazatriangulenium-silica microparticles singlet oxygen production was observed. The rate of

singlet oxygen production of the synthesized triazatriangulenium-silica microparticle conjugates

were compared by irradiation in the presence of green LED light and sunlight. Silica surface

modified with aptms alone and using this Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate

was cyclised,S1 shows maximum production of singlet oxygen and the least was observed in the

case of Silica surface modified with aptms and monoaza derivative was prepared with Tris(2,6-

dimethoxyphenyl)carbenium tetrafluroborate further two sides are cyclised with

ethanolamine,S2.The reason for the maximum production of singlet oxygen is due to the higher

amount of triazatriangulenium loaded on the silica surface as it is clear from the elemental

analysis. The least singlet oxygen production observed in the case of S2, may be due to the

quenching of the produced singlet oxygen by the unreacted amino groups present on the silica

surface. The same trend is observed in the production of singlet oxygen while irradiating these

synthesiszed triazatriangulenium microparticle conjugates in the presence of sunlight and in the

presence of green LED light.

The disinfection studies are also conducted in order to determine the

effectiveness of the synthesiszed triazatriangulenium-silica microparticle conjugates for the

waste water treatment. The Gram-negative bacteria underwent a 4 log decrease in survival upon

exposure to full spectrum visible light for 30 min during the irradiation in the presence of silica

surface modified with aptms alone and using this Tris(2,6-dimethoxyphenyl)carbenium

tetrafluroborate was cyclised particles. silica modified with aptms and monoaza derivative was

prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are

cyclised with ethanolamine shows only 0.09 log decrease in the inactivation of gram negative

bacteria. silica modified with aptms and mtms using this Tris(2,6-dimethoxyphenyl)carbenium

tetrafluroborate was cyclised shows only 0.38 log decrease in the inactivation of gram negative

bacteria. silica surface modified with aptms and mtms using this monoaza derivative was

prepared with Tris(2,6-dimethoxyphenyl)carbenium tetrafluroborate further two sides are

cyclised with ethanolamine shows only 1.5 log decrease in the inactivation of gram negative

bacteria. The extensive inactivation is probably a consequence of the tight interaction of the

cationic photosensitiser with the array of negatively charged moieties, including carboxylate

groups from teichoic and lipoteichoic acids, which are present on the outer surface of bacterial

cells. There are important advantages in using the microparticle conjugated triazatriangulenium

as exemplified by the present experiments. There is no release of the photosensitiser during the

incubation in water or the irradiation by visible light hence there is no diffusion into the

environment avoiding its pollution by chemical contaminants. Finally, it should be considered

that the efficacy of our protocol which involves generally mild conditions for experimental

parameters.