Accepted Manuscript

Title: Synthesis and characterization of chitosan-TiO2 :Cu

nanocomposite and their enhanced antimicrobial activity with
visible light

Author: A.V. Raut H.M. Yadav A. Gnanamani S.

Pushpavanam S.H. Pawar

PII: S0927-7765(16)30682-8
DOI: http://dx.doi.org/doi:10.1016/j.colsurfb.2016.09.028
Reference: COLSUB 8160

To appear in: Colloids and Surfaces B: Biointerfaces

Received date: 8-6-2016

Revised date: 5-9-2016
Accepted date: 21-9-2016

Please cite this article as: A.V.Raut, H.M.Yadav, A.Gnanamani, S.Pushpavanam,

S.H.Pawar, Synthesis and characterization of chitosan-TiO2:Cu nanocomposite and
their enhanced antimicrobial activity with visible light, Colloids and Surfaces B:
Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2016.09.028

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Synthesis and characterization of chitosan-TiO2:Cu nanocomposite
and their enhanced antimicrobial activity with visible light
A. V. Raut1, H. M. Yadav2, A. Gnanamani3, S. Pushpavanam4, and S. H. Pawar1*

1
Center for Interdisciplinary Research, D. Y. Patil University, Kolhapur, (M.S.) India.

2
Department of Materials Science & Engineering, University of Seoul, Seoul, 02504 South Korea.

3
Microbiology Division, CSIR-CLRI, Adyar, Chennai, (TN) India.

4
Department of Chemical Engineering, Indian Institute of Technology Madras (IIT Madras),

Chennai, (TN) India.

Corresponding Author:
Prof. Dr. S. H. Pawar.
Address: - Center for Interdisciplinary Research,
D. Y. Patil University, Kolhapur- 416 006, (M.S.) India.
Phone: +91-0231-260122
Fax: +91-231-2601595
*Email: shpawar1946@gmail.com

7
 Graphical Abstract

Highlights

 Synthesis of biocompatible organic inorganic chitosan-TiO2:Cu nanocomposite

 Nanocomposite used for enhancing the visible light antimicrobial activity

 Biocompatible material showing antibacterial effect and biocompatibility altogether

 Synergistic effect, without affecting each other presence; chitosan and Cu doped TiO2

leads to enhanced activities

8
Abstract: -

In the present investigation, novel strategy for the preparation of hybrid nanocomposite containing

organic polymer (Chitosan) and inorganic (TiO2:Cu) nanoparticles (NPs) has been developed and

demonstrated its biomedical application. The sol–gel and ultra-sonication method assisted for the

preparation of uniformly distributed Chitosan-TiO2:Cu (CS-CT) nanocomposite. The structural

properties of prepared CS-CT nanocomposite were studied by XRD and FTIR techniques. The

XPS was used to estimate elemental composition of the nanocomposite. Thermal properties were

studied using TGA. TEM and SEM analysis showed the non-spherical nature of NPs with the

average mean diameter 16 nm. The optical properties were analyzed with UV–visible diffuse

reflectance spectroscopy to confirm optical absorption in the visible region of light. Where CS-CT

showed 200% enhanced light mediated photocatalytic antimicrobial activity against

microorganism (Escherichia coli and Staphylococcus aureus) as compared with control. The

antimicrobial activity of CS-CT nanocomposite in presence of light is found to be enhanced than

that of its components, this is due to synergistic effect of organic and inorganic material

complimenting each other’s activity. The •OH radicals release studied by PL Spectroscopy on the

surface of nanocomposite was used to examine antibacterial activity. Cytotoxicity assessment of

CS-CT on human fibroblast cells was performed by MTT assay.

Keyword: - CS-CT nanocomposite; visible light antimicrobial activity; TiO2:Cu; hydroxyl radicle;

mechanisms of microbial adhesion; cell disruption; biocompatible.

9
1. Introduction

Multidrug resistance bacteria (MDR) emerging from nosocomial infection has alarmed

healthcare system worldwide to find new solutions to current problem [1,2]. Despite the advances

of science and technology, discovery of new drug remains a slow, expensive, and with a low rate

of new therapeutic discovery, taking decades with a financial burden over thousand million US

dollar [3]. Hence, innovative idea and different strategies are required to face this problem with

more efficient antimicrobial agents against these MDR microorganism.

Worlds urgent need is to develop a new class of composite materials that physically

integrates inorganic catalytic nanoparticles (NPs) and biologically active molecules which can

replace antibiotics and be effective against resistant bacterium [4]. The advances in nanoscience

and nanotechnology fields, the organic and inorganic material at nano level can be blend together

to give new hybrid conjugate materials which can display catalytic properties and unique

recognition [5,6].

TiO2 is extensively used in several industrially relevant processes, in systems ranging from

environmental applications to clean energy and from cosmetics to paint [7]. However the use of

TiO2 in biomedical applications is relatively new. The wide use of TiO2 is based on its

exceptionally efficient photo activity, high chemical stability, and low cost [8]. Photocatalytic

antimicrobial performance of TiO2 in the presence of UV light is well known and is documented

in many studies [9–12]. UV irradiation is quite less effective, harmful, energy-intensive and has

limited penetration in water. Current technological developments have created new metal doped

materials which can exterminate microbes in the presence of light e.g. Cu[13], Ni[14],Fe[15] etc.

doped TiO2. Metal ion doping can enhance the interfacial charge transfer and restricts the electron-

hole recombination and hence improves the photocatalytic activity of TiO2 under visible light.

10
 Furthermore, there are many organic molecules such as polyvinyl alcohol, polyvinyl

chloride, polyvinyl acetate, chitosan (CS) and cellulose; which are biologically active and act as

antimicrobial [16]. CS is one of the biologically active molecules and has been well studied by

researchers due to its promising properties like biocompatibility, natural antimicrobial activity,

non-immunogenic and muco-adhesiveness[17]. Besides it is naturally abundant, nontoxic and

biodegradable material [17]. The free amino functional groups present within CS impart the

biopolymer with a positive charge in acidic solutions and the ability to bind with many negatively

charged materials and surfaces. CS and its derivatives show antimicrobial properties towards

bacteria, viruses, and fungi [18–22]. CS shows both antimicrobial activity against both Gram-

positive and Gram-negative bacteria [23,24]. Moreover, the existence of polycationic CS is

beneficial for TiO2 or other inorganic materials. Both of these can bind to the negatively charged

cell surface through electrostatic interaction [25].

In the present study, we report a facile two-step method for the preparation of a well

dispersed, stable organic-inorganic hybrid Chitosan-TiO2:Cu (CS-CT) nanocomposite. The

prepared organic–inorganic composites show superior additive characteristics as compared to

individual organic and inorganic materials. Structural conformation of CS-CT was studied by XPS,

XRD, TGA and FTIR techniques. The efficiency of charge carrier trapping, migration, transfer

and separation were studied by PL spectroscopy. The influence of CS-CT nanocomposite under

visible light shows 200% enhanced antimicrobial activity on microbial cells e.g. Escherichia coli

(E. coli) and Staphylococcus aureus (S. aureus). Hydroxyl radicle which causes bacterial cell death

was probed by PL using coumarin. Also, the cytocompatibility of nanocomposite was evaluated

with human fibroblast cell by MTT assay.

2. Materials and methods

11
 CS (DA=05 %) was purchased from Sigma Aldrich. Gram-positive bacteria S. aureus

ATCC 6538, and Gram-negative bacteria E. coli ATCC 25922 were procured from the National

Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune, India. All

other chemicals and media components were analytical grade and obtained from Hi-media

Laboratories Pvt. Ltd (Mumbai, India) unless specified. All glassware’s used in the experiments

were washed with double distilled water, then autoclaved at 121°C for 20 min.

2.1. Synthesis of TiO2:Cu (CT) NPs

TiO2:Cu (CT) NPs were prepared by a sol–gel method reported previously [13]. In brief, a

stoichiometric amount of titanium (IV) isopropoxide was mixed with glacial acetic acid and stirred

for 5 min, followed by addition of 200 mg aqueous solution of sodium dodecyl sulfate and stirred

for 1h. To achieve the desired concentration of Cu2+ ion of 3.0 mole % as a dopant in the TiO2 host

lattice, a dopant precursor CuSO4·5H2O of required stoichiometric amount of was dissolved in 10

mL distilled water. The mixture was stirred vigorously for 2h with the solution of dopant. By

adding ammonia the pH was adjusted to 10. The filtration and washing was done with 50 mL of

ethanol after solution was stirred at 60 °C for 3h. The catalyst was calcinated in air at 500 °C for

5h, after drying at 110 °C in the oven. This procedure resulted in the synthesis of faint bluish CT

NPs.

2.2. Synthesis of CS–TiO2:Cu nanocomposite (CS-CT)

The prepared CT NPs (1 mg) as above was added to 5 mL of 1 % CS solution water and

mixed by using vortex for 1 min till solution become light turbid. The mixture was then

ultrasonicated for 30 min using a bath sonicator equipment (37 ± 3 kHz, 150 W; TOSHCON

Ultrasonic Cleaner, TPI Pvt. Ltd., Mumbai, India) to obtain well dispersed CS-CT nanocomposite.

12
2.3. Characterizations

X-ray diffractograms were obtained using a Bruker AXS D8 Advance X-ray diffractometer

under: 40 kV and 40 mA with Cu Kα1 radiation at 1.54184 Å and scan range of 2°/min. The

surface compositions of the sample was determined, using a Physical Electronics 5600 Multi-

technique system with monochromatic Al Kα radiation. The vacuum inside the chamber was 1.1

x 10-8 Pa. The surface morphology of the bacteria was examined by using a scanning electron

microscope (SEM) images recorded using a SEM (FEI Quanta 200 FEG, Oregon, USA), operated

at 20 kV, and equipped with an energy dispersive X-ray spectrometry (EDS) system. To investigate

the thermal stability of the samples thermogravimetric analysis (TGA) was performed on a

NETZSCH STA 449 F3 Jupiter thermogravimetric analyzer, Chennai, India. All Samples were

heated from 25 to 800 °C at a scanning rate of 5 °C min−1 under a dynamic nitrogen atmosphere.

Surface UV–visible diffuse reflectance spectra of all samples were measured by using a UV-VIS

Spectrophotometer 3092 - LabIndia Analytical, Thane (West) Maharashtra, India in the range 200–

800 nm. The photoluminescence (PL) spectra of the sample were recorded by using JASCO F.P.-

750 Model, spectrofluorometer, Easton, USA.

2.4. Photocatalytic antimicrobial properties of CS-CT nanocomposite

For the photocatalytic inactivation test, two pathogenic microorganisms, Gram-positive

(Staphylococcus aureus ATCC 6538) and Gram-negative (Escherichia coli ATCC 25922) were

used as test bacterium. The S. aureus and E. coli were obtained from National Collection of

Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune, India and maintained in

a nutrient medium. A multilamp borosilicate glass reactor was used to carry out photocatalytic

inactivation of microbes with eight fluorescent tubes (Philips 8 W T5, > 400 nm). The incident

light intensity ∼0.5 mW cm−2 was measured using a light meter (Agronic Lx-101). For bactericidal

13
activity, 1 mg catalyst CT was stirred with bacterial suspension in saline solution (5 mL, 0.9 %

NaCl at pH 7.0) and CS-CT nanocomposite and irradiated with visible light in a photo reactor. 100

µL test suspension samples were withdrawn in aliquots at regular time intervals and spread on

freshly prepared Mueller–Hinton agar plates. These plates were incubated at 37 °C for 24h in an

incubator. The standard plate count method was used to determine the viable number of cells as

cfu mL−1. All the samples were used for dark experiments without light irradiations in similar

condition as mentioned above. The same irradiation conditions were used for control runs without

photocatalyst. All experiments were performed under sterile conditions.

2.5. Cytotoxicity

The cytotoxicity of the prepared nanocomposite and separate organic and inorganic

particles were studied on NIH 3T3 embryonic mouse fibroblast cells. These cells were cultured

and maintained in DMEM supplemented with 10 % Fetal Bovine Serum (FBS), 200 mM

Glutamine, 2 mg/mL sodium bicarbonate and 1x antibiotic and antimycotic solution. The medium

was replaced periodically. The cells were cultured in tissue culture flasks and incubated at 37±2

°C in a humidified atmosphere of 5 % CO2. 0.05 % Trypsin was used to detach the cells.

Cell viability assessment study was carried out in pre-coated 12 well culture plates. In brief,

prepared stock solutions of samples were added to culture plates and subsequently oxidized with

periodate and subjected to air drying at 40 °C. The control wells were free from the nanocomposite

or organic and inorganic particles. The dried plates were then surface sterilized with 70 % alcohol

for 30 min and then UV sterilized for 1h. A cell density of 3 × 104 cells per well was seeded and

incubated with the growth medium for the period of 6, 12, 24 and 48h. The cell viability was

quantified by MTT assay. With regard to the MTT assay, the culture medium of each well was

14
replaced with MTT (5 mg/mL diluted in serum-free medium) and incubated at 37 °C for 4h. After

the removal of MTT solution, dimethyl sulfoxide was added and the medium was then left at room

temperature for two minutes. The absorbance was then measured at 570 nm using a plate reader

(Epoch, BIOTEK).

2.6. Statistical analysis

Statistical data analysis was performed using ‘Student t-test’ with P<0.05 as the minimal

level of significance. Calculations were done using the software Xl stat Version 5.0.

3. Results and discussion

The CT NPs synthesized by sol gel method was ultra-sonicated with aq. 1% CS solution to

acquire the CT-CS nanocomposite solution. The existence of CS increases the viscosity and as CT

is hydrophobic and could easily be dispersed in CS solutions for long periods [26]. Each solution

was prepared freshly before the experiment and some solution was dried in oven at 40 °C for other

characterizations.

3.1 X-ray diffraction analysis

The crystalline phases of raw material CS, CT and CS-CT were analyzed by X-ray

diffraction (XRD) (Fig. 1a). CS is polymorphic and occurs in the α-form. The crystalline

reflections of CS was indexed with (110) plane at form 2θ = 19.88°, CS-CT and CS both shares

this 2θ and confirmed the existence of CS in CS-CT nanocomposite[27]. The XRD pattern of CT

exactly matches with JCPDS card no. 21-1272 confirming that all the sharp peaks belong to the

anatase phase of TiO2. It can be observed that CT and CS-CT nanocomposite overlaps primary

sharp peaks at 2θ = 25.3°, 37.9°, 47.8°, 55.1°, and 62.6° which confirms the presence of CT in the

CS-CT nanocomposite without traces of rutile and brookite phase. In the structure of CS-CT

15
nanocomposite, there was not much change observed when compared with CT, indicating that the

CS-CT nanocomposite synthesis did not altered the characteristic structure of CT.

3.2 FT-IR analysis

Fig. 1b shows FT-IR spectra of the pure CS, CT and CS-CT nanocomposite. The

characteristic bands at 2990 to 3450 cm−1 are assigned to the N–H stretching with hydrogen bonded

amino groups, the band at 1638 cm−1 for C=O stretching of the carbonyl group (typical saccharide

absorption), and the band at 900 to 1360 cm−1 for –NH2 group in the CS molecule [28]. The bands

at 2923 cm−1 are attributed to the symmetric stretching of CH2 of CS[29]. The bands change

compared to the pure CS in the region from 670 to 400 cm−1 due to the presence of M–O [29]. The

broad peaks appearing at 3600 and 2800 cm-1 are assigned to stretching vibration of the single

bond hydroxyl group on the surface of CT [13]. The band changes at 1068 cm−1 and 1020 cm−1 is

accredited to the M–OH bond at OH group of CS [30]. The hydrogen bond and protonation of the

amino groups is observed by change in band around 1408 cm−1 [29]. FTIR analysis result reveals

that the M–O–M inorganic network was bonded with CS macromolecules by hydrogen bonding

as well as covalent bonding in CS-CT nanocomposite.

3.3 X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy analysis of CS-CT nanocomposite was performed and

the survey spectrum is shown in Fig. 2a. The photoemission bands corresponding to C 1s (285 eV),

N 1s (402eV), Ti 2p (461.0 eV), O 1s (534.9 eV) and Cu 2p (936.1 eV) were observed. As

expected, three main elements: carbon, oxygen and nitrogen were present in the survey spectrum

representing CS. Fig. 2b shows the C 1s one of the high-resolution spectra of the studied materials.

Here the C 1s core level spectrum of CS membranes reveals four peaks. The 285.0 eV peak was

16
assigned to C–H and C–C bonds in the CS backbone. The glucosamine rings evident peak observed

at 285.5 eV which corresponds to C–NH2. The peak present at 286.7 eV was assigned to C–O, C–

OH and C–N–C=O and the peak at 288.1 eV to O–C–O and N–C=O chemical binding [31]. Fig.

2c displays the N 1s XPS spectra of CS, with three different peaks centered at 399, 400 and 402

eV. These peaks correspond to C-NH2, C-NHC=O, and C-N+, respectively. It was found that there

was decrease in the protonated amine peak in CS-CT and increase in the amide peak [32]. This

provides the supporting evidence for the formation of new covalent functionalization between the

CS and CT, these data are consistent with FTIR [32]. Some marginal changes are in the heights of

other peaks, which manifest new few covalent bonds formation. Fig. 2d shows the core level Ti

2p spectra of CT sample. For TiO2, Ti 2p3/2 and Ti 2p1/2 peaks are observed at 461.00 and 466.75

eV, respectively. The splitting between the Ti 2p1/2 and Ti 2p3/2 is 5.75 eV, indicating a normal

matrix. Fig. 2e the Cu 2p3/2 and 2p1/2 core-levels peaks are located at 936.1 and 955.8 eV,

respectively, indicating a normal state of Cu2+ in the sample.

3.5 Thermal gravimetric analysis

Thermal stability of the CS and CS-CT nanocomposite was measured using a thermal

gravimetric analysis (Supplementary Fig. 1s). For pure CS, weight loss took place in three clear

stages. The first stage observed at 30 °C and ended at 140 °C with a weight loss of 17 %, which

was assigned to the loss of water in CS films. The second stage observed at 140 °C and went on

to 230 °C with a weight loss of 10 %, which is assigned to more strongly linked structural water.

The third stage observed at 230 °C and reached a maximum at 380 °C with a weight loss of 47 %,

which corresponded to the decomposition of CS and vaporization and elimination of volatile

products [29,33]. The TG curve of the CS-CT nanocomposite also shows three distinct weight-

loss stages. The first two steps from 30 °C to 140 °C and 140 °C to 340 °C was characterized with

17
an 8 % and 31 % weight loss inclusive of water loss this is endorsed to introduce M–O network in

the CS-CT nanocomposite correlate with the IR spectrum analysis respectively. The third step at

340–550 °C is caused by decomposition of the residual organic materials[34]. CS completely

decomposes at around 400 °C, permits to conclude that residue weight at 800 °C is of CT the

remnant of CS-CT nanocomposite. It can be seen that CS-CT exhibited better thermal stability

than the CS [35].

3.6 Electron microscopy studies

SEM micrographs illustrat the morphology of CS-CT in Fig. 3.a. The CS-CT particles were

found to be freely distributed, providing rough surface area to attach microbial cell. In Fig. 3.a it

can be clearly seen that CS-CT nanocomposite show uneven nanocomposite clusters with rough

surface and spherical primary CS-CT particle. The elemental analyses were done by EDX

(Supplementary Fig. 2s) which reveals the presence of elements viz., C and N in addition to Cu,

Ti and O, in CS-CT [36]. This proves that CS and CT were well mixed together. Fig. 3.b shows

TEM images of CS-CT nanocomposite. The TEM image of the nanocomposite reveal a network

of anatase NPs with sizes, range of 6-22 nm having mean average particle size distribution of 16

nm. The average particle size distribution diameter is measured by Image-J software

(Supplementary Fig. 3s). TEM investigations gave evidence that the CT NPs are uniformly

distributed in the CS clusters with uniform particles size.

3.7 UV–visible diffuse reflectance spectroscopy

The UV–visible diffuse reflectance spectra of CS, CT and CS-CT are shown in the Fig. 4s.

The recorded UV–visible diffuse spectra are in the range between 300-600 nm at room

18
temperature. CT NPs shifts its absorption edge to longer wavelength in the range 400-600 nm

while CS has it absorption peak in the range 300-400 nm. The selected concentration of Cu2+

dopant in TiO2 generates oxygen vacancies due to charge compensation effect which allows the

CT NPs shift towards visible range. In agreement with the previous reports, the concentration of

CT has band gap of 2.60 eV[13,37]. Whereas the CS-CT nanocomposite’s shows absorption is in

between 400-500 nm. The observed red shift for CS-CT was attributed to the existence of CT. This

emphasize that visible absorption phenomenon of CS-CT is closely associated with CT host lattice

and in presence of high amount of CS, significant changes can be seen.

3.8 PL spectroscopy

The efficiency of charge are studied by carrier trapping, migration, transfer and separation

with the help of PL spectroscopy. This also reveal the rate of electron hole pairs in composite with

semiconductor particles by defects on the surface of CT NPs. These signals are recognized on the

surface positions of oxygen vacancies [38–40]. Supplementary Fig. 5s shows the PL spectra of

CS, CT and CS-CT by employing an excitation wavelength of 290 nm. The PL emission peaks in

the region 400-500 nm with measurable intensity were detected for CS, CT and CT-CS (Fig. 5s).

The PL spectra at 441 nm represent peak of CS, CT and CS-CT nanocomposite and peak at 480

nm are due to excitonic PL of CT and CS-CT, signifying the existence of surface oxygen vacancies

and defects in the sample [38]. From Supplementary Fig. 5s we witness a decrease in the PL

intensity in CS-CT and CT. This is due to the fact that PL intensity is sensitive to polycationic

polyglucosamine polymer (chitosan) presence and metal doping. In the presence of Cu2+

concentration in CT intensity decreases which specifies a lower recombination rate of electron–

hole pairs and hence higher separation efficiency [41]. Metal dopants allows the electron transfer

from the excited state (CB) to the new levels [39]. Photo assisted killing is enhanced photocatalytic

19
activity is achieved due to decreased in recombination rate and more photo charge generated

carriers. Indeed, CS-CT demonstrate even lower intensity compared to CT which indicates that

both CT and CS-CT possess different optical properties. This difference in the optical property can

be due to simultaneous photocatalytic oxidation of the adjacent CS sub layer [42,43]. The oxidized

CS shows lower PL intensity as CS sub-layer can be photo catalytically oxidized in CS-CT

nanocomposite, indicating a clean red-shifted response. As a result, CS can play a significant role

in extending the range of the optical sensitivity, displaying dramatic changes in the optical

response of the photo catalytically-oxidization of the CT in CS-CT nanocomposite [43].

3.9 Photocatalytic antimicrobial activity and its mechanism

To study photocatalytic antimicrobial activity of CS-CT, experiment were performed as

reported previously [13]. Briefly the antimicrobial effect of CT and CS-CT were carried out in the

presence and absence of light. For dark experiment the microbes (E.coli and S. aureus) were

supplemented with the CS-CT nanocomposite, CS and CT in the respective tubes. The

antimicrobial effect in absence of light is shown in Fig. 4.a & 4b. CS shows influence on inhibiting

the bacteria even in dark which is reflect in the effect of the CS-CT nanocomposite. It can been

clearly seen that the S. aureus bacteria killed slightly more rapidly compare to E. coli. Our results

are in good agreement with several previous reports [44–46]. The polycationic properties of CS

helps to attack the negatively charged bacterial cell wall and cause cell wall leakage, leading to the

death of bacteria [47]. This is the main mechanism of antimicrobial activity of CS which is

universally well accepted [44,46]. No antimicrobial effect was seen in both the bacteria in the

presence of CT NPs in dark. This confirms that CT is almost inactive in absence of light whereas

CS and CS-CT shows inhibitory activity.

20
 The effect of visible light on bacteria showed no loss in bacterial cell number. This proves

that light doesn’t have any adverse effect on the microorganism grown in Muller Hinton medium.

However, in the presence of visible light CT and CS-CT showed a dramatic antimicrobial effect.

The percent survival of bacteria by CT and CS-CT with the function of time is shown in the Fig.

4-c & 4-d. Antimicrobial activity for E.coli was achieved in 240 min by CT and 120 min by CS-

CT (Fig. 4.c); whereas for S. aureus 100 % bacterial inactivation time was 300 min by CT and 150

min by CS-CT (Fig. 4.d). A 200 % enhanced activity was shown by a blend of CS-CT NPs. This

shows that the antimicrobial activity of CT is complimented in the presence of CS. In

photocatalytic inactivation experiments, many researchers reported reactive oxygen species (ROS)

plays vital role in bactericidal activity. The photo oxidative action of ROS accumulation can

damage cellular constituents and disrupt cell functions. Photocatalysis initially damages the

surface of the bacterial cells with an attack of ROS, before inducing breakage off the cell

membranes occur at weak points [9,48,49]. The CS also adds fuel in the process of cell damage as

the negatively charged microbial cell membrane interacts with the positively charged CS

molecules and leads to the leakage of proteinaceous and other intracellular constituents [47]. This

reaction compliments the photo oxidative action of ROS produced by CT in the presence of visible

light and hence CS-CT provides enhanced antimicrobial activity. According to some researchers

CS does not act as bactericidal, but it acts as a bacteriostatic [50]. From the graph we can see CS

after some time become static, but the ROS produced by CT causes the bactericidal activity. A

previously reported study reveals that the inactivation of E. coli is much slower in comparison to

S. aureus this also overcomes by CS-CT nanocomposite.

21
3.10 Scanning electron microscopy of bacterial cell.

Scanning electron microscopy of E.coli bacterial cell in Fig. 5 confirms the interaction of

NPs and inhibitory action of CS-CT nanocomposite. The E.coli cells morphology changes after

exposing the cells to light and nanocomposite for 2 h in dilute saline solution (Fig. 4 c & d). Fig.

5 b is the inverse and focused image of Fig. 5.a which noticeably shows the nanocomposite-cell

attachment and cell destruction clearly. The black arrow shows the nanocomposite which are

attached to the E.coli cell wall. Fig. 5 c, d shows control bacteria, irradiated with visible light

without nanocomposites. Before contact with nanocomposite E.coli cell are smooth and intact (Fig.

5 c) and after 2 h of contact in presence of light the cells deform in many places and destruct the

cell membrane (Fig. 5 a). The color difference in the inverse images in Fig. 5 b and d clearly shows

the good cell integrity. In Fig. 5 b the cell ruptures and the internal fluid comes out. Consequently

the internal and external space of the cell having same composition. While in Fig. 5 d cell is intact

and the cell components present inside the cell which seems to be dark in color. These results in

the presence of light show clearly that the cell membrane rupture due to photo-oxidation process

of nanocomposite.

3.11 PL spectroscopy to study free radicals

Reactive oxygen species (ROS) is an important factor in the photo induced antimicrobial

activity of the nanocomposite. Hydroxyl radical is one of the example of ROS, which is a

chemically reactive molecule containing oxygen, and damages the bacterial cell membrane. Here

the coumarin based fluorescence probe method is used [51]. The released •OH radicals is studied

by PL technique for its role in bacterial inactivation. The free radicals (e.g. hydroxyl radicals)

22
generated in the process of photocatalytic activity by CT, a component of CS-CT creates an

electron hole pair. The CS-CT nanocomposite in coumarin solution (1 × 10−3 M) is magnetically

stirred and irradiated with visible light. The sample was excited at 332 nm and fluorescence

emission spectrum form coumarin solution was measured for every 3 min during irradiation. The

emitted fluorescence spectrum of CS-CT nanocomposite with coumarin solutions are shown in the

supplementary Fig. 6s. A Gradual increase in the fluorescence generated by •OH at 448 nm was

observed only when coumarin solution was irradiated with CS-CT nanocomposite. The gradual

increase reveals the steady release of •OH radical from the CT host lattice. The copper content in

TiO2 enhances the transfer of photo generated electron which do not get affected by CS in

nanocomposite. Moreover reduction in the recombination of photo-generated charge carriers and

prolong separation does not get affected in presence of CS which are responsible for the

enhancement of photocatalytic activity [13,51].

3.12 Possible mechanism of nanocomposite for killing bacteria

The activation of photocatalytic nanoparticle present in nanocomposite involves several

steps such as adsorption-desorption, electron-hole pair production, recombination of electron pair,

and chemical reaction[52]. The proposed mechanism of photocatalytic antimicrobial activity of

nanocomposite is explained as follows (Fig. 6):

When the photocatalyst (CT) is irradiated with photons of energy equal to or more than

band gap energy of CT, the electrons (e−) are excited from the valence band (VB) to the conduction

band (CB) with the simultaneous creation of holes (h+) in the VB:

− +
𝐶𝑇 + ℎ𝑣 → 𝑒𝐶𝐵 + ℎ𝑉𝐵 ……………………………………………………………………. (1)

23
 Where hv is the energy essential to transfer the electron from VB to CB. The electrons

generated through irradiation could be readily trapped by O2 present over the photocatalyst surface

or the dissolved O2 (present on oxidized chitosan) to give superoxide radicals (·O2−):

−
𝑒𝐶𝐵 + 𝑂2 → ∙𝑂2− ………………………………………………………………………........ (2)

Accordingly, ∙O2− could react with H2O (present on chitosan surface) to produce

hydroperoxy radical (∙HO2) and hydroxyl radical (∙OH), which are strong oxidizing agents to

decompose the bacterial cell wall:

∙𝑂2− + 𝐻2 𝑂 → ∙𝐻𝑂2 + ∙𝑂𝐻 …………………………………………………………........... (3)

Simultaneously, the photo induced holes could be trapped by surface hydroxyl groups (or

H2O) on the organic and inorganic surface to give hydroxyl radicals (∙OH):

+
ℎ𝑉𝐵 + 𝐻2 𝑂 → ∙𝑂𝐻 + 𝐻 + ………………………………………………………………….. (4)

Finally ∙OH radical react with microbial surface and the microbial cell wall get

breakdown by oxidation to yield cell inner content and cell debris as follows:

∙𝑂𝐻 + 𝑀𝑖𝑐𝑟𝑜𝑏𝑖𝑎𝑙 𝑐𝑒𝑙𝑙 𝑤𝑎𝑙𝑙 + 𝑂2 → 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 (𝑐𝑒𝑙𝑙 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑎𝑛𝑑 𝑑𝑒𝑏𝑟𝑖𝑠) …………....... (5)

Meanwhile, recombination of positive hole and electron could take place which could

reduce the photocatalytic activity of prepared CS-CT nanocomposite:

− +
𝑒𝐶𝐵 + ℎ𝑉𝐵 → 𝐶𝑇 ……………………………………………………………………………. (6)

3.13 Cytotoxicity

The percent viability of NIH 3T3 cells after treatment with the CT, CS and CS-CT

nanocomposite were analyzed using MTT assay (Fig. 7).The results of the cytotoxicity evaluation

24
demonstrated the CS-CT nanocomposite shows more than 85 % of the cells viability for the

experimental period of 12, 24, 48 and 72 h. The CT alone shows cell viability of around 65 % and

CS alone shows cell viability more than 92 %. These result shows the toxicity of CT nanoparticle

decreases when present in nanocomposite. The evaluation of nanocomposite toward mammalian

cells is essential for its application in relation to human e.g. wound healing, burn wound covering

etc. The results indicated that CS-CT nanocomposite has low toxicity toward mammalian cells and

is promising as antimicrobial agent for many application in relation with humans.

Conclusions

In summary, we have successfully prepared an effective visible light based photocatalytic

antimicrobial CS-CT nanocomposite with the help of sol–gel and ultra-sonication method. XRD

and FTIR confirmed the CS-CT nanocomposite characteristic featuring nanocrystalline TiO2 in

tetragonal anatase phase. XPS analysis confirmed the substitution of Ti4+ cations by Cu2+ cations

in the TiO2 of CS-CT. SEM and TEM analysis revealed the non-spherical NPs size with an average

mean diameter of 16 nm. Present study is an attempt to utilize the visible light instead of only the

UV range for disinfecting microbes. The presence of CT played a vital role in altering

physiochemical and photocatalytic antimicrobial activity of CS-CT. The optical band gap energy

of CS-CT NPs shifted from UV region to visible light region as compared to CS. The CS-CT

shows best result and are 200 % more effective compared to positive control for

photocatalytic antimicrobial application. In two component system where CT kills the

bacteria by photo generated reactive radicals and CS destruct the cell wall by electrostatic

interaction and oxidative stress. Hence, the synergistic effects is the reason behind the enhanced

antimicrobial activities where, two components helps each other by their individual killing

mechanism. Considering the preparation process and visible light activity at low dopant

25
concentrations, CT photocatalyst in nanocomposite is found to be reasonably applicable for

biomedical applications. Also the nanocomposite shows good cytocompatibility which could be

possibly applicable for broad range of applications. This paper provides a protocol for the synthesis

of a new hybrid material which has photocatalytic antimicrobial activity in the visible light.

Acknowledgements

Authors are very grateful to Board of Research in Nuclear Sciences (BRNS), Mumbai and

Department of Science and Technology (DST), Delhi for providing the funds to carry out the

research activities. Authors are also thankful to Dr. M.S.R. RAO, IIT- Madras, for providing XPS

analysis in Nano Functional Materials Technology Centre at Indian Institute of Technology

Madras.

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a b

Fig. 1. (a) XRD patterns and (b) FT-IR spectra of CS-CT nanocomposite, CT and CS

a b

35
 c d

Fig. 2: - XPS spectra of CS-CT nanocomposite of (a) CS-CT full survey spectrum, (b) C 1s, (c)

N 1s (d) Ti 2p and (e) Cu 2p.

36
 a b

Fig. 3. (a)- SEM micrograph of CS-CT nanocomposite and (b)- TEM image showing embedded

CT in CS-CT nanocomposite.

(a) (b)

(c) (d)
Fig. 4: - Antimicrobial activity of CS, CT and CS-CT nanocomposite against (a) E. coli and (b)

S. aureus in the absence of light and (c) E. coli and (d) S. aureus in the presence of visible light.

37
 Fig. 5: - SEM images of (a) bacteria with nanocomposite (b) inverse magnified image bacteria

with nanocomposite (c) bacteria without nanocomposite (d) inverse magnified image of bacteria.

38
Fig. 6:- Possible mechanism of antimicrobial activity in the presence of nanocomposite

39
Fig. 7: - Cell cytotoxicity by MTT assay of CS, CT and CS-CT at different time interval.

40