Accepted Manuscript A simple and effective method to synthesize fluorescent nanoparticles using tryptophan and light and their lethal effect against bacteria Rafael Jun Tomita, Ricardo Almeida de Matos, Marcelo Afonso Vallim, Lilia Coronato Courrol PII: DOI: Reference:

S1011-1344(14)00245-0 http://dx.doi.org/10.1016/j.jphotobiol.2014.07.015 JPB 9797

To appear in:

Journal of Photochemistry and Photobiology B: Biology

Received Date: Revised Date: Accepted Date:

9 April 2014 13 July 2014 22 July 2014

Please cite this article as: R.J. Tomita, R.A. de Matos, M.A. Vallim, L.C. Courrol, A simple and effective method to synthesize fluorescent nanoparticles using tryptophan and light and their lethal effect against bacteria, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.07.015

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A simple and effective method to synthesize fluorescent nanoparticles using tryptophan and light and their lethal effect against bacteria

Rafael Jun Tomita , Ricardo Almeida de Matos, Marcelo Afonso Vallim, Lilia Coronato Courrol * Universidade Federal de São Paulo – UNIFESP – São Paulo – Brazil

*Corresponding Author: Instituto de Ciências Ambientais, Químicas e Farmacêuticas (ICAQF), Departamento de Ciências Exatas e da Terra (DCET), Universidade Federal de São Paulo (UNIFESP) - campus Diadema, Rua Prof. Artur Riedel, 275 CEP 09972270 - Diadema, SP, Brazil, e-mail: [email protected]

A simple, environmentally friendly and cost-effective method was used to synthesize silver nanoparticles using tryptophan and light. To prepare the nanoparticles, the following components were used: deionized water, silver nitrate, light and tryptophan. The effects of the tryptophan concentration and light exposure time on the formation of tryptophan silver nanoparticles (Tnnps) were studied. The synthesized Tnnps were characterized using transmission electron microscopy (TEM), absorption and fluorescence spectroscopy and zeta potential measurements. The synthesized Tnnps were nearly spherical, with sizes of approximately 17 nm. In addition, the antibacterial activity of Tnnps was determined by monitoring the growth curves of strains of E.coli, P.aeruginosa, S.epidermidis, S.marcescens, and E. faecalis using the microdilution test. The Minimum Inhibitory Concentration (MIC) for 4 of 5 tested bacteria was determined to be between 20.0 and 17.5 µg/mL for 48 h and between 22.5 and 20.0 µg/mL for 72 h.

1. Introduction Silver has a strong antimicrobial potential and has been used since the times of antiquity1, 2. However, with the discovery of antibiotics, the use of silver as an antimicrobial agent has declined. Because of the recent observation of the increase of the antimicrobial effects of silver when in the form of silver nanoparticles, interest in the use of silver as a potential antimicrobial agent has made an extraordinary

1

comeback3, 4. The bactericidal efficacy of silver nanoparticles has been investigated by many researchers3-12. Microorganisms are unlikely to develop resistance against silver, in contrast to antibiotics, as silver attacks a broad range of targets within the microbes9. There are various theories modeling the antimicrobial effect of silver nanoparticles 1, 4, 6, 13-16

.

Due to the nanoparticles size, a large surface area comes into contact with the

bacterial cells to provide a higher percentage of interaction when compared to bigger particles4. Yen et al.17 reported that smaller silver nanoparticles (radius around 3 nm) are more cytotoxic than larger particles (25 nm) at a concentration of 10 µg/mL, highlighting the importance of the nanoparticle size. The bactericidal potential of nanoparticles is also influenced by their shapes. Pal et al.9 reported the effect of spherical, rod and triangular nanoparticles, synthesized by citrate reduction, against E. coli. Triangular nanoparticles were found to be more effective against this pathogen than spherical nanoparticles, which are more active than rod-shaped nanoparticles. The synthesis of silver nanoparticles is carried out by several physical and chemical methods that include photochemical reduction18, 19, chemical reduction20, laser ablation3, sonochemical methods21 and microwave radiation22, 23. For the photochemical reduction of silver salts, very small colloidal particles can be produced, and the reduction of metal ions can be controlled through the control of the exposure time24. There are other factors that are also responsible for defining the particles sizes in photochemical reactions like the reactant concentration, presence of stabilizers,25 and the reaction medium26. The chemical and physical methods used to synthesize silver nanoparticles frequently raise questions regarding environmental risks due to the use of toxic, hazardous chemicals27. The synthetic methods also use organic solvents to counterbalance the hydrophobicity of the capping agents28. In addition, there are other various biochemical routes demonstrated in the literature 29. Several studies have described the controlled synthesis of metal nanoparticles with different sizes and shapes mediated by biomolecules that are nontoxic and minimize

environment

damages.

Tryptophan 30, 31

silver nanoparticles synthesis in several reports

has

already

been

used

for

, nevertheless, this is the first report

that uses a photochemical reduction. This paper describes the synthesis of silver nanoparticles mediated by a tryptophan/light system and the antibacterial activity of the resultant nanoparticles. The characterization of the synthetized silver nanoparticles was

2

done by optical/fluorescence spectroscopy, zeta potential measurements and transmission electron microscopy.

2. Materials and Methods Silver nanoparticles generation and evaluation Silver nitrate (AgNO3, 99%, CAS# 7761.88.8) and tryptophan (CAS 73-22-3) were purchased from Sigma-Aldrich. No other chemical reagents were used in the synthesis.The silver nanoparticles were obtained by mixing the reagents in water solution, followed by vigorous stirring for 5 minutes. The concentration rate study of silver nitrate and tryptophan aqueous solution was performed according to the conditions presented in table 1. The transparent solutions were illuminated by a xenon lamp (Cermax 400 Watts, not focused and placed 10 cm from the sample reservoir ~3.6W/cm2). The Xe lamp power measurement was done condensing its output in a broadband power meter (Spectra Physics 407A), using a lens and a calibrated beam splitter. The effects of the exposure time on the nanoparticle synthesis were studied, and the results are presented in table 2. UV-vis absorption spectra were recorded by a Shimadzu Multispec-1501 UV-vis spectrophotometer using 10-mm quartz cells. The emission spectra were obtained by exciting the samples, inside a 1-mm optical path cuvette. The emissions of the samples were analyzed using a Fluorimeter Jobin Yvon exciting samples at 280 nm. Emission spectra were obtained in the range of 300 nm to 550 nm. The silver nanoparticles zeta potential dependence on the solution pH of silver nanoparticles was measured by the analytical instrument Zetasizer NanoZS. To construct a curve of zeta potential as a function of pH, the adjustment to different pH values was performed using standard solutions of HCl ( 5 mo/L) and NaOH ( 5 mol/L ). The morphology of the tryptophan nanoparticles (Tnnps) was determined by transmission electron microscopy using a LEO 906E instrument from Zeiss, Germany (6 µA and 80 kV). For the measurements, a drop of silver nanoparticles (5 µL) dispersed in bi-deionized water was placed onto a carbon-coated copper grid. The excess liquid was removed using a paper wick, and the deposit was dried in air for 5 min prior to imaging. The images were captured by a Megaview III camera and processed using the iTEM universal TEM imaging platform (Olympus Soft Imaging Solutions GmbH, Germany).

3

Broth Microdilution Assay for the Determination of the Minimum Inhibitory Concentration (MIC) The preparation of E.coli, P.aeruginosa, S.epidermidis, S.marcescens, and E.faecalis strains were performed by a 1:20 dilution in a LB (Luria-Bertani) broth. Microtiter plates (96 wells) were used for the broth microdilution assays to ascertain the MIC for each tested strain. Two independent assays were conducted according to the guidelines of the National Committee for Clinical Laboratory Standards (CLSI, M100-S9). The following minor modifications were implemented: the target microorganisms were grown in test tubes overnight in 3 mL of the respective medium at 30 °C and agitated in a rotary shaker (150 rpm). After a 1:20 dilution in a LB (Luria-Bertani) broth, the expected density for the broth microdilution method was 5 × 105 CFU/mL, which is equivalent to approximately 100 colonies isolated on the plates containing a medium solid culture. Finally, a 90-µL aliquot of this suspension was used in each well of a 96well plate acrylic (ELISA), resulting in a concentration of 5 × 104 CFU/mL on a well acrylic plate. The concentration was confirmed by viability counts on the LB plates. The microtiter plates were subsequently incubated at 30°C for 24 h, 48 h and 72 h. Finally, the absorbance at 530 nm was measured in a plate reader (Epoch, Bio-Tek, Winooski, VT, USA). The threshold for the MIC was set at a minimum of an 80% growth inhibition. All of the tests were performed in triplicate in 100 µL of reaction volume. Evaluation of the antibacterial activity of silver nanoparticles The concentration of the silver nanoparticle samples ranged from 15 to 30 µg/mL (10.0 µL stated on each well). A positive growth control containing only culture medium and the strains under study was also prepared. The LB liquid culture medium was used as negative control in the study. After preparation of the experiment, the plates were incubated at 37°C for 24 h, 48 h and 72 h and analyzed using a microplate reader (Biotek - Epoch Microplate Spectrophotometer). All of the tests were performed in triplicate.

3. Results and Discussions 3.1) Synthesis and characterization of tryptophan silver nanoparticles (Tnnps)

4

During light exposure, a modification of the solution color was noted, which was observed to change from colorless to a brownish/yellow due to the presence of silver nanoparticles in suspension (Figure 1).

Figure 1. Color of a silver colloidal suspension before (A) and after (B) illumination with white light from a Xe-lamp for 5 minutes.

3.1.1) Variations of the silver nitrate concentrations The absorbance of the silver colloidal solutions was measured for the solutions prepared with different AgNO3 concentrations. The results are shown in figure 2. With low silver nitrate concentration (0.83 mM), a small absorption band at approximately 440 nm was observed. The peak was low in intensity and very broad. According to the literature, broad peaks in the beginning of the formation of nanoparticles are attributed to very small particles (seeds)

32

. With an increase of the silver concentration, the

absorption band increased in intensity. The minimum observed at 320 nm is based on the inter-band transition of the silver nanoparticles33. The absorption peak at approximately 450 nm occurred due to the SPR (surface plasmon resonance) effect, indicating the formation of silver nanoparticles. A growth of

the absorbance

was

observed

when

the concentration of

silver

nanoparticles increased, and more particles were formed during the same time. At higher silver concentrations, the absorption peak was red-shifted and its shape became broader. This broadening can be attributed to the appearance of silver ions clusters in the solution. The red shift observed in the UV vis spectra could also be attributed to the increase in particle size of the slilver nanoparticles34.

Figure 2. Optical absorption spectra of silver nanoparticles formed with illumination for 1 minute by a Xe lamp as a function of the silver concentration.

3.1.2) Xenon light exposure time Without xenon illumination, no changes were observed in the part of the spectra where a characteristic silver absorbance peak should be observed (see figure 3). The formation of the nanoparticles is possible because the absorption of light

5

(spectral distribution: 185 nm to 2000 nm) promotes the reduction of silver ions (Ag+) in metallic silver (Ag0) (in tryptophan/silver nitrate solution) and because the tryptophan suppresses the agglomeration of silver particles, thereby stabilizing the colloidal suspension. The use of white light illumination using a xenon lamp is important to catalyze this reduction because tryptophan by itself is not an adequate reducing agent for the reduction of silver, which has a reduction potential of 0.799 V.35 Simultaneous to this reduction, heating the solution via the absorption of the IR spectrum of the lamp enhanced the aggregation process toward larger silver clusters. The effect of the xenon lamp light exposure time is shown in figure 3. This figure shows that with an increase of the exposure time to light from the Xe lamp, the absorption band increased in intensity. The exposure time was continued for 15 min to determine whether silver nanoparticles aggregate to form larger clusters or to form clusters of other geometries; if such clusters formed, the shifting of the peak could be observed. The increase in the absorption intensity of the UV-Vis spectra at a constant wavelength is an indication that the reaction leads towards completion till 15 min. The increase in the width of the absorption spectra indicates agglomeration of the particles36, but the absence of shift in the SPR peak indicates that the particle size is not modified34. The AgNO3 solution did not present any change in color after illumination by Xe lamp showing that the tryptophan is required for reduction of Ag+ to Ag0.

Figure 3. Optical absorption spectra of silver nanoparticles produced using different Xe-lamp light illumination times.

3.1.3) Tnnps fluorescence Tryptophan exhibits fluorescence due to its aromatic ring. The fluorescence spectra obtained by exciting samples of different silver concentrations are shown in figure 4. From this figure, it is possible to correlate the formation of silver nanoparticles with the reduced amount of tryptophan in the solution. The decrease of the tryptophan fluorescence with the increase in the silver concentration indicates that a total conformational change is occurring due to a reaction with the silver. Note that fluorescence is very sensitive to environmental factors.

Figure 4. Tryptophan fluorescence spectra obtained under excitation at 280 nm in function of silver concentration.

6

3.1.4) TEM images and zeta potential A Transmission Electron Microscope was used to characterize the size, shape and morphology of the synthesized Tnnps, and the micrographies of the synthesized particles ([AgNO3] = 5.41 mM, [Tryp] = 12 mM and 1 min Xe) are shown in Figs. 5a and b. The Tnnps are nearly spherical with sizes ranging from 5 to 40 nm, as observed in figure 5c. Figure 5. (A and B) TEM images of synthesized Tnnps (5.41 mM, 1 min Xe). (C) The average sizes of the nanoparticles.

The zeta potential provides important information regarding the stability of the synthesized colloidal system. As the zeta potential is obtained indirectly by measuring electrokinetic charge of the particles, it can determine the full charge of the colloid surface, considering that the electric double layer system of the present Tnnps varies as a function of pH. Generally, a suspension that exhibits an absolute zeta potential of less than 20 mV  is considered unstable and will result in the precipitation of particles in suspension37, whereas the absolute zeta potential higher than 20 mV  is stable. The zeta potential for silver nanoparticle synthesized with 5.41 mM and 1 min of exposure to the Xe-lamp light is shown in Fig. 6. The nanoparticles synthesis resulted in an unstable suspension (zeta potential below 20 mV), in an acidic medium. However, increasing the solution pH, stability was reached (as seen in figure 6).

Figure 6. Zeta potential of Tnnp (5.41 mM, 1 min Xe) as a function of pH.

The irradiation with light from the Xe lamp induces three effects in the reaction medium, especially in tryptophan molecules: 1) increase in kinetic energy and consequently heating; 2) population of higher energy levels and 3) formation of induced dipole moment. The combination of these three factors facilitates the oxidation (loss of electrons) of the tryptophan molecules, and these electrons are reduce the ionic silver, resulting in the formation of metallic silver clusters that agglomerate into silver nanoparticles.

7

3.2) Antibacterial activity

A study on the sensitivity of bacteria to exposure to Tnnps was performed. This study was performed according to the microdilution test38 and provided results for the inhibition of growth of certain microorganisms. The microdilution test provided quantitative results concerning the inhibition of bacterial growth, as obtained by reading the plates in a spectrophotometer. The data from the experiments performed in triplicate are shown in Figures 7, 8 and 9, referring to the incubation periods of 24 hours, 48 hours and 72 hours, respectively. AgNO3 solution precipitates in the culture medium and the LB broth preclude the use of this compound as an antimicrobial in the broth microdilution assay. Thus, the assay was only used to test the antibacterial power of silver nanoparticles because they did not precipitate when they came in contact with the LB broth components. The silver nanoparticles solutions were used at concentrations of 30.0, 27.5, 25.0, 22.5, 20.0, 17.5 and 15.0 µg/mL.

Figure 7: Growth inhibition versus AgNP (5.41 mM, 1 min Xe, pH = 7.0) as shown in a concentration graph: the broth microdilution method, read after 24 hours of incubation.

Figure 8: Growth inhibition versus AgNP (5.41 mM, 1 min Xe, pH=7.0) as shown in a concentration graph: the broth microdilution method, read after 48 hours of incubation.

Figure 9: Growth inhibition versus AgNP (5.41 mM, 1 min Xe, pH=7.0) as shown in a concentration graph: the broth microdilution method, read after 72 hours of incubation.

The results indicate that the silver nanoparticles strongly inhibited the development of Gram positive and Gram negative bacteria. After 24 hours of incubation, inhibition of all of the tested strains remained near 100% at all of the tested concentrations of silver nanoparticles, except for E. faecalis, the inhibition of which in the presence of the highest concentration of the antimicrobial agent was close to 90% at 30 µg/mL. After 48 hours of incubation, there was a decrease in the inhibitory effect at

8

lower concentrations of the silver nanoparticles (17.5 µg/mL and 15.0 µg/mL) for E. coli, P. aeruginosa, S. marcescens and S. epidermidis. However, higher concentrations of antimicrobial nanoparticles continued to exert strong growth inhibition to all of the tested bacteria over 48 hours. Similar results were found for the 72-hour incubated period; however, at this incubation period, there was a decreased inhibitory effect at low concentrations (including 20.0 µg/mL) of the Tnnp solution. Thus, at higher concentrations, the growth inhibition continued to be extremely effective for the tested bacteria species. Except for E. faecalis, which exhibited a lower inhibitory effect of the silver nanoparticles, these data are consistent with observations made previously for the 24- and 48-hour incubation periods. The mechanism of the inhibitory action of silver nanoscale compounds has not been fully elucidated. It is believed that the inhibitory action occurs mainly due to the strong electrostatic interaction of the surface charged particles as well as through the release of Ag+ ions from the nanoparticles in aqueous media, with diverse microbial structures. According to the literature, the interaction of Ag+ ions can occur with the thiol groups of proteins, leading to denaturation of the proteins located in the cell membrane, thereby causing structural changes with direct consequences on the respiratory chain that would lead to cell death. Another hypothesis is that the nanoparticles are capable of penetrating the inner bacterial cell can interact with DNA molecules through the phosphorus groups, leading to loss of the capacity for cell replication. Some authors recently suggested that the inhibitory action occurs due to free radicals derived from the particle surface that are capable of damaging the cell membrane. The efflux pumps are recognized as one of the main forms of antibiotic resistance and metal ion mechanism. The ATP-dependent mechanism essentially consists of pumping the antimicrobial ions that are present in the cytoplasm to the extracellular medium. Cells that exhibit resistance to silver nanoparticles often have lower amounts of Ag+ in the cytoplasmic half compared to the susceptible parental cells 5, 12, 39

. The MIC for 4 of 5 tested bacteria was determined to be between 20 µg/mL and

17.5 µg/mL for 48 h and between 22.5 µg/mL and 20 µg/mL for 72 h.

9

4. Conclusions In this paper, silver nanoparticles were synthesized using a green chemistry method based on tryptophan and light. The preparation conditions were optimized using the results of absorption and fluorescence spectroscopy, and the antimicrobial effect of the resulting nanoparticles was tested.

Through this study, the stoichiometric ratio

of the reagents resulting in the best molar ratio of approximately 2.0 between tryptophan and silver nitrate was determined. The

formation

of

nanoparticles

was

verified

satisfactorily

using

spectrophotometry in the UV-visible region because the maximum absorbance band due to the surface plasmon resonance at approximately 440 nm was observed, which is typical for the silver nanoparticles, as reported in the literature. Images obtained using a transmission electron microscope indicated that the nanoparticles synthesized exhibited a diameter of ~ 17 nm on average and had a spherical shape. The zeta potential measurements indicated that the pH of the synthesis is not ideal because the suspended particles have an insufficient charge to characterize a thermodynamically stable system and should thus undergo further treatment with alkaline solutions. The antimicrobial sensitivity test performed using the broth microdilution method provided quantitative data on the inhibitory effect of silver nanoparticles on microorganisms, which, in concentrations greater than 22.5 µg/mL, revealed that the inhibition was close to 100% for periods up to 72 hours. The MIC for 4 of 5 tested bacteria was determined to be between 20.0 µg/mL and 17.5 µg/mL for 48 h and between 22.5 µg/mL and 20.0 µg/mL for 72 h. Thus, the silver nanoparticles synthesized based on the concepts of the green chemistry methodology exhibited promising results regarding the development of the inhibitory action to different bacteria species. Fluorescent tryptophan silver

nanoparticles can be used

in advanced

environmental treatments, including air, water, or surface disinfections, and personal hygiene. For Tnnps to be used in the field of infectious disease treatment, however, further investigation is required.

10

5. Acknowledgements The National Council for Scientific and Technological Development (CNPq) is acknowledged for their financial support, Sylvia Carneiro is acknowledged for providing the electron microscope support and Victor Hildebrand Rosas is acknowledged for the zeta potential measurement support.

11

Tables Table 1: Concentration rate study of silver nitrate and Tryptophan. Tryptophan (mmol.L-1)

AgNO3 (mmol.L-1)

[Tryptophan/ AgNO3]

Xe-lamp illumination times (min)

11.75

-

-

1

12.38

0.83

14.92

1

12.24

3.41

3.59

1

12.03

5.41

2.22

1

12.10

11.77

1.08

1

12.41

23.72

0.52

1

12.11

47.34

0.25

1

[Tryptophan/ AgNO3]

Xe-lamp illumination times (min)

Table 2: Study of Xe exposure time. Tryptophan (mmol.L-1)

AgNO3 (mmol.L-1)

12.03

5.41

2.22

30’’

12.03

5.41

2.22

1’

12.03

5.41

2.22

3’

12.03

5.41

2.22

5’

12.03

5.41

2.22

10’

12.03

5.41

2.22

15’

12.03

5.41

2.22

12

A

B

Figure 1. Color of a silver colloidal suspension before (A) and after (B) illumination with white light from a Xe-lamp for 5 minutes.

13

1.5

1' Xe [Ag] 47.34 mM [Ag] 23.72 mM [Ag] 11.77 mM [Ag] 05.41 mM [Ag] 03.41 mM [Ag] 00.83 mM

Optical Density

450 nm

1.0

0.5

300

400

500

600

700

800

Wavelength (nm)

Figure 2. Optical absorption spectra of silver nanoparticles formed with illumination for 1 minute by a Xe lamp as a function of the silver concentration.

14

[Ag] = 5.41 mM

2.5

15' Xe (dilution: 50%) 10' Xe (dilution: 50%) 05' Xe (dilution: 50%) 03' Xe (dilution: 50%) 01' Xe (without dilution: 100%) 30'' Xe (without dilution: 100%) Without Xe (without dilution: 100%)

Optical Density

2.0

1.5

1.0

0.5 300

400

500

600

700

800

Wavelenght (nm)

Figure 3. Optical absorption spectra of silver nanoparticles produced using different Xe-lamp light illumination times.

15

exc = 280 nm

[Ag] 00.00 mM [Ag] 00.83 mM [Ag] 03.41 mM [Ag] 05.41 mM [Ag] 11.77 mM [Ag] 23.72 mM [Ag] 47.34 mM

Intensity x 105(CPS)

0.02

0.01

0.00 300

350

400

450

500

550

Wavelength (nm)

Figure 4. Tryptophan fluorescence spectra obtained under excitation at 280 nm in function of silver concentration.

16

Particle size

Frequency

Average: 16.65 nm D.P.: 8.28 nm

10

A

20

30

Diameter (nm)

B

C

Figure 5. (A and B) TEM images of synthesized Tnnps (5.41 mM, 1 min Xe). (C) The average sizes of the nanoparticles.

17

40

20 Model: Boltzmann y = A2 + (A1-A2)/(1 + exp((x-x0)/dx)) Chi^2/DoF = 9.06211 R^2 = 0.99776 A1 24.95994 ±2.00513 A2 -35.29881 ±0.74267 x0 3.62827 ±0.06336 dx 0.82544 ±0.07418

Zeta Potential /mV

10 0 -10 -20 -30 -40 -50 2

4

6

8

10

12

pH

Figure 6. Zeta potential of Tnnp (5.41 mM, 1 min Xe) as a function of pH.

18

Figure 7: Growth inhibition versus AgNP (5.41 mM, 1 min Xe, pH = 7.0) as shown in a concentration graph: the broth microdilution method, read after 24 hours of incubation.

Figure 8: Growth inhibition versus AgNP (5.41 mM, 1 min Xe, pH=7.0) as shown in a concentration graph: the broth microdilution method, read after 48 hours of incubation.

Figure 9: Growth inhibition versus AgNP (5.41 mM, 1 min Xe, pH=7.0) as shown in a concentration graph: the broth microdilution method, read after 72 hours of incubation.

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Highlights ► Synthesis of spherical silver nanoparticles using tryptophan. ► The reduction of silver ions can be controlled through the control of the exposure time. ► Fluorescence can be observed. ► For the concentrations >25mg/mL the growth inhibition continued to be extremely effective for the tested bacteria species.

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Keywords: Tryptophan; silver nanoparticles; photoreduction; MIC; bacteria.

A simple and effective method to synthesize fluorescent nanoparticles using tryptophan and light and their lethal effect against bacteria.

A simple, environmentally friendly and cost-effective method was used to synthesize silver nanoparticles using tryptophan and light. To prepare the na...
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