alexa Detection of Pathogenic Escherichia coli (E. coli) Using Robust Silver and Gold Nanoparticles | OMICS International
ISSN: 2157-7048
Journal of Chemical Engineering & Process Technology
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Detection of Pathogenic Escherichia coli (E. coli) Using Robust Silver and Gold Nanoparticles

Boken J1*, Dalela S2, Sharma CK3 and Kumar D4
1Department of Physics, Banasthali Vidyapith, Rajasthan, India
2Department of Pure and Applied Physics, University of Kota, Kota, Rajasthan, India
3Assistant Professor, Department of Bioscience & Biotechnology, Banasthali University, Rajasthan, India
4Department of Chemistry, Banasthali Vidyapith, Rajasthan, India
Corresponding Author : Boken J
1Department of Physics, Banasthali University, Banasthali
Rajasthan-304022, India
Tel: +91-992810802
E-mail:[email protected]
Received July 23, 2013; Accepted September 27, 2013; Published September 30, 2013
Citation: Boken J, Dalela S, Sharma CK, Kumar D (2013) Detection of Pathogenic Escherichia coli (E.coli) Using Robust Silver and Gold Nanoparticles. J Chem Eng Process Technol 4: 175. doi:10.4172/2157-7048.1000175
Copyright: © 2013 Boken J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

The paper deals with differently synthesized robust metal nanoparticles using chemical synthesis approach for detection of a new pathogenic strain E. coli in water sample. The reactions have been completed using aqueous metal salt solution and trisodium citrate as a capping agent to obtain nanoparticles. The nanoparticles and their interaction against microorganisms have been characterized using UV-Vis spectroscopy and Transmission Electron Microscopy (TEM). For different concentration of sodium borohydride, surface plasmon resonance peaks have been obtained at 390 nm to 402 nm for silver nanoparticles and 518 nm to 524 nm for gold nanoparticles. It has been observed to be shifted from 520 nm to 527 nm and 395 nm to 408 nm for gold and silver nanoparticles after the interaction with E. coli respectively. Silver and gold nanoparticles have been synthesized at 80°C exhibited strong surface plasmon resonance peaks respectively at 433 nm and 529 nm. Remarkable shift have been noticed in surface plasma resonance peaks for small amount of E. coli added in the solution.

Keywords
E. coli strain of JB-26; Metal Nanoparticles; Silver and Gold nanoparticles; Surface Plasmon Resonance
Introduction
Escherichia coli (E. coli) have been the members of a large group of bacterial germs that inhabit the intestinal microorganism of humans and other warm blooded animals. The presence of E. coli in water is strong indication of sewage or animal waste contamination and as few as 10 cells can cause serious human illness and even death. That’s why presence of E. coli in foodstuffs and drinking water is a chronic worldwide problem. The world wide food production industry is worth about billion and the demand for economic biosensors to detect waterborne pathogens and pollutants in food stuffs, growing day by day [1]. Recently, metal nanoparticles as antimicrobial properties have been of great interest because of their unique optical and electrical properties and reported to have excellent prospects on biological and chemical sensing [2]. It has been estimated that due to bacterial cause thousands of food borne illness and hundreds of hospitalized and deaths taken place every year. It has been very important to monitor pathogenic E. coli strain and stop their growth using simple methods [3]. Mostly, water quantity standards have been set at 103 coliforms per 100 mL and then health goal for total coliforms at zero for drinking water [4]. In addition, human population growth and climate change are expected to increase in the number of species as well as concentrations of waterborne pathogens in surface water [5].
Bionanotechnology has emerged up as integration between biotechnology and nanotechnology for developing environment friendly and biosynthetic technology for preparation of nanomaterials [6]. The importance of nanomaterials for science and technology has been highly increased in past years. The metallic nanoparticles have become more attractive because of their fascinating properties such as optical, physicochemical, electronic and photonic etc. properties due to their high surface area to volume ratio. Mostly the properties have been affected by their size, morphology and size distribution of nanoparticles. Nanoparticles have been synthesized by growing, shaping or assembling the materials by physical, chemical and biological methods.
Nowadays, silver has been currently used to control bacterial growth in a variety of applications. In fact, it is well known that silver ions and silver based compounds are highly toxic to microorganism which shows strong biocidal effects on as many species of bacteria including E. coli [7]. The effect of silver ions on bacteria has been observed by the structural and morphological changes. Silver ions and silver salts have been used or decades as antimicrobial agents in various fields because of their growth inhibitory capacity against microorganisms. Silver nanoparticles have been synthesized by various methods such as wet chemical method, electrochemical [8], photochemical [9], laser ablations [10], γ-radiations method [11] etc. Silver nanoparticles have been synthesized through reduction of silver nitrate by reduction of sodium borohydride along with the stabilizing agents in aqueous solution. Stabilizing agent has been used for protecting the growth of nanoparticles by aggregation [12]. Silver nanoparticles have been prepared by chemical reduction method in which silver nitrate is reduced by sodium borohydride in aqueous medium at room temperature [13]. Although various methods have been developed to synthesize stable and monodispersed particles but chemical route method has been found to be most suitable as it is easy, versatile and economical. By controlling the reaction parameters such as temperature, pH, stabilizing agents, reactant concentration, oxidizing or reducing agents etc, variety of particles of different shape and size have been produced. Now a day using wet chemical method, it have been become possible to produce not only spherical metal nanoparticles and nanoshells but also nanodisks, multipods, triangular nanoprisms etc. When spherical nanoparticles have been transformed into one of these shapes the surface plasmon resonance has been strongly affected [14].
Gold nanoparticles have been employed in multiple application involving biological systems, it has remarkable binding properties, which makes it attractive for attaching ligands to enhance various bimolecular interactions [15]. It has been well known that inorganic nanoparticles can act as antifungal and antibacterial agents and thus have the ability to interact with microorganisms [16]. Due to its various shapes and sizes, it has been very difficult to predict the positive and negative effects and its mode of action in environment and within living microorganisms. A very small size of nanoparticles has been reported to modify the physiochemical properties of materials which can lead to adverse biological effects on living cells [17]. In our present study, simple methods for the detection of E. coli and its interaction with gold and silver nanoparticles have been presented. To understand the interaction of E. coli with silver and gold nanoparticles UV-VISNIR spectroscopy and TEM have been used to discuss the surface morphology and optical properties of the solutions.
Materials and Methods
Materials
Silver nitrate (AgNO3), trisodium citrate (Na3C6H5O7), sodium borohydride (NaBH4), tetrachloroauric acid (HAuCl4) of Sigma Aldrich make have been used for synthesizing silver and gold nanoparticles. All the solutions have been freshly prepared for the synthesis of nanoparticles especially NaBH4 aqueous solution that has been ice bathed before use. Distilled water passed through a Millipore system (resistivity = 18.2 Ω) has been used for all the solution preparation and throughout the experiments. All glassware has been first rinsed with aqua regia solution and then thoroughly with distilled water, which is followed by Millipore water.
For identification of E. coli stain present in the ground water of near Banasthali area, water sample has been sent to Imtech, Chandigarh and finally we have obtained pure culture of E. coli for sensing purposes.
Synthesis of robust metal nanoparticles at room temperature
For the fabrication of silver nanoparticles sodium borohydride and trisodium citrate have been used as reducing and capping agent respectively. In the synthesis of silver nanoparticles without heating method, silver nitrate solution (0.2 mM in 10 mL), trisodium citrate (2 mM in 10 mL) have been used as a metal precursor and stabilizing agent, respectively. Sodium borohydride solution (0.1 M) in 0.1 mL to 1 mL of water (to obtain different samples) have been added drop wise to the above silver nitrate solution and further stirred at 15 min. As soon as the freshly prepared sodium borohydride have been added in the solution, the colour of solution has been turned light yellow to golden yellow. The final solution has been observed to change in its colour from light yellow to golden yellow colour while increasing the concentration of sodium borohydride solution. All the reactions have been carried out at room temperature.
Similarly, the gold nanoparticles have been synthesized by adding trisodium citrate (10 mM in 10 mL) in tetrachloroauric acid (2 mM in 10 mL) solution and further stirred for 15 min. After 15 min sodium borohydride (1 mM) solution in 1 mL to 10 mL of water have been added drop wise in the chloroauric acid solution to obtain gold nanoparticles. The final solution has been turned from light ruby red to dark ruby red in colour with increased concentration of sodium borohydride.
Synthesis of robust metal nanoparticles at 80°C
A typical synthesis procedure has been used to obtain silver and gold nanoparticles using trisodium citrate as capping as well as stabilizing agent. In this method, silver nitrate (2 mM in 50 mL), which have been heated at 80°C for 15 min then trisodium citrate (20 mM in 50 mL) have been added to precursor solution and resulting mixtures refluxed at 80°C for 30 min. After 15 min, initially clear solution of silver nitrate has been observed to be turned pale yellow and then brownish yellow in colour, which indicate the formation of silver nanoparticles. Similarly, the synthesis of gold nanoparticles have been performed using tetrachloroauric acid (2 mM in 50 mL), which has been heated at 80°C for 15 min then trisodium citrate (10 mM in 50 mL) has been added to this solution, which finally refluxed at 80°C for 30 min. After 15 min, initially clear solution of chloroauric acid has been found to turn light red, which indicate the formation of gold nanoparticles.
Characterization Techniques
The particle size and surface morphology of the nanoparticles have been examined using TEM of TECNAI make G20 operated at an accelerating voltage of 220 keV. All the samples for TEM have been deposited on carbon coated copper grid by placing the drops of diluted samples of gold and silver. Optical absorption measurements have been performed using a Perkin-Elmer Lambda 750 UV-VIS-NIR spectrophotometer with pre-aligned tungsten, halogen and deuterium sources. The resolution of the spectrophotometer is 0.17-5.00 nm for UV-VIS and 0.20-20.00 nm for NIR.
Results
Silver and gold nanoparticles have been synthesized by wet chemical method using trisodium citrate as well as sodium borohydride at different reaction temperatures. It is well known from the literature that trisodium citrate merely serves as a capping agent because it is weak reducing agent and cannot perform reduction at room temperature. Sodium borohydride being a strong reducing agent, reaction takes place almost instantly and very small particles are obtained [18]. Reduction using sodium borohydride has been done at room temperature while trisodium citrate needs higher temperature ~80°C for the reduction. The sizes and shapes of the nanoparticles have been tuned, selecting the reducing and capping agents. NaBH4 acts as a strong reducing agent and trisodium citrate merely serve as surface passivating agent that’s why it is good capping agent.
A simple mechanism for the formation of gold nanoparticles using a metal precursor which is given below:
AgNO3→ Ag+ + NO-3
Na3C6H5O7→3Na++C6H5O73-
HAuCl4+Na3C6H5O7→3Au++C6H5O7-+6NaCl+3Cl2
Similarly, the chemical reactions of the precursor leading to the simply formation of silver nanoparticles which is given below: AgNO3→Ag++NO3-
Na3C5H5O7→3Na++C6H5O37-
3AgNO3 + Na3C6H5O7→3Ag++C6H5O7-+ 3NaNO3
Simple mechanisms of silver and gold nanoparticles formation are clear from the above equations. When silver nitrate is dissolved in water and it breaks in Ag+ and NO3- whereas tri sodium citrate breaks into Na+ and C6H5O7-. While silver particles nucleate, the negatively charged citrate ions from a layer around the nucleation site and act as a capping agent for controlling the shape and size of the particles. At lower concentration of borohydride, formation of silver nuclei will be low so silver nuclei can be combine with other silver ions to from large particles in the solution mixture. On the other hand at higher concentration of borohydride, rate of formation of silver nuclei will be higher and nuclei growth will be low which leads to the smaller particles. Similarly this mechanism is valid for gold nanoparticles.
Optical properties of silver and gold nanoparticles and its correlation with NaBH4 concentration:
In Figure 1, UV-Vis absorption spectra of particles (a) gold and (b) silver nanoparticles have been shown. All the spectra show prominent surface plasmon resonance peaks, characteristics of spherical nanoparticles at 529 nm for gold and 433 nm for silver nanoparticles, as shown in Figure 1a and 1b. In silver nanoparticles, the absorption spectrum is showing a hump at 322 nm along with the intense peak at 433 nm. The presence of hump at lower wavelength may owe its presence either due to origin of higher order multipole modes or due to incomplete reduction of silver ions.
Wine red colour of colloidal solution of gold nanoparticles is due to strong absorption of gold nanoparticles. Peak is arising due to absorption of energy via the collective oscillations of free electrons (dipolar plasmon) and interband transitions (HOCO to LOCO). Surface plasmon resonance gets coupled when the gap between the particles decreases.
The optical absorption spectra have been also recorded for the solution, which have been obtained gold and silver nanoparticles to study the interaction between metal nanoparticles and E. coli. In the absorption spectra one can clearly observe a tendency of particle size increment with the increase in concentration of E. coli.
It is clear from the Figure 2a that a red shift can be seen for absorption peak recorded for the silver nanoparticles after having interaction with different concentrations of E. coli.
Similarly, for the gold nanoparticles red shift is also occurred as shown in Figure 2b. As the size of the gold nanoparticles increases, light can no longer polarize the nanoparticles homogeneously, and higher order modes at lower energy dominate. This causes a red shift, broadening and with less amplitude of the surface plasmon band. Therefore, small gold nanoparticles aggregates will make their surface plasmon combine and result in the color change from red to purple.
Hence we can say that the surface plasmon resonance plays an important role in the determination of optical absorption spectra of metal nanoparticles which shifts to a longer wavelength with increase in particles size. More and more particles begin to aggregate due to which HUMO-LUMO gap between the particles decreases. Less energy is required to excite the electron which leads to red shift in surface plasmon resonance wavelength. So red shift and broadening were due to both surface plasmon coupling and aggregation between closely spaced nanocomposites.
If the naked clusters are in contact with one another agglomerisation occurs, particle size increases and quantum properties are lost. All the HOCO’s merge, as do all LOCO’s to form band structure.
However, TEM image of silver nanoparticles show the size of particles are less than 10 nm as can be seen from Figure 3. For this size of particles, higher order plasmon modes cannot be expected. In Figure 4, a TEM image represents the size of gold nanoparticles before and after the interaction of E. coli in the solution. Here we can see that the particles size have been increased after the interaction of E. coli.
Gold and silver nanoparticles have been synthesized using NaBH4 in aqueous medium. Since it is a strong reducing agent and reaction proceeds at room temperature. The concentration of NaBH4 solution has been varying from 1 mL to 10 mL for the gold nanoparticles and 0.1 mL to 1 mL for silver nanoparticles synthesis to examine the effect of borohydride concentration on the resulting particles size. After the interaction of E. coli, we can see the particles size increased. This can be concluded from a continuous red shift in the absorption spectra having increasing concentration of borohydride solution.
All the spectra show prominent surface plasmon resonance peaks, characteristics of spherical nanoparticles in the 518 nm to 524 nm ranges for gold nanoparticles and 390 nm to 402 nm for silver nanoparticles respectively. We clearly observed a tendency to form a larger particle size with increasing concentration of NaBH4 in the reaction, which could be concluded from a continuous red shift in the absorption spectra with an increasing NaBH4 concentration. The size of gold and silver nanoparticles can be tuned by the NaBH4 concentration within certain range.
The absorption spectra in Figure 5a show the prominent surface plasmon resonance peaks, characteristics of nanoparticles in 518 nm to 524 nm ranges for gold nanoparticles with NaBH4 (different concentration of water e.g. 3 mL, 5 mL and 10 mL) and for the same concentration of borohydride the surface plasmon resonance peaks shifted at 520 nm to 527 nm after the interaction of E. coli as shown in Figure 5b.
We also obtained the absorption spectra for silver nanoparticles at 390 nm without interaction and with interaction of E. coli the peak shifted at 395 nm in Figure 6a. Similarly for another concentration of NaBH4 we obtained in Figure 6b, the absorption peak at 402 nm for silver nanoparticles and after the interaction of E. coli the peak also shifted at 408 nm. This shows that as we increase the concentration of NaBH4 in the solution then absorption peaks of silver will be increased, hence the red shift will occurs.
One more interesting result we would like to quote that after interaction with E. coli bacteria the surface plasmon resonance peaks shifted at 520 nm to 527 nm for gold nanoparticles, which may be ascribed due to particle size increase with interaction of E. coli with nanoparticles and it can be expected that a large number of E. coli bacteria coagulate around silver and gold nanoparticles and cluster formation can be expected. To confirm the effect of silver and gold nanoparticles, a comparative study of silver and gold nanoparticles activity against E. coli bacteria were performed. In case of silver nanoparticles, surface plasmon resonance peaks shifted at 395 nm to 408 nm after interaction with E. coli.
TEM images of silver and gold nanoparticles show with and without interaction of E. coli in the solution are as shown in Figures 7 and 8.
In Figures 3b, 4b and 8b, represents the TEM images of nanoparticles show the cluster formation after the interaction of E. coli bacteria, it may be due the size of the nanoparticles has large surface area to come in contact with the bacterial cell. Hence, it will have a higher percentage of the interaction than larger size particles. The smaller nanoparticles interact with bacteria and produce electronic effects which enhance the reactivity of nanoparticles. Finally we can conclude that the bacterial effect of nanoparticles is size dependent and which can be used to determine the presence of the E. coli bacteria in the solution.
Conclusions
Gold and silver nanoparticles have been synthesized by wet chemical method using tri sodium citrate and sodium borohydride. Tri sodium citrate cannot perform reduction at room temperature because it is weak reducing agent so that it needs higher temperature (~ 80°C) for the reduction. While increasing the concentration of sodium borohydride in the reactant, continuous red shifts have been observed in the absorption spectra. The size of gold and silver nanoparticles could be tuned by the borohydride concentration within certain range. After the interaction of E. coli, the size of particles has been found to increase with increasing in the concentration of sodium borohydride. The size of the nanoparticles implies that it has a large surface area to come in contact with the bacterial cells. Hence, it will have a higher percentage of the interaction than bigger particles. Thus we can say that the bacterial effect of silver nanoparticles and gold nanoparticles are size dependent and can be used for detection of bacterial impurities in the water.
Acknowledgement
One of the authors (J. Boken) is thankful to Department of Science and Technology (DST), New Delhi for financial assistance vide grant no. F.No.SR/ WOS-A/PS-35/2010. Authors are also thankful to Vice Chancellor of Banasthali Vidyapith for extending the facilities of “Banasthali Centre for Education and Research in Basic Sciences” sanctioned under CURIE programme of the DST, New Delhi. The authors would also like to thank AIIMS (All India Institute of Medical Sciences, New Delhi) for providing TEM facility.
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