Langmuir 2008, 24, 11995-12000
11995
Rapid Estimation of Bacteria by a Fluorescent Gold Nanoparticle-Polythiophene Composite Biswa Ranjan Panda,† Atul Kumar Singh,‡ Aiyagari Ramesh,*,‡ and Arun Chattopadhyay*,†,§ Department of Chemistry, Department of Biotechnology, and Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati 781 039, Assam, India ReceiVed July 9, 2008. ReVised Manuscript ReceiVed August 6, 2008 Herein we present a facile method for rapid quantitation of bacterial cells over several logarithmic dilutions. The quantitation is based on loss of the fluorescence intensity of a positively charged Au nanoparticle-polythiophene composite in the presence of bacterial cells. The present method allowed estimation of both Gram-positive and Gram-negative bacteria with cells as low as 1000. Transmission electron microscopic investigations revealed attachment of the composite with bacteria with no discernible change in the morphology of the cells. Further, dynamic light scattering experiments indicated preferential attachment of smaller composite particles over larger ones, which were also attached at higher bacterial concentrations. The ease of operation with minimal sample manipulation steps, high sensitivity, quantitative detection, and its generality offer specific advantages over conventional methods.
Introduction Bacterial contamination is a major health hazard especially in the context of food safety, environmental monitoring, and the pharmaceutical industry. It assumes even greater significance in case of pathogens as the presence of even a single cell may lead to serious health risk.1 Thus, rapid quantification of bacteria is imperative for clinical diagnosis, food safety, therapeutic strategies, and for reducing potential infections. Conventional microbiological methods for estimating low numbers of bacterial cells mostly encompass enrichment of the target bacteria in the sample.2 Most of these methods are arduous, time-consuming, and require prior knowledge of the bacterial species to select an appropriate growth medium. On the contrary, molecular methods based on biorecognition events such as immunoassays and polymerase chain reaction are capable of achieving high sensitivity and specificity, circumventing additional culturing of bacterial cells.3,4 However, like the conventional microbiological techniques, a prior knowledge of appropriate surface antigens and target genes is required to detect the bacterial cells. Recently, many attempts have been made to augment the sensitivity of bacterial estimation without the need for target amplification and enrichment. A number of immunoassays have been developed based on bioconjugated nanoparticles, kinetic exclusion assays and on-chip microfluidic platforms.5-7 However, the majority of these methods require sophisticated instrumentation, which makes the process cost prohibitive. Ideally, analytical * Corresponding authors. E-mail:
[email protected] (A.R.); arun@ iitg.ernet.in (A.C.). † Department of Chemistry. ‡ Department of Biotechnology. § Centre for Nanotechnology.
(1) Nataro, J. P.; Kaper, J. B. Clin. Microbiol. ReV. 1998, 11, 142–201. (2) Deisingh, A. K.; Thompson, M. J. Appl. Microbiol. 2004, 96, 419–429. (3) Geng, T.; Morgan, M. T.; Bhunia, A. K. Appl. EnViron. Microbiol. 2004, 70, 6138–6146. (4) Matsuda, K.; Tsuji, H.; Asahara, T.; Kado, Y.; Nomoto, K. Appl. EnViron. Microbiol. 2007, 73, 32–39. (5) Zhao, X.; Hilliard, L. R.; Mechery, S. J.; Wang, Y.; Bagwe, R. P.; Jin, S.; Tan, W. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15027–15032. (6) Su, F.; Endo, Y.; Saiki, H.; Xing, X.; Ohmura, N. Biosens. Bioelectron. 2007, 22, 2500–2507. (7) Boehm, D. A.; Gottlieb, P. A.; Hua, S. Z. Sens. Actuators, B 2007, 126, 508–514.
tools meant for estimation of bacterial cells should be designed to achieve rapid and high sensitivity with minimal sample manipulations. A reasonable solution from the point of sensitivity and speed is the use of fluorescence, which is noninvasive, with an obvious advantage of zero background interference and is hence very sensitive toward its microenvironment. The advent of nanoscale science and technology in recent years has witnessed significant application of nanomaterials in biodiagnostics and therapeutics. For example, functionalized inorganic nanomaterials such as quantum dots and Au nanoparticles (NPs) have been used for detection and quantification of DNA, RNA, and proteins.8-11 These materials are also being used for development of sensors for glucose and other biomolecules.12,13 Further, Ag NPs as well as a composite of Ag NPs and chitosan have been used as bactericides.14 Interestingly, there is no report of use of either metal NPs, quantum dots, or their polymer composites in sensing and estimation of bacteria. This is important considering the sensitivity and rapidity that these materials could provide in the quantification of bacteria present in a medium. In the present article, we describe a new method for bacterial quantification based on reduction of the fluorescence emission intensity of a polymer nanocomposite by the bacterial cells. The composite consists of Au NPs embedded in polythiophene, which has been synthesized in our laboratory recently.15 The salient features of the method include (1) its ease of operation with minimal sample manipulation steps, (2) high sensitivity and quantitative estimation over several logarithmic dilutions, and (3) the generality in detection of different bacterial cells. Our results show that the composite could estimate as low as 1000 (8) Han, M. S.; Lytton-Jean, A. K. R.; Oh, B.; Heo, J.; Mirkin, C. A. Angew. Chem., Int. Ed. 2006, 45, 1807–1810. (9) Li, H.; Rothberg, L. Anal. Chem. 2005, 77, 6229–6233. (10) Liu, J.; Lu, Y. Angew. Chem., Int. Ed 2006, 45, 90–94. (11) Fortina, P.; Kricka, L. J.; Surrey, S.; Grodzinski, P. Trends Biotechnol. 2005, 23, 168–173. (12) Majumdar, G.; Goswami, M.; Sarma, T. K.; Paul, A.; Chattopadhyay, A. Langmuir 2005, 21, 1663–1667. (13) Zayats, M.; Baron, R.; Popov, I.; Willner, I. Nano Lett. 2005, 5, 21. (14) Gogoi, S. K.; Gopinath, P.; Paul, A.; Ramesh, A.; Ghosh, S. S.; Chattopadhyay, A. Langmuir 2006, 22, 9322–9328. (15) Panda, B. R.; Chattopadhyay, A. J. Colloid Interface Sci. 2007, 316, 962–967.
10.1021/la802171b CCC: $40.75 2008 American Chemical Society Published on Web 09/24/2008
11996 Langmuir, Vol. 24, No. 20, 2008
Panda et al.
bacterial cells suspended in saline solution. Transmission electron microscopy (TEM) measurements and dynamic light scattering based particle size analyses indicated that both Gram-positive and Gram-negative bacterial cells were attached to the composite in solution, leading to the loss of fluorescence.
Materials and Methods Chemicals and Growth Media. HAuCl4 (17 wt % solution of HAuCl4 in dilute HCl; 99.99%) and thiophene (99+%) were obtained from Sigma-Aldrich Chemicals, U.S.A. Nutrient broth (NB), brain-heart infusion (BHI), and de Man Rogosa and Sharpe (MRS) growth medium were purchased from HiMedia, Mumbai, India. Au Nanoparticle-Polythiophene Composite Preparation. A homogeneous mixture of thiophene (99+%, Sigma-Aldrich) of 30 mM strength was prepared by sonicating the requisite amount of thiophene in 10 mL of water at 35 kHz and room temperature (∼28 °C). This was followed by addition of 50 µL of 1.72 × 10-2 M HAuCl4 (prepared from 17 wt % solution of HAuCl4 in dilute HCl; 99.99%, Sigma-Aldrich, U.S.A.). The above mixture was incubated in a mechanical shaker operating at 100 rpm and 25 °C for a day. Initially, the solution was dark purple in color, which upon further shaking turned light yellow to a colorless solution of pH ∼ 3.0. In addition, a small amount of a dark precipitate was also obtained at the end of the reaction, which was not used herein. The solution was decanted and then used for the experiments with a series of diluted bacterial samples. The concentration of protonated species in the composite was considered as the concentration of the composite, the details of which are available in ref 15. Bacterial Strains. The bacterial strains used in the present investigation included Gram-positive strains of Pediococcus acidilactici CFR K7, Lactobacillus plantarum MTCC 1325, and Enterococcus faecalis MTCC 439 and Gram-negative strains of Escherichia coli MTCC 433, Enterobacter aerogenes MTCC 2822, and Pseudomonas aeruginosa MTCC 2488. The strains of lactic acid bacteria (LAB) were propagated in MRS medium under static condition at 37 °C. Ec. faecalis MTCC 439 was grown in BHI broth medium at 37 °C at 180 rpm for 12 h, whereas E. coli MTCC 433, Ps. aeruginosa MTCC 2488 and Eb. aerogenes MTCC 2822 were grown in NB medium at 37 °C at 180 rpm for 12 h. Fluorescence Measurements and Cell Viability Studies. Bacterial cultures were grown in the respective medium for 12 h as stated before. Cells were harvested from a 1.0 mL aliquot of the culture broth by centrifugation at 8000 rpm for 5 min. The cells were then washed twice with 0.85% saline solution to remove residual media components and then diluted 10, 50, 100, 500, 1000, 10 000, and 100 000 times (separately) in 0.85% saline solution. The bacterial cell suspensions and Au NP-polythiophene composite solution were mixed in a ratio of 2:1 (v/v), and the emission spectra of the samples were recorded in a fluorescence spectrophotometer (Cary Eclipse, Varian Inc., U.S.A.) following excitation at the wavelength of 345 nm. In another set of experiments, 105 CFU/mL bacterial cells (strain CFR K7 or MTCC 433) were mixed with varying concentrations of Au NP-polythiophene composite using variously diluted composite solutions. The fluorescence measurements were performed as stated before. The effect of polythiophene-Au NP composite on the viability of the bacterial cells was studied by incubating 106 CFU/mL bacterial cells (strain CFR K7 or MTCC 433) in the composite solution for 30 min and 1 and 3 h, respectively. The cell viability was determined for the treated samples by standard serial dilution method, and the percent loss in viability was calculated by comparing those with untreated bacterial cells, incubated in sterile saline solution for the same period of time. TEM Analysis. For TEM analysis, 5.0 µL aliquots of the Au NP-polythiophene composite, cell suspensions of strain P. acidilactici CFR K7 and E. coli MTCC 433, and a mixture of bacteriaAu NP-polythiophene composite were drop-cast on five separate carbon-coated copper TEM grids followed by air-drying. The bacterial cells were taken at a concentration of 105 CFU/mL. Images of the
Figure 1. Decrease in fluorescence intensity of the Au NP-polythiophene composite with increase in bacterial cell number in the samples: (A) for P. acidilactici CFR K7 (Gram-positive) and (B) for E. coli MTCC 433 (Gram-negative) cells. The concentrations of cells are mentioned in the legends.
samples were recorded in a TEM (JEM-2100, JEOL, Japan) operating at an accelerating voltage of 80 keV. Particle Size Analysis. Quantitative analysis of bacteria added Au NP-polythiophene composite was studied by dynamic light scattering based particle size analyzer (LB-550, Horiba, Japan). Ten milliliter saline suspensions of P. acidilactici CFR K7 cells in the range of 105 and 104 CFU/mL were mixed with Au NP-polythiophene composite solutions (2:1 v/v). The particle size distributions for polymer composite and bacterial cells, as well as their mixtures, were then analyzed using the above equipment.
Results and Discussion The polymer nanocomposite used herein comprised Au NPs and chlorinated polythiophene, which has been developed recently in our laboratory. The water dispersible composite exhibits intense fluorescence that depends on the pH of the solution. A single intense emission peak is obtained with the maximum occurring at 435 nm, when the polymer-nanocomposite solution at pH 3.0 is excited at 345 nm. This emission is due to the protonated form of the composite (AH+). On the other hand, when the pH of the solution is increased to 6.0, the emission spectrum consists of two weaker peaks occurring at 390 and 465 nm.15 These peaks are due to completely deprotonated form of the composite (A). At intermediate pH values, mixtures of the two species with concentrations corresponding to equilibria between the two forms exist. Further details of the composite and its fluorescence properties have been published elsewhere.15 Interestingly, we observed that the fluorescence of the composite was highly sensitive to the presence of bacteria in the medium and the change in fluorescence was maximum when the pH of the composite solution was kept at near 3.0. For example, when the emission intensity of a 2:1 mixture of the aqueous solution of the composite and the various diluted bacterial cell suspensions (with pH of the whole mixture being 3.75) were recorded, systematic decrease in the intensity was observed, with the increase in bacterial population in the medium. Typical representative results, corresponding to the change in fluorescence in the presence of P. acidilactici CFR K7 (Gram-positive) and E. coli MTCC 433 (Gram-negative) bacteria are shown in Figure 1. As is clear from the figure, the changes in fluorescence in the
Estimation of Bacteria by a Fluorescent Composite
Langmuir, Vol. 24, No. 20, 2008 11997
presence of both types of bacteria were similar and quantitative over several 10-fold dilutions of bacterial cells. The decrease in fluorescence intensity of the composite with increasing bacterial concentration provided an excellent opportunity for quantitative and rather accurate estimation of the bacterial concentration. It may be mentioned here that the absorbance at 345 nm (i.e., excitation wavelength) was not much affected by the presence of bacteria except at the highest bacterial concentration. For example, the absorbance was about 0.01 for the polymer-nanocomposite only as well as for the composite in presence of up to 105 CFU/mL of bacterial cell concentration. On the other hand, the value was observed to be 0.02 at 106 CFU/mL. Thus it may be considered that the fluorescence emission was not masked by the presence of bacteria and thus the decrease was due to interaction of the composite with the bacteria, which enabled the present method of quantitation of bacterial cells. We have accounted for the decrease in emission intensity in terms of the effective concentration of the species AH+ (i.e., the protonated composite) in the sample upon addition of bacterial cells. The relationship between the decrease in fluorescence intensity of the composite and bacterial cell concentration could be established from the well-known relationship between the intensity of fluorescence and the concentration of the fluorescent species and which is derived below. The dependence of intensity of steady-state fluorescence, Ifc, and the concentration of the species, c can be written as16
Icf ) κFλI0{1 - e-2.3εlc}
(1)
Here κ is a proportionality constant, Fλ is the steady-state fluorescence intensitysat the emission maximumsper absorbed photon at the excitation wavelength, I0 is the intensity of incident light, and ε is the molar extinction coefficient at the excitation wavelength. When the sample is excited at a fixed wavelength keeping all other optical configurations unchanged, the above equation can be further simplified into
Icf ) B{1 - e-2.3εlc}
(2)
where B ) κFλI0. For the pure sample, with concentration C0, eq 2 can be written as
ICf 0 ) B{1 - e-2.3εlC0}
(3)
In the presence of the bacterial cells, when the effective concentration of the fluorescent species decreases due to sequestration by bacterial cells, eq 3 can be written as
ICf ) B{1 - e-2.3εl(C0-x)}
(4)
where x is a function of bacterial cell number and has the dimension of concentration and c is the concentration of the remaining fluorophores unaffected by the presence of bacteria (c ) c0 - x). Equations 3 and 4 can be combined and approximated as
log(ICf 0 - ICf ) ) log D + 2.3εlx
(5)
where D ) Be-2.3εlC0. Interestingly, when the logarithm of difference in fluorescence intensities (at 435 nm) between that of the pure composite and that in the presence of different amounts of bacteria was plotted against the logarithm of the number of bacterial cells a linear (16) Valeur, B. In Molecular Fluorescence; Wiley-VCH: Weinheim, Germany, 2002; pp 50-51.
Figure 2. Decrease in fluorescence intensity (at 435 nm) of Au NP-polythiophene composite as a function of bacterial cell numbers: (A) treatment with P. acidilactici CFR K7; (B) treatment with Lactobacillus plantarum MTCC 1325; (C) treatment with Enterococcus faecalis MTCC 439; (D) treatment with E. coli MTCC 433; (E): Pseudomonas aeruginosa MTCC 2488; (F) Enterobacter aerogenes MTCC 2822.
relationship was obtained. This was true for all six types of bacteria comprising of Gram-positive strains, P. acidilactici CFR K7, L. plantarum MTCC 1325, Ec. faecalis MTCC 439, and Gram-negative strains, E. coli MTCC 433, Eb. aerogenes MTCC 2822, and Ps. aeruginosa MTCC 2488. The results are shown in Figure 2. Clearly, the linear relationship being valid for all the six types of bacteriasthree of which are Gram-positive and the other three being Gram-negative bacteriasdemonstrates the efficacy of the method in quantitative estimation of bacteria and the generality of the approach. On the other hand, when a calibration curve was obtained from the fluorescence of the composite alone at various dilutions,
11998 Langmuir, Vol. 24, No. 20, 2008
Panda et al.
Figure 3. Calibration curve indicating fluorescence intensity (at 435 nm)asafunctionofmolarconcentrationoftheAunanoparticle-polythiophene (AuNP-PTh) composite solution.
the emission intensity was linear with concentration indicating that mere dilution of the composite did not lead to any structural or fluorescence change of the species (Figure 3). Hence, the change in fluorescence was exclusively due to interaction between the composite and the bacteria. Presumably, there was no equilibrium between the bacterial cells and the composite as observed in the normal quenching process. Also important to mention here is that the lowest number of bacterial cells that can be estimated using the present scheme is 1000 (Figure 2), indicating the sensitivity of the method. Remarkably, the method allows estimation of logarithmic dilutions of bacterial cell numbers, which in the present case is on the order of 103-106 cells. Furthermore, on the basis of the above results eq 5 could be rewritten as
log10(ICf 0 - ICf ) ) log10 D + εlKb log10 Nb
(6)
Here, Kb is a phenomenological constant having the dimension of concentration and Nb is the number of bacteria present in the medium. From the slopes of the graphs in Figure 2, the values of Kb for the six bacterial species were found to be 4.20 × 10-3 ML-1 for CFR K7, 3.63 × 10-3 ML-1 for MTCC 1325, 4.41 × 10-3 ML-1 for MTCC 439, 4.00 × 10-3 ML-1 for MTCC 433, 2.82 × 10-3 ML-1 for MTCC 2488, and 4.04 × 10-3 ML-1 for MTCC 2822 species, respectively. The nearly equal values of Kb for all the species indicate that the interactions between the bacteria and the composite for all cases are similar and may depend primarily on the number of bacteria present in the medium. The quantitative nature of the interaction is startling and thus provides a good mean for their accurate estimation. It may be mentioned here that in all cases of measurements the bacteria were suspended in saline and then mixed with composite (pH ∼ 3.0) in a 2:1 ratio and the resultant pH of the mixture was ∼3.75. Since the working pH was acidic, it was pertinent to investigate the effect of the Au NP-polythiophene composite on the viability of bacterial cells. In order to pursue this objective, strain CFR K7 or MTCC 433 at a concentration of 106 CFU/mL was incubated with the composite for 30 min, and 1 and 3 h, respectively. The percent loss in viability was calculated by comparing the number of cells with untreated bacterial cells, incubated in sterile saline solution for the same period of time. The results indicated that there was no loss in viability for the bacterial cells until 30 min of incubation, whereas upon prolonged exposure there was significant loss of their viability. For example, there was 14.0% and 30% loss in the cell viability after 1 and 3 h, respectively, when incubated in the medium containing the composite. It is important to mention that during estimation of bacterial cells by the present method cells were mixed with the composite and fluorescence spectra were recorded immediately (in 5 min). Thus, the exposure time of the cells to the composite was minimal, and hence it can be assumed that there was no loss in the viability of cells during
Figure 4. Interaction of 105 CFU/mL bacterial cells of P. acidilactici CFR K7 strain with varying concentrations of the Au NP-polythiophene composite. The top graph (with square points) represents fluorescence of Au NP-polythiophene composite only; the bottom graph (with triangular points) represents fluorescence of the composite in the presence of bacteria (both measured at 435 nm).
the period of fluorescence measurement for estimation of bacterial cell numbers. Further experiments were carried out with different concentrations of the composite, while keeping the number of cells constant. For this 105 CFU/mL bacterial cells of P. acidilactici CFR K7 strain was treated with variously diluted composite solution and fluorescence spectra were recorded. As is shown in Figure 4, the difference in fluorescence intensity of the composite for treated and untreated samples was constant at all concentrations of the composite. In other words, the linearity of concentrationdependent fluorescence of the composite was maintained in the presence as well as absence of the bacteria. The results further indicate the quantitative nature of the interaction between the cells and the composite. Also, the presence of the bacteria did not change the fluorescence properties of the remaining composite, which were free in the medium and did not interact with the cells. Otherwise, the linearity of the concentration-dependent fluorescence would not have been followed. Further, the graphs also indicate that the concentration of the composite used herein was well above that needed for interaction with the bacteria and the remaining free composite with its fluorescence led to quantitative estimation of bacterial cells. The Au NP-polythiophene composite, at pH 3.0, is in the completely protonated form (AH+), and bacterial cell surfaces are known to be negatively charged possibly due to the presence of either teichoic acid in Gram-positive bacteria17 or the outer membrane lipopolysaccharide present in Gram-negative bacteria.18 It is plausible that the positively charged composite interacts with the negatively charged cell surfaces of the bacteria leading to the reduction of fluorescence intensity of the composite. This could be similar to the interaction between chitosansa known antimicrobial agentsand the bacteria where the cell surfaces of the bacteria get attached to the positively charged chitosan leading to annihilation of the bacteria.19 However, the role of Au NPs present in the composite in the attachment of the bacteria cannot be completely ruled out, for which further investigations are required. A schematic representation of the charge-based (17) Berry, V.; Gole, A.; Kundu, S.; Murphy, C. J.; Saraf, R. F. J. Am. Chem. Soc. 2005, 127, 17600–17601. (18) Wilson, W. W.; Wade, M. M.; Holman, S. C.; Champlin, F. R. J. Microbiol. Methods 2001, 43, 153–164. (19) Lin, C.-H.; Lin, J.-C.; Chen, C.-Y.; Cheng, C.-Y.; Lin, X.-Z.; Wu, J.-J. J. Appl. Polym. Sci. 2005, 97, 893–902.
Estimation of Bacteria by a Fluorescent Composite
Langmuir, Vol. 24, No. 20, 2008 11999
Scheme 1. Schematic Representation of Interaction between Positively Charged Composite and Bacteria Leading to Loss of Fluorescence
interaction between the composite and the bacterial cells leading to loss of total fluorescence is shown in Scheme 1. The interaction between the bacteria and the composite was further probed using TEM. Investigation of drop-coated composite on a TEM grid revealed the presence of small NPs of Au, with diameters in the range of 5-10 nm, interspersed uniformly in the polymers that were fibrilar in shape (Figure 5A). A micrograph of the bacteria P. acidilactici CFR K7 is shown in Figure 5B. When the bacteria-treated composite solution was similarly observed through TEM, then the cells of P. acidilactici CFR K7 (Figure 5C) or E. coli MTCC 433 (Figure 5D) being attached to the composite could be observed. The number of bacterial cells used for TEM measurements for all samples was 105 CFU/ mL. Interestingly, there was neither any indication of the bacteria being lysed nor was there any significant deformation of the bacteria upon attachment with the composite. The micrograph of P. acidilactici CFR K7 bacteria, as shown in Figure 5B, compares well with those attached to the composite. The TEM images evidently support the idea that the composite is strongly interacting with the bacterial cell wall. At a working pH of 3.75, the ionization of functional groups such as -COOHswhich may otherwise render negative charge to the bacterial cell wall at higher pHsare relatively suppressed. Hence, it may be plausible that in addition to the ionic interactions, multiple weak interactions originating from dipole-dipole, ion-dipole, and
Figure 6. Particle size distributions of Au NP-polythiophene composite (A); 105 CFU/mL cells of P. acidilactici CFR K7 (B); a mixture of the composite and bacterial cells (C and D). Bacterial cell numbers are 104 CFU/mL in C and 105 CFU/mL in D, respectively.
van der Waals interactions might lead to the adsorption of the composite onto the cell surface. Further, the quantitative attachment of the polymer species onto bacterial cell surface was ascertained by dynamic light scattering experiments wherein the particle size distributions of the polymer composite, bacterial cells alone, and a mixture of cells and the composite were analyzed (Figure 6). It is clear from Figure 6A that the majority of the composite particles were submicrometer in size with a large number of particles being in the range of 100-300 nm in size, whereas the bacterial cells displayed an average size of about 3-4 µm (Figure 6B). When the composite and the bacterial cells (104 CFU/mL) were mixed together the particle size distribution characteristic for the smallersized composite diminished indicating preferential attachment of the smaller size composite species to bacterial cells (Figure 6C). Also, the overall relative particle size distribution at above 3 µm increased indicating the formation of composite-bacteria particles, in addition to the presence of free composite particles. The propensity of this diminution of particle size distribution for the composite was even more pronounced when a higher number of bacterial cells (105 CFU/mL) were added to the composite solution (Figure 6D). Here, the relative number of particles above 4 µm was also significant. Collectively, the results clearly indicate a quantitative interaction between the polymer composite and the bacterial cells and demonstrate the preferential attachment of smaller size polymer species onto the bacterial cell surface. Thus, selection of smaller size Au NP-polythiophene composite can possibly enhance the sensitivity of bacterial quantitation. On the other hand, relatively larger size particles (composite) were also attached to the bacteria especially when the concentration of the bacteria was higher. However, the change in fluorescence intensity seems to be independent of the size distribution of the composite as several samples prepared under identical conditions produced small differences in particle size distribution and also that did not seem to affect the present way of estimating bacterial concentrations.
Conclusion
Figure 5. TEM images of Au NP-polythiophene composite (A), P. acidilactici CFR K7 (105 CFU/mL) (B), P. acidilactici CFR K7 (105 CFU/mL) strain mixed with the composite (C), and (D) is of 105 CFU/ mL E. coli MTCC 433 strain treated with the composite.
Sensing and quantification of bacterial cells is not only technologically challenging but also scientifically intriguing. Although it is important to find routine and facile methods of estimating bacteria in a laboratory or industrial setup, understanding the modus operandi of such methods is equally important to innovate and formulate sensing platforms, which would be amicable for automation and serve as the next-generation gold standards in rapid microbiological methods. In the present case, a new composite of monochlorinated Au NP-polythiophene
12000 Langmuir, Vol. 24, No. 20, 2008
that is highly fluorescent, while being dispersed in aqueous solution as small particles, has been found to be useful in measuring bacterial cells with high accuracy over a wide range of concentrations along with the ability to estimate reasonably smaller number of bacteria. The method allows quantification of different kinds of bacteria too, opening the possibility of wider applications. It is interesting to observe that interaction between the bacteria and the composite appears to be independent of size (molecular weight) of the polymer. This possibly indicates that the interaction of bacteria with the composite leads to the quenching of the fluorescence from the electronic excited state, which possibly is in units smaller than the size of the polymer. Also, the positively charged composite fluorophore may interact with the negatively charged cell surface thereby losing its fluorescence completely. The remaining fluorescence observed could be due to free composite that is not attached to the cells and thus provide a quantitative estimation of the number of
Panda et al.
bacterial cells. This is important in a sense that in future if systematic changes in the backbone of the polymer could be made, and thus the nature of emission as well as interaction could be controlled, then sensing of individual bacterial species as well as their detection in a quick and precise manner could be carried out. This requires a clear understanding of the interaction between the composite and the bacteria, which is one of our future goals. Acknowledgment. We thank the Department of Science and Technology (SR/S5/NM-01/2005 and 2/2/2005-S.F.), Department of Biotechnology (BT/PR9988/NNT/28/76/2007), and Council of Scientific and Industrial Research (01(2172)/07/EMR-II), Government of India for financial supports. We also thank CIF (IIT Guwahati) for help in measurements. LA802171B
