Colorimetric Detection and Fingerprinting of Bacteria by Glass

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Langmuir 2007, 23, 4682-4687

Colorimetric Detection and Fingerprinting of Bacteria by Glass-Supported Lipid/Polydiacetylene Films Yogesh Scindia, Liron Silbert, Roman Volinsky, Sofiya Kolusheva, and Raz Jelinek* Department of Chemistry and the Ilse Katz Center for Nanotechnology, Ben Gurion UniVersity, Beer SheVa, Israel 84105 ReceiVed December 14, 2006. In Final Form: February 7, 2007 Glass-supported films of lipids and polydiacetylene were applied for visual detection and colorimetric fingerprinting of bacteria. The sensor films comprise polydiacetylene domains serving as the chromatic reporter interspersed within lipid monolayers that function as a biomimetic membrane platform. The detection schemes are based on either visible blue-red transitions or fluorescence transformations of polydiacetylene, induced by amphiphilic molecules secreted by proliferating bacteria. An important feature of the new film platform is the feasibility of either naked-eye detection of bacteria or color analysis using conventional scanners. Furthermore, we find that the degrees of bacterially induced color transformations depend both on the bacterial strains examined and the lipid compositions of the films. Accordingly, bacterial fingerprinting can be achieved through pattern recognition obtained by recording the chromatic transformations in an array of lipid/PDA films having different lipid components.

Introduction The demand for new diagnostic and sensing technologies that can serve as alerts for bacterial contamination has significantly increased in recent years because of incidents of food poisoning, bioterrorism alerts, and anthrax scares. Numerous technologies for bacterial detection have been developed.1-5 There are, however, limitations to existing bacterial detection techniques as rapid and generic approaches. Specifically, many methods employed for pathogen sensing provide results after relatively long time spans (several hours to days in the case of culturebased methods).6 Other currently employed technologies often involve complex detection mechanisms that require specialized instrumentation, trained personnel, and the need for active operation (addition of reagents, initiation of chemical reactions, etc.), which overall do not make possible applications in settings other than laboratory environments.5,7 Furthermore, a prerequisite for many detection methods is a detailed understanding of the biochemical and structural properties of the bacterial species sought, limiting applications in the case of unknown pathogens or variants. Here we describe a bacterial detection platform based on Langmuir-Schaeffer films containing lipids and polydiacetylene (PDA). PDA has attracted significant scientific and technological interest in recent years because of its unique chromatic properties. Specifically, PDA was shown to self-assemble into organized vesicles and films, forming an ene-yne conjugated framework that absorbs light in the visible region of the electromagnetic spectrum and consequently appearing intensely blue.8,9 Fur* To whom correspondence should be addressed. E-mail: [email protected]. (1) Hobson, N. S.; Tothill, I.; Turner, A. P. F. Biosens. Bioelectron. 1997, 61, 279-286. (2) Deisingh, A. K.; Thompson, M. Analyst 2002, 127, 567-581. (3) Perkins, E.; Squirrell, D. Biosens. Bioelectron. 2000, 14, 853-859. (4) DeMarco, D. R.; Lim, D. V. J. Rapid Methods Autom. Microbiol. 2001, 9, 241-257. (5) Gfeller, K. Y; Nugaeva, N; Hegner, M. Biosens. Bioelectron. 2005, 21, 528-533. (6) Ivnitski, D.; Abdel-Hamid, I.; Atanasov, P.; Wilkins, E. Biosens. Bioelectron. 1999, 4, 599-624. (7) Canhoto, O. F.; Magan, N. Biosens. Bioelectron. 2003, 18, 751-754. (8) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem. 1988, 27, 113-158. (9) Okada, S.; Peng, S.; Spevak, W.; Charych, D. Acc. Chem. Res. 1998, 31, 229-239.

thermore, it was shown that external perturbations, primarily affecting the reorganization of the pendent polymer side-chains as a result of enhanced surface pressure, give rise to stressinduced structural transformations of the PDA backbone, resulting in dramatic blue-red transitions.9 PDA also exhibits interesting fluorescence properties; no fluorescence is emitted by the initially polymerized blue-phase PDA, whereas the red-phase PDA strongly fluoresces at 560 and 640 nm.10 Several studies have demonstrated the construction of PDAbased vesicles for bacterial detection.11-13 Chromatic signals in these investigations were generated through the incorporation of recognition elements in the PDA matrixes, which bound to specific markers on the surface of bacterial cells. Recent reports have further shown that thin films of lipids and PDA can, on a simple level, mimic membrane surfaces allowing both the occurrence and consequent detection of membrane events induced by soluble biological analytes.14,15 The important feature of such systems in the context of biosensor development is the observation that the structural transformations of PDA (corresponding to bluered/fluorescence transformations) could be induced by amphiphilic biological analytes, essentially making the polymer a “built-in optical reporter” within the mixed lipid/PDA assemblies. The chromatic reactions induced in lipid/PDA films by amphiphilic membrane-active molecules are the primary phenomenon exploited in this work. The detection scheme is based upon bacterially secreted amphiphilic compounds that bind to the lipid/PDA film surface, thereby inducing chromatic transformations in PDA. We demonstrate that the chromatic changes are visible to the naked eye, can be recorded spectroscopically (UV-vis and fluorescence), or can be analyzed by conventional color scanning of the films combined with simple image analysis. Furthermore, we show that film arrays incorporating different (10) Takayoshi, K.; Masahiko, Y.; Shuji, O.; Hiro, M.; Hachiro, N. Chem. Phys. Lett. 1997, 267, 472-480. (11) Ma, Z.; Li, J.; Liu, M.; Cao, J.; Zou, Z.; Tu, J.; Jiang, L. J. Am. Chem. Soc. 1998, 120, 12678-12679. (12) Rangin, M.; Basu, A. J. Am. Chem. Soc. 2004, 126, 5038-5039. (13) Guangyu, M.; Cheng, Q. Langmuir 2005, 21, 6123-6126. (14) Volinsky, R.; Gaboriaud, F.; Berman, A.; Jelinek. R. J. Phys. Chem. B 2002, 106, 9231-9236. (15) Volinsky, R.; Kolusheva, S.; Berman, A.; Jelinek, R. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 9, 1393-1407.

10.1021/la0636208 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/20/2007

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lipid compositions makes bacterial fingerprinting possible through distinct color patterns corresponding to tested bacteria. Experimental Section Materials. 10,12-Tricosadiynoic acid (TRCDA) was purchased from GFS chemicals (Powell, OH) and was purified by dissolution in chloroform followed by filtration through a 0.45 µm nylon filter. The filtrate was solidified by evaporation of the solvent to obtain the purified form of TRCDA. Lipids 1,2-dimyristoyl-sn-glycero3-phosphocholine (DMPC), l-R-dioleoylphosphatidylethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), sphingomyelin, and cholesterol were purchased from Sigma. Chloroform (CHCl3) was HPLC grade (Frutarom Ltd). Ultrapure water used throughout this study had a resistivity of 18.3 MΩ obtained by using a Barnstead D7382 water purification system (Barnstead Thermolyne Corporation, IA). Preparation of Lipid/PDA Films. Film construction was described in detail in previous studies.14,15 Briefly, stock solutions (2 mM) of TRCDA and lipids were prepared by dissolving the appropriate amounts of material in CHCl3. TRCDA/lipid mole ratios in all films were 9:1, except in the TRCDA/sphingomyelin/cholesterol film where the mole ratio was 90:7:3. Lipid/TRCDA mixtures (50 µL) were spread on a water subphase (pH 6.5) within a computerized Langmuir trough (model 622/D1, Nima Technology Ltd, Coventry, UK). Following 20 min of equilibration, the film was compressed at a constant barrier speed of 10 Å2 molecule min-1. The equilibration time and compression rate outlined were found to produce optimal films. The compressed films were kept for 5 min at a constant surface pressure of between 16 and 22 mN/m, depending upon film composition. Following equilibration, the films were irradiated with 254 nm light for 10 s (1.8 mW/cm2) and transferred to a glass surface (cleaned thoroughly by placing in ethanol/water) by horizontally placing the slide on the film surface and lifting slowly. Bacterial Growth. Bacterial strains Salmonella seroVar typhimurium (strain CS093) 1a (provided by A. Porgador, Ben Gurion University), Bacillus cereus, E. coli K-12 strain C600 pMRInv, (provided by E. Gazit, Tel Aviv University), and E. coli XL1 (provided by D. Bar-Zvi, Ben Gurion University) were used in this study. The bacteria were grown aerobically at 37 °C on a sterilized solid Luria Bertani (LB) medium composed of 13.5% yeast extract, 27% peptone, 27% NaCl, and 32.5% agar at pH 7.4. The E. coli C600 pMRInv strain was grown in LB medium containing 30 µg/ mL kanamycin. After overnight growth, a colony from each bacterial strain was taken and added to 0.5 mL of sterilized growth medium consisting of peptone and NaCl. After suspension, two 200 µL aliquots were taken from this solution, and was each diluted to 1 mL using the growth medium. One of these solutions was used to monitor bacterial growth at the desired time points through measuring the concentration of the bacteria by UV-vis spectroscopy (600 nm). The other solution was used for chromatic film analysis. Specifically, at distinct time intervals 50 µL aliquots were extracted from the growth solution and centrifuged at 3700 rpm for 7 min, and the supernatant solutions were placed on the glass-supported films for colorimetric analysis. Ultraviolet-Visible (UV-Vis) and Fluorescence Spectroscopy. Spectra obtained from the glass-supported films were measured by mounting the films vertically in the beam of a Jasco V-550 spectrophotometer (Osaka, Japan) (UV-vis measurements) or a FL920 spectrofluorimeter (Edinburgh, UK) (fluorescence spectroscopy). In the fluorescence experiments, excitation was at 488 nm, and emission was recorded at 540 and 640 nm. Fluorescence Microscopy. Fluorescence images of the film were obtained by placing 20 µL of the bacterial growth supernatant on the film surface, followed by 30 min of incubation at 33 °C. After incubation, the films were washed with distilled water, dried gently with filter paper, and examined on an Olympus IX70 microscope (Japan) with a PlanFl20x/0.50 objective. Excitation was from 470 to 490 nm, and emission was at a wavelength >520 nm. Atomic Force Microscopy (AFM). AFM measurements were carried out under ambient conditions using a Dimension 3100 (Digital

Figure 1. Lipid/PDA film structure. AFM image of a section of a glass-supported DMPC/PDA film showing the bright elevated PDA domains within the DMPC background. The height profile (indicated by a straight line in the image) is depicted on the right, showing the expected height of a PDA trilayer. Instruments, CA) mounted on an active antivibration table. A 100 µm scanner and a microfabricated silicon oxide ultralever (NSC12[\]50 type Ultrasharp, Thermomicro) with intergrated pyramidal tips were used. The 512 pixel × 512 pixel images were taken in tapping mode with a scan size of up to 30 µm at a scan rate of 1 Hz. Digital Colorimetric Analysis (DCA). The colorimetric response of the sensor films was measured using colorimetric image analysis methodology developed in our laboratory.16 Briefly, following deposition of the bacterial supernatant solutions (15 µL) on the film surface and drying, the film was placed in a special film adaptor and scanned on an Epson Perfection 4990 Photo Scanner in transmission mode at an optical resolution of 2400 dpi and a color depth of 24 bits. Digital colorimetric analysis (DCA) was carried out by cropping the sample spots in the scanned images, and the color-change values were calculated using MATLAB mathematical software. Specifically, the extent of red intensity in each pixel can be calculated as the red chromaticity level (r)17 r)

R R+G+B

where R (red), G (green), and B (blue) are the three primary color components. For a defined surface area within a lipid/PDA film, we define a quantitative parameter denoted the red chromaticity shift (RCS) that represents the total blue-red transformations of the pixels encompassed in the area RCS )

rsample - r0 × 100% rmax - r0

in which rsample is the average red chromaticity level of all pixels in the scanned surface, r0 is the average red level calculated in a blank surface (blue film initially prepared prior to treatment), and rmax is the average red chromaticity level of the maximal blue-red transition, an area of the film in which the most pronounced blue-red transition was induced (positive control usually achieved by the addition of an organic solvent such as ethanol). In essence, RCS is the normalized change in the red chromaticity level within the film surface on which the tested sample was deposited.

Results and Discussion Chromatic Bacterial Sensing with Lipid/PDA Films. Solidsupported films comprising phospholipids and PDA have been shown to exhibit unique chromatic properties, undergoing visible blue-red transformations and fluorescence transitions in response to binding of varied biological analytes.14,15 Films were prepared by spreading the lipid/diacetylene mixtures at the air/water interface, followed by compression and equilibration, deposition onto glass substrates by the Langmuir-Schaeffer technique, and polymerization (Experimental Section). Figure 1 depicts a representative AFM image of a DMPC/PDA film. Importantly, (16) Volinsky, R.; Kolusheva, S.; Sheynis, T.; Kliger, M.; Levy, M.; Jelinek, R. Biosens. Bioelectron., 2006, in press. (17) Pratt, W. K. Digital Image Processing, 3rd ed.; Wiley-Interscience: New York, 2001.

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Figure 2. Bacterially induced color transformations on lipid/PDA films. (A) Scanned DOPE/PDA film prior to color change. (B) Scanned DOPE/PDA film after bacterially induced (E. coli XL1) color changes. Reddish spots appear within areas on which drops of supernatant solutions were placed, and extraction was carried out at the indicated times (hours) after the initiation of growth. LB corresponds to the growth media. The volume of each supernatant solution placed on the surface was 15 µL.

at the surface pressures in which the films were transferred onto the glass substrate, both lipids and the diacetylene molecules form distinct domains exhibiting different organizations (Figure 1). Specifically, the lipid molecules form a fluid monolayer, and the diacetylene monomers are compressed into rigid trilayer structures, confirmed by the height profile depicted in AFM analysis, which are dispersed within the phospholipid monolayer.18The trilayer organization reduces the relative surface area occupied by the PDA domains (Figure 1). The domain organization depicted in Figure 1 confers important structural and functional properties to the lipid/PDA as a biosensing platform. Specifically, the creation of separate diacetylene and phospholipid areas rather than a homogeneous mixture of the two components facilitates the polymerization of the diacetylene trilayers, which gives the film its distinctive blue appearance. The rigid PDA moieties further serve as “scaffolding”, imparting high stability to the lipid/PDA film even after exposure to ambient conditions for long periods of time. In addition, the lipid monolayer essentially constitutes a biomimetic membrane platform that is designed to promote the binding of biological analytes to the film surface. The thrust of the bacterial detection scheme described here is the induction of chromatic transformations in the lipid/PDA films upon film-surface interactions of bacteria and bacterially released amphiphilic substances. Figure 2 depicts scanned images of a glass-supported DOPE/PDA film before (Figure 2A) and after (Figure 2B) the deposition of supernatant solutions extracted at different times after the initiation of bacterial growth (E. coli XL1). Specifically, the scanned image in Figure 2B was recorded following the separation of the pellet (containing the bacterial particles) and supernatant (containing bacterially released substances within the growth media), placing small drops (15 µL) of the supernatant on the film surface, incubating at 33 °C for 30 min, and washing and drying the film surface. The image (18) Gaboriaud, F.; Golan, R.; Volinsky, R.; Berman, A.; Jelinek, R. Langmuir 2001, 17, 3651-3657. (19) Miller, M. B.; Bassler, B. L. Annu. ReV. Microbiol. 2001, 55, 165-99. (20) Thanassi, D. G.; Hultgren, S. J. Curr. Opin. Cell Biol. 2000, 12, 420430. (21) Batdorj, B.; Dalgalarrondo, M.; Choiset, Y.; Pedroche, J.; Metro, F.; Prevost, H.; Chobert, J. M.; Haertle, T. J. Appl. Microbiol. 2006, 101, 837-48. (22) Nikaido, H. and Vaara M. In Escherichia coli and Salmonella typhimurium; Neidhardt, F. C., Ed.; American Society for Microbiology: Washington, DC, 1987; pp 7-22. (23) Kolusheva, S.; Shahal, T.; Jelinek, R. Biochemistry 2000, 39, 1585115859. (24) Katz, M.; Tsubery, H.; Kolusheva, S.; Shames, A.; Fridkin, M.; Jelinek, R. Biochem. J. 2003, 375, 405-413.

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in Figure 2B clearly shows that blue-red transformations occurred on the film surface. In particular, Figure 2B demonstrates that more pronounced color changes were induced by suspensions that contained higher concentrations of bacteria (i.e., longer times after the beginning of growth). Importantly, similar color transitions were observed after placing on the films the total bacterial suspensions (without separation between bacterial particles and supernatant). However, the use of the bacterial supernatant rather than the total bacterial suspensions gave “cleaner” color transformations, reducing masking by bacterial particles attached to the film surface. Also, an analysis of the resuspended pellets (containing only the bacterial particles) gave rise to less pronounced colorimetric transformations (data not shown). Overall, the observation that supernatant solutions of the bacterial growth media induced color transformations on the lipid/PDA film (Figure 2) indicates that the color changes are most likely due to film-surface interactions of bacterially secreted membrane-active compounds. This conclusion is supported by a recent analysis of bacterially induced chromatic transformations in lipid/PDA vesicles.25 Indeed, the release of varied amphiphilic substances by bacteria into their environments is widely encountered in bacterial fauna. Bacterially secreted substances have been identified in processes that have essential functional roles, such as overcoming host-defense mechanisms, allowing colony proliferation, or facilitating bacterial communication.19,20 Bacteriocins, for example, are cytolytic peptides that induce membrane leakage and are released by wide variety of bacterial species.21 Secretion of pore-forming exotoxins is also abundant, and endotoxins such as lipopolysacharides, often released by gram-negative bacteria, strongly interact with membrane components of host cells.22 Importantly, previous studies have shown that lipid/PDA assemblies are highly sensitive to varied peptides and toxins and undergo chromatic transformations following the binding of such molecules.23,24 Specifically, the interactions of the secreted compounds with the lipid/PDA surface occur either through electrostatic attraction to the polymer surface and/or binding to and insertion of hydrophobic peptides and proteins into the biomimetic lipid layers within the films. Overall, these interactions give rise to the structural (and chromatic) transformations in polydiacetylene. The bacterially induced changes in the lipid/PDA films are also manifested spectroscopically (Figure 3). Figure 3A features UV-vis spectra of DOPE/PDA films placed in contact with supernatant solutions extracted at different times after the initiation of bacterial growth (S. typhimurium). Figure 3A demonstrates that the spectral component at around 650 nm progressively decreases while the signal at approximately 500 nm increases as the bacteria proliferate in the growth media. This spectral change corresponds to the blue-red transformation of the film. The blue-red changes in the lipid/PDA films following interactions with the bacterial supernatant solutions go hand in hand with the induction of fluorescence emission (Figure 3B). The spectra depicted in Figure 3B demonstrate that significantly higher fluorescence emission signals are recorded after supernatants extracted from solutions containing greater bacterial concentrations were placed on the film. The enhanced fluorescence emission is ascribed to the transition of the nonfluorescence blue PDA phase to the fluorescent red PDA, induced by bacterially secreted compounds present in the supernatant solutions. Fluorescence microscopy can also be applied to the detection of bacterially induced chromatic changes. The microscopy images (25) Silbert, L.; Ben Shlush, I.; Israel, E.; Porgador, A.; Kolusheva, S.; Jelinek, R. Appl. EnViron. Microbiol. 2006, 72, 7339-7344.

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Figure 3. Bacterially induced film transformations of DOPE/PDA film analyzed by visible spectroscopy and fluorescence. Experiments were carried out with supernatant solutions of S. typhimurium, extracted at different times after the initiation of growth. (A) UV-vis spectra. (B) Fluorescence emission (excitation at 488 nm). (C) Fluorescence microscopy images showing the borderline between the supernatant solution and the untreated film area (excitation at 488 nm, emission at wavelengths >520 nm). In all experiments, the following conditions applied: (i) only medium added, (ii) 1 × 107 bacterial cells in solution, (iii) 1 × 108 bacteria, and (iv) 1 × 109 bacteria.

in Figure 3C highlight the contrast between the bright (fluorescent) film surface onto which bacterial suspensions were added and the darker, nonfluorescent background. The fluorescence microscopy images that depict very small areas within the bacterially covered film surface demonstrate qualitatively that there is a clear difference between the control surface and the surface after the deposition of bacterial solutions. The use of fluorescence microscopy could facilitate the examination of a large number of samples at minute volumes. The bacterially induced chromatic transformations occur within minutes after the deposition of bacterial supernatants on the film. Figure 4 depicts the kinetic profile of the fluorescence transformation induced in a DOPE/PDA film by a supernatant extracted from a suspension containing 108 S. typhimurium bacterial particles. The curve in Figure 4 indicates that a significant fluorescence signal appears almost instantly after the addition of the bacterial solution, reaching a plateau after approximately 30 min. The evolution of the chromatic transformation is most likely determined by the diffusion and binding of the bacterially released substances on the film surface. Colorimetric Bacterial Fingerprinting. The bacterially induced color transitions can be recorded and analyzed by conventional color scanning and digital image analysis (Figure 5). Recent studies have demonstrated that digital colorimetric

Figure 4. Kinetic curve of bacterially induced fluorescence emission in a DOPE/PDA film. Fluorescence emission at 555 nm (excitation at 488 nm) was recorded at 33 °C after placing the supernatant extracted from a solution containing 108 bacterial cells (S. typhimurium). (, LB medium; 0, bacterial supernatant.

analysis (DCA) of the blue-red transformations on lipid/PDA films is a powerful and simple method for the evaluation of color changes occurring on such films.16 DCA measures the abundance and average intensity of transformed red pixels within a defined film surface area (generally the surface covered by the sample

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Figure 5. Color response induced by several bacterial strains on different films. (A) color transition (RCS) curves of different bacteria placed on a sphingomyelin/cholesterol/PDA film (7:3:90 mole ratio). 1, LB medium; b, B. cereus; (, E. coli C600 pMRInv; 9, E. coli XL1; and 2, S. typhimurium 1a. (B) RCS values calculated for aliquots extracted 7 h after the start of growth (bacterial concentration of 1 × 109/mL, see Supporting Information), placed on films with different lipid compositions. (i) E. coli XL1, (ii) B. cereus, (iii) S. typhimurium, and (iv) E. coli C600 pMRInv.

tested) and thus provides a quantitative value for the colorimetric transformation occurring on the film. The DCA analysis is applied using a conventional desktop scanner and readily available image analysis software16 (Experimental Section). Figure 5A depicts red chromaticity shift (RCS) curves calculated after placing supernatant aliquots of several bacterial strains, extracted at different times after the initiation of bacterial growths, on a sphingomyelin/cholesterol/PDA film (mole ratio 7:3:90). RCS values are essentially a measure of the blue-red transformations occurring on the film surface, with higher values corresponding to a more pronounced red appearance of the film (greater blue-red transformation) and low values reflecting a less pronounced color transition (Experimental Section). Thus, each data point in Figure 5A mirrors the extent of the blue-red transformation induced by a specific bacterial sample on the film. As expected, bacterial proliferation in the growth media gave rise to more pronounced blue-red transitions in later stages of the growth; this result was apparent for all bacterial species examined. However, Figure 5A also demonstrates a divergence

among the RCS curves, reflecting a variation in the extent of blue-red transformations induced by each bacterium. For example, E. coli XL1 suspensions gave rise to faster and consistently more pronounced blue-red transformations (squares in Figure 5A) compared to Bacillus cereus (circles in Figure 5A). Importantly, the concentrations of the different bacterial species examined were similar throughout the experimental time frame (Supporting Information). Accordingly, the divergence of the RCS curves in Figure 5A is intimately related to the underlying mechanism of colorimetric changes (i.e., the interaction of bacterially secreted substances with the lipid/PDA film surface). Indeed, because each bacterial strain exhibits a distinct profile of metabolic pathways and secreted substances, it is expected that different interactions and consequent colorimetric transitions would be induced by each bacterium. The divergence of the RCS curves in Figure 5A is the key to the colorimetric bacterial fingerprinting method. Specifically, the colorimetric reactions of the tested bacterial strains can be

Colorimetric Bacterial Fingerprinting

Figure 6. Colorimetric bacterial fingerprinting. The color combination for each bacterium reflects the RCS values recorded 7 h after the start of growth at a bacterial concentration of 1 × 109/mL (Figure 5B). The RCS color key is shown on the left. (i) DOPE/PDA (1:9 mole ratio); (ii) sphingomyelin/cholesterol/PDA (7:3:90); (iii) DMPC/ PDA (1:9); and (iv) POPG/PDA (1:9).

recorded on films having different lipid compositions. Figure 5B, for example, depicts the RCS values calculated for the four bacterial strains deposited on films having different lipid compositions. The diagram in Figure 5B features the RCS data calculated 7 h after the initiation of growth, a time point in which the bacteria are in the stationary growth modes (concentration of 1 × 109 particles/mL). Importantly, the choice of this time point reflects the experimentally significant differences in the RCS values induced by the bacterial species examined (Figure 5A). Figure 5B confirms that the extent of blue-red changes induced on the films varied between both of the bacterial strains and among the lipid compositions examined. For example, E. coli XL1 induced approximately 70% RCS (reflecting the pronounced blue-red transformation of the film surface) when placed on a DOPE/PDA film but only 30% RCS on a DMPC/ PDA film. S. typhimurium, however, gave rise to 70% RCS in the DOPE/PDA film and 40% RCS following deposition on the DMPC/PDA film. The variations in color changes among both bacterial strains and film compositions were anticipated. Specifically, because the color transformations correspond to interactions of the secreted molecules with the lipid/PDA film surface, the distinct pool of molecules released by each bacterial species to its surroundings should interact in a rather distinctive way with films having different lipid head groups, sizes, structures, surface charge, organization of the lipid molecules, and so forth, overall leading to disparities in the colorimetric response depending upon film composition.

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The bar diagram in Figure 5B can be also represented as a means of pattern recognition or bacterial fingerprinting utilizing an array of different lipid/PDA films (Figure 6). Figure 6 presents, in a color-code display, the cumulative RCS values recorded for each bacterial species deposited on the films 7 h after the initiation of growth. Figure 6 shows that distinct colorimetric fingerprints are obtained for the four bacterial species examined. The fingerprints are dependent upon the bacterial concentration selected; when the color induced on the films by other bacterial concentrations is tested, the bacterial fingerprints might not be unique. However, the significant result depicted in Figure 6 is that at a certain time point in the bacterial growth curves (identified here as 7 h after the initiation of growth) the supernatant solutions will provide unique patterns, or fingerprints, for the bacterial species examined. It is plausible that a selection of an optimal array of different lipid compositions would produce individual colorimetric signatures for many bacterial strains.

Conclusions Colorimetric technology exhibits important strengths as a bacterial-sensing and discrimination platform. An important feature of the system is that bacterial detection is not achieved through the use of specific recognition elements but rather is achieved through the affinity of bacteria and bacterially secreted compounds for phospholipid bilayers. In particular, the presence of phospholipids is critical for achieving bacterial discrimination; experiments investigating the response of pure PDA films to bacterial secretions from different strains yielded no experimentally significant differences among the different strains. The sensing platform also exhibits practical benefits. Film preparation is straightforward and inexpensive, and the films can be stored for long periods of time before use. The new film assay facilitates bacterial detection at a threshold concentration of around 106 particles/mL, a concentration that is reached after short time spans under regular bacterial growth conditions. The colorimetric transformations occur over a wide bacterial concentration range and can be detected by the naked eye or through conventional desktop scanners. Analysis can be carried out by employing either supernatant solutions or the total nonseparated bacterial suspensions. Utilization of the colorimetric bacterial film sensor in high-throughput screening and parallel analysis of metabolic disruption should be feasible. Acknowledgment. R.J. is grateful to the Human Frontiers Science Program for generous financial support. We are grateful to Roxana Golan for her assistance in the AFM experiments. Supporting Information Available: Bacaterial growth curves. This information is available free of charge via the Internet at http://pubs.acs.org. LA0636208