Flow Cytometric Analysis To Detect Pathogens in Bacterial Cell

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Anal. Chem. 2008, 80, 864-872

Flow Cytometric Analysis To Detect Pathogens in Bacterial Cell Mixtures Using Semiconductor Quantum Dots Megan A. Hahn,† Peter C. Keng,‡ and Todd D. Krauss*,†

Department of Chemistry, University of Rochester, Rochester, New York 14627, and Department of Radiation Oncology, University of Rochester Medical Center, Rochester, New York 14642

Compared to a common green organic dye, semiconductor quantum dots (QDs) composed of CdSe/ZnS core/ shell bioconjugates display brighter fluorescence intensities, lower detection thresholds, and better accuracy in analyzing bacterial cell mixtures composed of pathogenic E. coli O157:H7 and harmless E. coli DH5r using flow cytometry. For the same given bacterial mixture, QDs display fluorescence intensity levels that are ∼1 order of magnitude brighter compared to the analogous experiments that utilize the standard dye fluorescein isothiocyanate. Detection limits are lowest when QDs are used as the fluorophore label for the pathogenic E. coli O157: H7 serotype: limits of 1% O157:H7 in 99% DH5r result, corresponding to 106 cells/mL, which is comparable to other developing fluorescence-based techniques for pathogen detection. Finally, utilizing QDs to label E. coli O157: H7 in cell mixtures results in greater accuracy and more closely approaches the ideal fluorophore for pathogen detection using flow cytometry. With their broader absorption spectra and narrower emission spectra than organic dyes, QDs can make vast improvements in the field of flow cytometry, where single-source excitation and simultaneous detection of multicolor species without complicating experimental setups or data analysis is quite advantageous for analyzing heterogeneous cell mixtures, both for prokaryotic pathogen detection and for studies on eukaryotic cell characteristics. Flow cytometry is a powerful analytical tool employed by molecular biologists and toxicologists for purposes such as quantizing bacterial amounts in food or water samples, assessing cell surface antigens with immunofluorescence, analyzing cell cycles, and testing cell viability.1-4 Essentially, this technique is a highly automated fluorescence microscope: cells pass single file * To whom correspondence should be addressed. Phone: (585) 275-5093. Fax: (585) 276-0205. E-mail: [email protected]. † University of Rochester. ‡ University of Rochester Medical Center. (1) Steen, H. B. J. Microbiol. Methods 2000, 42, 65-74. (2) Grogan, W. M.; Collins, J. M. Guide to Flow Cytometry Methods; Marcel Dekker, Inc.: New York, 1990. (3) Holmes, K.; Lantz, L. M.; Fowlkes, B. J.; Schmid, I.; Giorgi, J. V. In Current Protocols in Immunology; Coligan, J. E., Bierer, B. E., Margulies, D. H., Shevach, E. M., Strober, W., Eds.; Wiley & Sons: New York, 2001; pp 5.3.15.3.24.

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through a focused laser beam, and each cell is individually characterized by its own unique scattering and fluorescence properties. As the cells pass through the beam, they scatter the light from the excitation source and, if labeled with fluorophores, emit. This scattering triggers the numerous photomultiplier tubes (PMTs) to detect fluorescence from the scatterers. Scatter of the light from the excitation source on the same axis as the beam (i.e., forward scatter, or FS) is indicative of cell volume; scatter of the light from the excitation source at a direction orthogonal (90°) to the laser beam path (i.e., side scatter, or SS) is indicative of cell complexity and granularity.5 These scattering characteristics are true for all cell types, prokaryotic as well as eukaryotic. In addition to this scattering, multicolor fluorescence from labeled cell regions can be detected through the use and proper placement of optical filters. Multiparameter flow cytometry is possible, with analysis of up to 17 fluorescence parameters,6 but analyses utilizing 2 scattering and 3- or 4-color fluorescence parameters are more common.7 What makes this technique so valuable is the fact that several scattering and fluorescence parameters of a single cell can be analyzed simultaneously and quite rapidly. In fact, 10 000 cells can be characterized in less than 1 min.5 Flow cytometry, a high-throughput analytical technique that couples rapid analysis of large sample volumes with sensitive detection, is common in the research areas of oncology, immunology, and toxicology.8 Used for quantitative analysis of cell subpopulations, flow cytometry can analyze heterogeneous cell mixtures and can even characterize cells of the same type that possess different biochemical or biophysical properties using the proper fluorescent markers or probes, the most common of which are antibodies conjugated to dyes (e.g., fluorescein isothiocyanate (FITC)) or fluorescent phycobiliproteins (e.g., phycoerythrin), and small molecules that bind to nucleic acids (e.g., propidium iodide (4) June, C. H.; Moore, J. S. In Current Protocols in Immunology; Coligan, J. E., Bierer, B. E., Margulies, D. H., Shevach, E. M., Strober, W., Eds.; Wiley & Sons: New York, 2004; pp 5.5.1-5.5.20. (5) Holmes, K. L.; Otten, G.; Yokoyama, W. M. In Current Protocols in Immunology; Coligan, J. E., Bierer, B. E., Margulies, D. H., Shevach, E. M., Strober, W., Eds.; Wiley & Sons: New York, 2001; pp 5.4.1-5.4.22. (6) Chattopadhyay, P. K.; Price, D. A.; Harper, T. F.; Betts, M. R.; Yu, J.; Gostick, E.; Perfetto, S. P.; Goepfert, P.; Koup, R. A.; De Rosa, S. C.; Bruchez, M. P.; Roederer, M. Nat. Med. 2006, 12, 972-977. (7) Roederer, M. In Current Protocols in Immunology; Coligan, J. E., Bierer, B. E., Margulies, D. H., Shevach, E. M., Strober, W., Eds.; Wiley & Sons: New York, 2002; pp 5.8.1-5.8.10. (8) Shapiro, H. M. Practical Flow Cytometry, 2nd ed.; Alan R. Liss, Inc.: New York, 1988. 10.1021/ac7018365 CCC: $40.75

© 2008 American Chemical Society Published on Web 01/11/2008

(PI) and Hoechst 33342). Most flow cytometric analyses study characteristics of eukaryotes;2-4 however, studies with fluorescently labeled bacteria have also been performed. Bacteria are more difficult to analyze using flow cytometry: with their dimensions of ∼1 order of magnitude smaller than typical eukaryotes (resulting in a 1000-fold smaller volume) and with their lack of organelles (resulting in a lower cellular complexity), prokaryotes display weaker scattering signals. Nevertheless, this technique has been used to detect the Escherichia coli O157:H7 serotype through the use of FITC-labeled antibodies9-12 and cyanine dimerlabeled bacteriophages.13,14 Despite the common incorporation of organic dyes in flow cytometry, there are certain serious disadvantages for their use in such techniques. Often a flow cytometer is equipped with only a single-wavelength excitation source, which emphasizes a severe limitation with dyesstheir narrow absorption spectra. Therefore, for multicolor analyses, either supplementary excitation sources must be added to excite the different emitting species, which can complicate experimental setups, or fluorescence resonance energy transfer (FRET) conjugates must be developed (vide infra), which are not necessarily straightforward. While possessing high fluorescence quantum yields and larger extinction coefficients than dyes like FITC, the phycobiliproteins can also be conjugated to antibodies for labeling of surface antigens.15 Despite having numerous chromophores per protein, these phycobiliproteins still suffer from the same disadvantages as dyes when used in flow cytometry. These proteins absorb bluegreen and green light (e.g., phycoerythrins), green and yellow light (e.g., phycocyanins), or orange and red light (e.g., allophycocyanins); therefore, there is no possibility of attaining blueemitting species, and even green fluorescence (i.e., 530-nm emission) from phycobiliproteins is difficult to obtain because of their limited regions of absorption. Unlike dyes, the phycobiliproteins suffer from significant conversion to triplet states, resulting in phosphorescence and, therefore, longer observation times in flow cytometry. Such longer acquisition times are undesirable because they lead to greater electronic noise levels and hence poorer detection limits. In contrast, quantum dots do not suffer from such switching to triplet states and only fluoresce, consequently having much shorter lifetimes than the phosphorescence of the phycobiliproteins. In addition, simultaneous excitation of many different phycobiliproteins in the same analysis is impossible unless FRET conjugates are made incorporating other smaller dye molecules that can absorb the emitted light from the protein and, thus, emit themselves. With their molecular weights ranging in the hundreds of thousands, these proteins require nontrivial processing in order (9) Yamaguchi, N.; Sasada, M.; Yamanaka, M.; Nasu, M. Cytometry A 2003, 54, 27-35. (10) Tanaka, Y.; Yamaguchi, N.; Nasu, M. J. Appl. Microbiol. 2000, 88, 228236. (11) Tortorello, M. L.; Stewart, D. S.; Raybourne, R. B. FEMS Immunol. Med. Microbiol. 1998, 19, 267-274. (12) Kusunoki, H.; Kobayashi, K.; Kita, T.; Tajima, T.; Sugii, S.; Uemura, T. J. Vet. Med. Sci. 1998, 60, 1315-1319. (13) Goodridge, L.; Chen, J.; Griffiths, M. Int. J. Food Microbiol. 1999, 47, 4350. (14) Goodridge, L.; Chen, J.; Griffiths, M. Appl. Environ. Microb. 1999, 65, 13971404. (15) Shapiro, H. M. Practical Flow Cytometry, 4th ed.; Wiley & Sons: Hoboken, NJ, 2003.

to make such conjugates. Last, FRET conjugates differ in their ability to efficiently transfer energy, depending on the spectral overlap between the donor emission and the acceptor absorption. Despite the careful selection and proper placement of optical filters, overlap of multiple dye fluorescence spectra is common due to their broad and asymmetric emission; therefore, electronic color compensation must be performed to remove spectral overlap between detector channels, thereby complicating analyses. In fact, when used in multicolor analyses, especially those that utilize a very bright fluorophore along with a very dim fluorophore, this same compensation can actually result in false positives, false negatives, histogram shapes altered by artifacts, or a combination thereof.16 The problems in flow cytometry associated with the use of organic dyes or fluorescent proteins can potentially be overcome by replacing these fluorophores with semiconductor quantum dots (QDs). With their broad absorption spectra, QDs are not limited to specific excitation sources, thus proving to be quite an advantage in flow cytometric methods. In addition, color compensation is not needed due to the narrow emission spectra of QDs, which results in little overlap between detectors. Also, their larger absorption cross sections compared to dyes can help discern lower levels of fluorescence on cellular components having lower densities of antigens, receptors available for labeling, or both. Quantum dot labels also need no FRET conjugates because of their broad absorption spectra. In fact, QDs are already impacting eukaryotic cell studies: flow cytometry was used to study immunophenotyping of certain T-cell populations produced in humans.6 This work presents the results of using flow cytometry to analyze bacterial cell mixtures composed of pathogenic greenlabeled E. coli O157:H7 cells amid increasing amounts of benign red-labeled E. coli DH5R cells; this pathogen labeling and detection using green semiconductor QDs is compared to similar methods performed with the green organic dye FITC. We used standard biochemical techniques17 utilizing the strong and wellknown streptavidin-biotin interaction18,19 to selectively label pathogenic heat-killed E. coli O157:H7 cells with biotinylated antiE. coli O157:H7 antibodies and streptavidin-functionalized CdSe/ ZnS core/shell QDs that fluoresce green. The red fluorophore that was used to label nonpathogenic E. coli DH5R cells is PI, an intercalating dye that binds nucleic acids within dead cells or those having compromised membranes. Possessing another harmless strain of E. coli in addition to the pathogenic serotype, the cell mixtures are intended to simulate contaminated samples, where the presence of additional harmless bacteria is quite common and may complicate analytical detection schemes. E. coli O157:H7 cells were labeled with QDs and diluted into solutions containing increasing amounts of PI-labeled E. coli DH5R cells to determine the relative sensitivity of QD-labeled pathogens amid increasing amounts of innocuous cells detected by flow cytometry; these results were compared to labeling of E. coli O157:H7 with an equal amount of FITC (1×) and with a 10-fold greater amount of FITC (16) Sharrow, S. O. In Current Protocols in Immunology; Coligan, J. E., Bierer, B. E., Margulies, D. H., Shevach, E. M., Strober, W., Eds.; Wiley & Sons: New York, 1991; pp 5.1.1-5.1.8. (17) Harlow, E.; Lane, D. Using Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1999. (18) Green, N. M. Methods Enzymol. 1990, 184, 51-67. (19) Chaiet, L.; Wolf, F. J. Arch. Biochem. Biophys. 1964, 106, 1-5.

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(10×) than the QDs used in the equivalent experiment. Additional experiments were also performed in the hopes of increasing the detected percentages of labeled cells (e.g., fixing) and of better mimicking the analysis of bacterial mixtures (e.g., double labeling). EXPERIMENTAL SECTION Reagents. Enumerated samples (given in colony-forming units (cfu) per milliliter) of heat-killed E. coli O157:H7 and E. coli DH5R in phosphate-buffered saline (PBS; 10 mM, pH 7.4) were kindly provided by Agave BioSystems (Ithaca, NY). Cells were grown overnight past the logarithmic phase, yielding saturated stock solutions of ∼109 cfu/mL, and then heat-killed. Bovine serum albumin (BSA) and PI solution (1 mg/mL stock in water) were purchased from Sigma (St. Louis, MO). BacTrace affinity-purified anti-E. coli O157:H7 antibodies produced in goat were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD), and they were biotinylated with an EZ-Link sulfo-NHS-LC-biotinylation kit from Pierce Biotechnology (Rockford, IL) per the manufacturer’s instructions. These antibodies maintain their binding to surface antigens, despite the heat-killing treatment of the bacterial cells. Having fluorescence quantum yields of g40%,20 streptavidinfunctionalized CdSe/ZnS core/shell QDs (Qdot 525 streptavidin conjugate) and incubation buffer (Qdot incubation buffer) were purchased together from Quantum Dot Corp. (Hayward, CA). Streptavidin conjugated to FITC was purchased from Pierce Biotechnology. Every step of these procedures was carried out at ambient temperature and pressure. Bioconjugate-Labeled E. coli O157:H7 (Green). Approximately 1 × 108 E. coli O157:H7 cells in PBS (diluted from stock solutions of ∼109 cfu/mL) were pelleted and resuspended in blocking solution (1% BSA in PBS) for 30 min. A solution of biotinylated anti-E. coli O157:H7 antibodies (6.8 pmol) in a solution of 1% BSA in PBS was then added to the cells, and they were incubated for 2 h. Cell solutions were then rinsed twice by centrifugation and resuspended in PBS. Cell pellets were subsequently incubated with additional blocking solution for 30 min, and then a solution of streptavidin-modified fluorophore emitting at ∼525 nm (20 pmol of either streptavidin-conjugated QDs or FITC-conjugated streptavidin, 1×) in incubation buffer (2% BSA in 50 mM borate, pH 8.3 with 0.05% sodium azide as preservative) was added and reacted for 2 h in darkness. An attempt to produce a larger fluorescence signal was generated in an additional trial using 200 pmol of FITC-conjugated streptavidin (10×). Cells were rinsed twice by centrifugation and resuspended in 1% BSA in PBS for analysis with a standard flow cytometer as described below. The cultures of stock E. coli O157:H7 were labeled 32-35 months after harvesting for these flow cytometric experiments; however, cells from this same stock solution were labeled similarly and observed with epifluorescence microscopy 15 months after harvesting and found to be labeled properly and uniformly, with no evidence of cellular degeneration or debris. Also, the same stock was labeled and studied with fluorometry 22 months after harvesting and displayed the appropriate fluorescence spectra. Therefore, despite the stock pathogenic cells being quite aged, there is no evidence of compromised O157 antigens. The initial (20) Qdot Streptavidin Conjugates User Manual; Quantum Dot Corp.: Hayward, CA, 2005; Rev. 9.2, p 15.

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green-labeled E. coli O157:H7 stock solution was subsequently diluted into separate assays with increasing relative concentrations of PI-labeled E. coli DH5R cells, which were labeled as described below. All labeled bacterial solutions and mixtures were analyzed immediately after labeling (i.e., same day). PI-Labeled E. coli DH5r (Red). Approximately 1 × 108 E. coli DH5R cells in PBS (diluted from stock solutions of ∼109 cfu/ mL) were fixed overnight at 4 °C in a solution of 70% ethanol to permeabilize the bacterial cell membranes. Cells were then pelleted by centrifugation and resuspended with an aqueous solution of PI (30 nmol) that emits at ∼617 nm when bound to nucleic acids. The cultures of stock E. coli DH5R were labeled 17-20 months after harvesting for these flow cytometric experiments; however, cells from this same stock solution were labeled similarly and observed with epifluorescence microscopy 1 month prior to these experiments and found to be intact, displaying intracellular red fluorescence and exhibiting the proper PI emission spectra. Therefore, despite the stock innocuous cells being somewhat aged, there is no evidence of cellular degeneration or improper fluorescence labeling. Appropriate volumes of PI-labeled cells were mixed with the above green-labeled E. coli O157:H7 cells to generate bacterial solutions with increasing PI/QD (i.e., red/green, or R/G) ratios. Differences in the concentrations of the bacterial mixtures (causing a difference in the R/G ratio from what was expected) were accounted for with a slight variation on a common method used for cell enumeration21 and is further described in detail in the Supporting Information. Flow Cytometry. The above bacterial cell mixtures were analyzed with a Coulter EPICS Elite flow cytometer, using the 488-nm line of an Ar ion laser (15 mW) for excitation of all fluorophores (i.e., QD, FITC, and PI). Sheath fluid was composed of PBS containing 4% fetal bovine serum, and the sample flow rate was constant throughout the analysis of all bacterial mixtures. FS and SS were detected with a photodiode and PMT1, respectively, with a 488 ( 5 nm band-pass filter in front of PMT1. The green fluorescence signals (i.e., QD or FITC) were detected with PMT2 using a 525 ( 5 nm band-pass filter, and the red PI fluorescence was detected with PMT3 using a 575 ( 20 nm bandpass filter. PMT gains/voltages were set at 30/363, 50/345, 10/ 800, and 10/630 V for FS, SS (i.e., PMT1), PMT2, and PMT3, respectively. No electronic color compensation was performed, and 5000 fluorescence events from each bacterial mixture were collected for subsequent analysis. Proper gates for the detection both of only green-labeled E. coli O157:H7 cells (QD or FITC, gate D4) and of only red-labeled E. coli DH5R (PI, gates D1 and D2) fluorescence events were set with blank solutions containing an equivalent amount (1 × 108 cfu/mL) of unlabeled E. coli O157: H7 and unlabeled E. coli DH5R, respectively. These unlabeled cells were simultaneously prepared with the labeled cells and analyzed before the fluorescent bacterial assays; no antibodies or fluorophores were added to the blank solutions, only equal volumes of the appropriate buffer solution. As a result of their low fluorescence intensity signals detected by flow cytometry, thresholds for gate D3 (unlabeled cells) were set to include the majority of detected events (g97%) from these blank cell solutions. (21) Brando, B.; Barnett, D.; Janossy, G.; Mandy, F.; Autran, B.; Rothe, G.; Scarpati, B.; D’Avanzo, G.; D’Hautcourt, J.-L.; Lenkei, R.; Schmitz, G.; Kunkl, A.; Chianese, R.; Papa, S.; Gratama, J. W. Cytometry 2000, 42, 327-346.

Table 1. Red/Green Percentages for Cell Mixtures Using 1× QDs as Label uncorrected R/Ga

corrected R/Gb

measured R/Gc

measured unlabeledd

0./100. 50./50. 80./20. 90.0/10.0 95.0/5.0 98.00/2.00 99.000/1.000 99.500/0.500 99.800/0.200 99.9000/0.1000 100./0.

0./100. ((0.) 22/78 ((2) 63/37 ((1) 81.9/18.1 ((0.7) 94.8/5.2 ((0.1) 97.90/2.10 ((0.04) 99.572/0.428 ((0.009) 99.585/0.415 ((0.008) 99.844/0.156 ((0.005) 99.9697/0.0303 ((0.0006) 100./0. (undefined)

0.9/50.9 6.0/37.7 12.1/27.8 22.2/13.9 33.6/8.3 24.1/10.1 39.2/1.8 30.7/1.7 35.1/0.2 42.3/1.1 35.1/0.0

48.2 56.3 60.0 63.9 58.0 65.8 59.1 67.6 64.7 56.6 64.8

a Determined from simple dilutions, assuming equal concentrations of bacterial stock solutions and negligible errors in volume measurements. Determined with eqs S.1 and S.2 (Supporting Information), with absolute errors in G shown in parentheses and propagated using reported flow cytometer times ( 1 s. c Determined by flow cytometer, using defined gates (D1 + D2)/D4. d Determined by flow cytometer, using defined gate D3.

b

Table 2. Red/Green Percentages for Cell Mixtures Using 1× FITC as Label

Table 3. Red/Green Percentages for Cell Mixtures Using 10× FITC as Label

uncorrected R/Ga

corrected R/Gb

measured R/Gc

measured unlabeledd

uncorrected R/Ga

corrected R/Gb

measured R/Gc

measured unlabeledd

0./100. 50./50. 80./20. 90.0/10.0 95.0/5.0 98.0/2.0 99.00/1.00 99.50/0.50 99.80/0.20 99.900/0.100 100./0.

0./100. ((0.) 62/38 ((1) 74/26 ((1) 89.1/10.9 ((0.5) 94.7/5.3 ((0.3) 98.0/2.0 ((0.1) 99.19/0.81 ((0.04) 99.52/0.48 ((0.02) 99.82/0.18 ((0.01) 99.913/0.087 ((0.006) 100./0. (undefined)

0.2/8.1 9.6/5.0 26.3/2.5 39.2/2.0 51.3/1.3 58.6/0.7 60.8/0.7 59.9/0.3 64.2/0.1 55.0/0.1 54.0/0.0

91.7 85.4 71.2 58.8 47.4 40.7 38.5 39.7 35.7 44.9 46.0

0./100. 50./50. 80.0/20.0 90.0/10.0 95.0/5.0 98.00/2.00 99.00/1.00 99.500/0.500 99.800/0.200 99.900/0.100 100./0.

0./100. ((0.) 15/85 ((1) 51.7/48.3 ((1.0) 76.8/23.2 ((0.5) 92.4/7.6 ((0.2) 97.83/2.17 ((0.05) 98.79/1.21 ((0.03) 99.588/0.412 ((0.007) 99.826/0.174 ((0.004) 99.915/0.085 ((0.001) 100./0. (undefined)

0.4/8.5 3.3/6.4 10.2/5.4 19.7/4.5 50.4/2.1 61.9/0.9 67.3/0.7 60.5/0.5 71.7/0.2 62.3/0.1 65.2/0.1

91.2 90.3 84.4 75.8 47.5 37.2 32.0 39.0 28.1 37.6 34.8

a Determined from simple dilutions, assuming equal concentrations of bacterial stock solutions and negligible errors in volume measurements. b Determined with eqs S.1 and S.2 (Supporting Information), with absolute errors in G shown in parentheses and propagated using reported flow cytometer times ( 1 s. c Determined by flow cytometer, using defined gates (D1 + D2)/D4. d Determined by flow cytometer, using defined gate D3.

a Determined from simple dilutions, assuming equal concentrations of bacterial stock solutions and negligible errors in volume measurements. b Determined with eqs S.1 and S.2 (Supporting Information), with absolute errors in G shown in parentheses and propagated using reported flow cytometer times ( 1 s. c Determined by flow cytometer, using defined gates (D1 + D2)/D4. d Determined by flow cytometer, using defined gate D3.

All data were analyzed with the EPICS ELITE computer software EXPO (v. 2).

Briefly, cell enumeration using a single platform with flow cytometry involves first positively identifying the cells of interest in a given volume without the need for a reference population: for positive identification of our cells, green- or red-labeled cell solutions containing only E. coli O157:H7 or only E. coli DH5R were analyzed with the flow cytometer, 5000 events were collected at the same flow rate, and the time required to count those events was recorded. For analysis of the bacterial mixtures, the same sample flow and number of events were used, and a weighted average of the time needed to collect those events (also incorporating the volume dilutions used to make the mixtures) was used to correct for the R/G values. For example, a simple weighted average to derive the ratio of components in a theoretical mixture (99/1 E. coli DH5R to E. coli O157:H7 labeled with PI and QDs, respectively) is done by utilizing the simple volume dilutions of each cell type ((R/G)Theoretical) and the times to collect 5000 fluorescent events for the 0/100 E. coli O157:H7 (27 s) and for the 100/0 E. coli DH5R (50 s) samples; therefore, a theoretical time (TimeTheoretical) of 49.77 s is needed to collect 5000 events. This time is compared to the actual time needed (TimeActual) to collect 5000 events from the flow cytometer (117 s), and thus, an

RESULTS AND DISCUSSION Percentage of Green E. coli O157:H7 Detected. Because the concentrations of the initial stock solutions of E. coli O157: H7 and E. coli DH5R were not exactly equal, the actual R/G ratios of the resulting fluorescently labeled DH5R/O157:H7 cell mixtures must be corrected from what would be obtained from simple dilutions (and assuming equal initial concentrations). Along with values for the uncorrected and corrected R/G ratios, a complete list of the percentages reported by the flow cytometer of green events (gate D4), red events (sum of gates D1 and D2), and unlabeled events (gate D3) can be found in Tables 1, 2, and 3 for each series of experiments (1× QD, 1× FITC, and 10× FITC). The gate windows for fluorescence are illustrated in the rightmost columns in Figures 1 and 2. A variation from a single-platform technique using flow cytometric methods, which was tested by Brando et al. and found to be superior to dual-platform techniques,21 was used as the basis for our corrections to initial cell concentration (i.e., R/G ratios).

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Figure 1. Flow cytometric analysis comparing 0/100 R/G E. coli O157:H7 solutions (i.e., no E. coli DH5R present). Leftmost dot plots show scattering of bacterial solutions, with gate A indicated, thus discriminating bacterial cells from other debris or noise. Plots in second and third columns display single-parameter fluorescence histograms in green PMT2 channel and red PMT3 channel, respectively. Rightmost dot plots show dual-parameter results of red channel versus green channel. In the dot plots, the z-axis (perpendicular to page) represents the number of events with those particular intensity profiles, and boldface numbers indicate the amount of green-labeled events detected in gate D4. (A) 1× QD-labeled cells. (B) 1× FITC-labeled cells. (C) 10× FITC-labeled cells.

actual fractional amount ((R/G)Actual) of pathogenic cells (99.572/ 0.428) in the mixture can be deduced. (See eqs S.1 and S.2 in the Supporting Information.) A variation of this approach is a common way to analyze cellular mixtures,21 and a more detailed explanation can be found in the Supporting Information. It is important to note that this correction method supports the fact that at low pathogen (G) amounts (i.e., R/G g 95/5), the method of simple dilutions agrees well with the corrected G values. The flow cytometry results of the initial 0/100 R/G E. coli O157:H7 samples labeled with a green fluorophore are shown in Figure 1. Single-color histograms of each bacterial solution, plotted as counts versus log of green intensity (second column from left in Figure 1), demonstrate a distribution of fluorescence intensities. The maximum FITC fluorescence intensities are lower than the maximum of the QD intensity for the initial 100% green-labeled E. coli O157:H7 cell solutions, according to the exact locations of their respective green histogram peaks on the x-axis. The QDlabeled E. coli O157:H7 solution has a maximum log of fluorescence intensity at ∼1; the maxima of the fluorescence intensity peaks for both the 1× and the 10× FITC-labeling experiments are ∼0.3, practically 1 order of magnitude lower (Figure 1). Therefore, it is apparent that despite using a 1 order of magnitude 868 Analytical Chemistry, Vol. 80, No. 3, February 1, 2008

greater amount of FITC bioconjugate, QD-labeled cells are much brighter fluorescence tags for this common pathogen, which is quite an advantage in the difficult analysis of bacteria using flow cytometry. Because bacteria have dimensions on the order of only a few micrometers, any bound fluorophore must be extremely bright in order to be detected in the appropriate D4 gate. Theoretically, the percentage of green (QD- or FITC-labeled) E. coli O157:H7 detected by a flow cytometer should be equal to the actual amount of cells artificially spiked into the mixture. However, the initial solutions of green-labeled E. coli O157:H7 cells are not detected with 100% of fluorescent events located in gate D4 by the flow cytometer (Figure 1). Therefore, the amount of pathogenic cells in the subsequent mixtures are not detected as possessing their ideally accurate percentages, especially at lower R/G values (e90/10); for example, the corrected R/G ratio of 22/78 in the QD-labeling experiment is determined to have only 37.7% of green-labeled cells, instead of the appropriate 78% (Table 1). In fact, the amount of pathogenic cells in the 100% QDonly O157:H7 solution is determined to be only 50.9% by the flow cytometer in gate D4 (Figure 1A). The majority of the remaining events (48.2% of the total) are detected in gate D3, which was set according to background intensities of an unlabeled blank solution

Figure 2. Flow cytometric analysis comparing 98/2 R/G bacterial cell mixtures. Leftmost dot plots show scattering of bacterial solutions, with gate A indicated, thus discriminating bacterial cells from other debris or noise. Plots in second and third columns display single-parameter fluorescence histograms in green PMT2 channel and red PMT3 channel, respectively. Rightmost dot plots show dual-parameter results of red channel versus green channel. In the dot plots, the z-axis (perpendicular to page) represents the number of events with those particular intensity profiles. (A) 1× QD-labeled cells. (B) 1× FITC-labeled cells. (C) 10× FITC-labeled cells.

of E. coli O157:H7 cells. In contrast, FITC is not easily visible in the D4 channel: only 8.4% of the events in the equivalent 1× FITClabeling experiment containing only E. coli O157:H7 cells were determined to be positively green, while 91.3% events were detected as unlabeled cells that fell into gate D3 (Table 2, Figure 1B). Even using a 10× greater amount of FITC to label the harmful cells has little effect on the percentage of E. coli O157:H7 pathogens detected: only 8.5% are considered green, while 91.2% are considered unlabeled (Table 3, Figure 1C). These lower percentages of detected positive green events compared to what is expected are not inferring that these cells are not pathogenic; merely, the fluorescence intensities from these cells are lower than the threshold set for gate D4. Instead, the majority of events are registered in gate D3, which is the area containing the lowest fluorescence intensities of all bacterial cells (i.e., the unlabeled cells). Because the majority of FITC-labeled cells were detected in gate D3, these results indicate either that these cells did not have enough fluorophores on their surfaces or that the fluorophores themselves were not bright enough for the flow cytometer to record as positive green signals in gate D4. Because the 10× FITC-labeling experiment results in approximately the same detected percentage of green-labeled pathogenic

cells in the 100% green-only E. coli O157:H7 sample as in the comparable 1× FITC experiment, it is most likely not a matter of fluorophore number. It is also unlikely that the threshold for gate D3 is too high, because this gate was set with unlabeled bacterial cells so that the majority (i.e., g97%) of cells from the blank unlabeled samples were detected in this window. Further experiments were performed in order to increase the percentage of green events detected in gate D4 and are outlined in the next subsection. This dilemma obtained with all fluorophores in that the supposedly 100% green-only, fluorescently labeled pathogenic cells are not detected as consisting of the correct 100% of green fluorescent events in gate D4 has many possible explanations: (1) not every cell is labeled with fluorophores; (2) every cell does not have a high enough density of fluorophore on its surface to be detected by the instrument; (3) the total amount of fluorophore on each cell is not bright enough to register as a fluorescent event in the correct gate; and (4) the flow cytometer is not optimized for bacterial detection. Given previous results,22 the first explanation is unlikely: the majority of E. coli O157:H7 cells appear to (22) Hahn, M. A.; Tabb, J. S.; Krauss, T. D. Anal. Chem. 2005, 77, 4861-4869.

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have some amount of bound fluorophore, and it is also doubtful that certain cells would have preferential labeling over other cells of the same serotype. Although not likely, the second issue may be a concern, as it was revealed that while most pathogenic cells were fluorescent throughout the entire cell, some were fluorescent only around the bacterial cell wall.22 Given the necessary high cell concentrations and limited reagent amounts used to minimize nonspecific binding (vide infra), a lack of saturated sites on the cell surface may result. The third concern is very probable, and the fact that QDs are brighter than dyes on an individual fluorophore-to-fluorophore basis may explain their superior performance. The last explanation, related to (3), is also quite plausible, due to the small sizes of bacteria. Unfortunately, the flow cytometer used here is not optimized for bacterial cell analysis; instead, it is used primarily in the research milieu of radiation oncology, predominantly in cancer studies of cell cycle analysis and DNA content of mammalian cells. Detection of pathogenic bacteria could be improved upon by increasing the laser intensity through an increase of laser power, a decrease of laser spot size, or both. However, major changes to the experimental setup were not possible. For the given R/G ratio of 98/2 (Figure 2), the QD-labeled cells display a histogram in the green channel with a distinct peak, whereas the 1× and 10× FITC-labeled solutions have no discernible peaks in their respective green histograms and, rather, display only an increasing slope at very low intensities (i.e., the detection threshold of the flow cytometer for these bacteria). In fact, the 1× FITC trial has a distinct peak in the green channel at only an 80/20 R/G value, and the 10× FITC experiment has a discernible green histogram peak at only a 90/10 R/G ratio of labeled DH5R/ O157:H7 cells (data not shown). Accordingly, the brighter QDlabeled cell solution leads to higher R/G ratios (i.e., lower amounts of pathogenic cells) that can be detected by the flow cytometer. The detection limit when QDs are used for fluorescent labeling is 99/1 R/G (i.e., 1% E. coli O157:H7 cells in a mixture with 99% E. coli DH5R cells). This result contrasts greatly with the higher detection limits that occur when FITC is used as the label: 10% O157:H7 when an equal amount of FITC (1×) is used; and 5% O157:H7 using a 10× greater amount of FITC. Defined as the highest R/G ratio and, therefore, the lowest pathogen concentration in which a distinct peak in the green histogram is not discernible, these detection limits correspond to 106 (1× QD), 107 (1× FITC), and 5 × 106 (10× FITC) cfu/mL E. coli O157:H7. Despite having a higher detection limit than various fluorescencebased assays used currently to identify this specific pathogenic bacterium by itself (typically 102-104 cfu/mL),23 these preliminary results indicate that QDs can surpass their organic dye counterparts and, with instrumental optimization, should be able to detect lower levels of this common pathogenic bacterium. In fact, although detection limits are slightly higher here than those of 102-104 cells/mL in other studies of bacterial mixtures, where FITC was used as the fluorescent label for E. coli O157:H7 in the presence of other background bacteria,9-11,24 these unoptimized results rival those of newer techniques for pathogen detection (i.e., detection limits of 105-107 cfu/mL); such strategies include immunofluorescence-based biosensors, used alone or coupled with (23) Deisingh, A. K.; Thompson, M. J. Appl. Microbiol. 2004, 96, 419-429. (24) Nakamura, N.; Burgess, J. G.; Yagiuda, K.; Kudo, S.; Sakaguchi, T.; Matsunaga, T. Anal. Chem. 1993, 65, 2036-2039.

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Figure 3. Correlation between amount of pathogenic E. coli O157: H7 cells detected by flow cytometry (y-axis) and actual amount inoculated in bacterial mixture (x-axis) for green labeling done with 1× QDs (dotted with circles), 1× FITC (dashed with triangles), and 10× FITC (solid with squares), compared to theoretical line for ideal fluorophore (bold solid).

other methods, such as magnetic bead separation, matrix-assisted laser desorption/ionization mass spectrometry, and surface plasmon resonance.23,25-27 It is important to note that the green signals in the R/G dot plots from QDs (Figures 1A and 2A) are narrow enough to include only gate D4, while the red signals from PI are much broader and must include both gates D1 and D2 (Figure 2). This observation is why electronic color compensation must usually be done when more than one dye is used for fluorescent labeling and why it was not needed in these experiments. With their narrower emission, semiconductor QDs exhibit little spectral cross talk between channels, thus illustrating a significant advantage over their analogous organic dyes in the technique of flow cytometry. A comparison was made between the percentage of green (QDor FITC-labeled) E. coli O157:H7 cells detected by the flow cytometer versus the corrected percentage of green cells actually inoculated in the bacterial mixtures, which contain increasing amounts of nonpathogenic red-labeled E. coli DH5R (Figure 3). This theoretical line representing a 1:1 detected/actual percentage of pathogenic cells is indicative of the ideal fluorescent label for flow cytometry. The ideal fluorophore, which can be used to determine the actual amount of pathogens present in a sample, ultimately quantizes the number of cells in an assay with little error; in other words, the percentage of pathogenic cells spiked into a sample should be the percentage reported by the flow cytometer. Because of the higher percentages detected in the initial solutions and, therefore, subsequent mixtures than in the comparable 1× and 10× FITC-labeling experiments, utilizing QDs to label pathogenic bacteria better approximates the theoretical line of percent G reported by the flow cytometer versus percent G spiked into the bacterial mixture. The slopes of the actual experimental curves using 1× QDs, 1× FITC, and 10× FITC as fluorescent labels are 0.48 ( 0.03, 0.081 ( 0.006, and 0.078 ( 0.008, respectively. The trial using QDs as the fluorophore has the lowest percent error of 51.94%, while the percent errors in (25) Ochoa, M. L.; Harrington, P. B. Anal. Chem. 2005, 77, 5258-5267. (26) Bosch, J. A.; Veerman, E. C. I.; Turkenburg, M.; Hartog, K.; Bolscher, J. G. M.; Nieuw Amerongen, A. V. J. Microbiol. Methods 2003, 53, 51-56. (27) Chuang, H.; Macuch, P.; Tabacco, M. B. Anal. Chem. 2001, 73, 462-466.

the slopes of 1× and 10× FITC-labeled E. coli O157:H7 cells are quite high, 91.9 and 92.21%, respectively; therefore, QD-labeled cells are overall more indicative of the accurate number of pathogenic cells in the mixture. In other words, results obtained with QDs to quantify amounts of E. coli O157:H7 are accurate to within a factor of 2, whereas with 1× or 10× FITC, the amount of pathogenic bacterial cells in the mixture can be 1 order of magnitude inaccurate. There are important factors to consider when performing this type of flow cytometric analysis on bacterial cell mixtures. A high starting cell concentration is imperative. If the cell concentration is too dilute (e.g., the common concentration of 106 cells/mL for typical eukaryotic cell analysis), longer acquisition times are required to collect the preset number of events; as a result, higher electronic noise levels are also detected due to the higher operating voltages of the PMTs. Contributing to the total number of detected events, this noise lowers the detection threshold of the measurement, thereby leading to lower population percentages in the fluorescent cells of interest. Also, determining cell concentrations is important: a sufficient correction for the slight concentration differences can be performed as outlined in the Supporting Information,21 which yields relatively accurate G percentages at lower pathogen concentrations as those predicted by simple dilutions. Additional Labeling Experiments and Flow Cytometry of E. coli. Other experiments were attempted in order to optimize the percentage of QD-labeled E. coli O157:H7 detected in the pathogen-only solutions, hopefully obtaining the appropriate 100% detected green events in gate D4. Using UV excitation at 360 nm provided no advantage over 488-nm excitation on the intensities of the QD fluorescence signals, and attempting cell labeling with Hoechst 33342, a live cell stain that emits at 460 nm, was also unsuccessful. These results are not entirely surprising, as all of the cells used here were heat-killed, not live. Also, fixing cells with 70% ethanol improves the efficiency of labeling E. coli DH5R with PI, but fixing E. coli O157:H7 cells results in a lower detected percentage of green QD-labeled cells. The amount of PI-labeled E. coli DH5R measured using fixed cells was 10.8%, whereas only 6.6% of events was determined using untreated cells. Meanwhile, the amount of E. coli O157:H7 cells that was identified as positive green decreased 1 order of magnitude: 28.7% of untreated cells were found pathogenic, compared to only 2.3% with fixed cells. Therefore, QD labeling of E. coli O157:H7 was done using cells that were not permeabilized, whereas PI labeling was done with fixed E. coli DH5R cells. This observation can be explained by ethanol disrupting the bacterial cell membrane and, therefore, disturbing the surface antigens to which the biotinylated antibodies bind, thus leading to decreased streptavidin-conjugated QD attachment. Another strategy of labeling bacterial cell mixtures was attempted in order to reproduce a more realistic test on a contaminated sample. Cells of each type were mixed before labeling, and the reagents were added in the following order: biotinylated antibodies, fluorophore-modified streptavidin, fixing agent (2% paraformaldehyde (PFA) in PBS), and PI. With this approach, detected red fluorescence events would be indicative of total bacterial cell number, whereas detected green events and detected double-positive red and green events would indicate the

amount of pathogenic E. coli O157:H7 in the mixture. Such simultaneous labeling was not possible for this system. Necessary for allowing PI to permeate the bacterial cell membranes, the 2% PFA solution used for fixing cells resulted in loss of all green QD fluorescence. Because an aqueous solution of equal parts 2% PFA and QDs in the absence of any bacteria retains the proper QD emission profile, the PFA does not seem to quench or affect the fluorophore in any way. Theoretically, stabilizing the proteinprotein interactions used here, the PFA may have actually disrupted the streptavidin-biotin or antibody-antigen interactions, thus causing a lack of detectable green events. Other chemical fixatives, such as methanol, ethanol, and acetone, work by destroying the cell membrane structure,17 a quite undesirable effect in cases of labeling surface antigens on a pathogen. CONCLUSIONS Resulting in brighter intensities, lower detection thresholds, and greater accuracy, QDs are improved fluorophores over a comparable amount of FITC to detect green-labeled pathogenic E. coli O157:H7 cells among increasingly greater amounts of harmless PI-labeled E. coli DH5R cells analyzed by flow cytometry. In fact, using a 1 order of magnitude greater amount of FITC does little to improve the results; QDs are still the superior fluorophore for bacterial cell labeling and detection. As demonstrated in these findings, QDs provide greater fluorescence intensity levels for flow cytometric analyses of cell mixtures containing low concentrations of pathogenic bacteria, which is ordinarily quite difficult due to their smaller volumes compared to mammalian cells. As a result of their smaller size, bacterial cells cannot be labeled with as many fluorophores, so the labels that do bind must be extremely bright for positive identification of cells to occur. The use of semiconductor quantum dots could potentially revolutionize the field of flow cytometry. With their broad absorption spectra, QDs could simplify the excitation sources of flow cytometers: a single excitation source in the UV could simultaneously excite all visible colors of CdSe QDs from blue to red. Unlike the disadvantage of organic dyes, there would be no need to circumvent the problem of simultaneous excitation of numerous fluorophores using FRET conjugates or complicated experimental setups with multiple excitation sources. Another advantage provided by semiconductor QDs is a result of their narrow emission spectra. As can be seen in the flow cytometry data presented here, PI overlaps into the green channel more than QDs overlap into the red channel. Such spectral cross talk, all too common with the broad, asymmetric emission of dyes, is diminished, and color compensation would be unnecessary if only QDs were used in a flow cytometric analysis. In addition, the greater absorption cross sections of semiconductor QDs over FITC on a fluorophore-to-fluorophore basis is evident here in the enhanced fluorescence intensities of labeled cells. As soon as the modification of semiconductor QDs with particular proteins like streptavidin or antibodies becomes as straightforward as the protocols already established for the incorporation of their organic dye counterparts, QDs can certainly provide the needed component for more sensitive fluorescence assays for bacterial contaminants. ACKNOWLEDGMENT The authors thank Brian Adrian Warsop for data acquisition from the flow cytometer. This work was supported by the New Analytical Chemistry, Vol. 80, No. 3, February 1, 2008

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York Office of Science Technology and Academic Research (C030086), the Camille and Henry Dreyfus Foundation, and the Air Force Office of Scientific Research (FA9550-04-1-0430).

independent cell counting methods for these bacterial samples. This material is available free of charge via the Internet at http:// pubs.acs.org.

SUPPORTING INFORMATION AVAILABLE Detailed outline of method for correcting the concentrations of bacterial cell mixtures of E. coli O157:H7 and E. coli DH5R; discussion of cell aggregation and debris; and comments on

Received for review August 31, 2007. Accepted November 20, 2007.

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