Anal. Chem. 2004, 76, 4806-4810
Quantum Dot Biolabeling Coupled with Immunomagnetic Separation for Detection of Escherichia coli O157:H7 Xiao-Li Su and Yanbin Li*
Department of Biological and Agricultural Engineering, The University of Arkansas, Fayetteville, Arkansas 72701
A sensitive, specific, and rapid method for the detection of E. coli O157:H7 was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with immunomagnetic separation. Magnetic beads coated with anti-E. coli O157 antibodies were employed to selectively capture the target bacteria, and biotin-conjugated anti-E. coli antibodies were added to form sandwich immuno complexes. After magnetic separation, the immuno complexes were labeled with QDs via biotin-streptavidin conjugation. This was followed by a fluorescence measurement using a laptop-controlled portable device, which consisted of a blue LED and a CCD-array spectrometer. The peak intensity of the fluorescence emission was proportional to the initial cell concentration of E. coli O157:H7 in the range of 103-107 CFU/mL with a detection limit at least 100 times lower than that of the FITC-based method. The total detection time was less than 2 h. Neither E. coli K12 nor Salmonella typhimurium interfered with the detection of E. coli O157:H7. Quantum dots (QDs) such as CdSe-ZnS core-shell nanocrystals are a brand new class of fluorescent markers with several important advantages compared to conventional fluorescent dyes.1,2 First, conventional dyes impose stringent requirements on the choice of appropriate optical systems including excitation sources and optical filters for the fluorescence measurement because of their narrow excitation and broad emission spectra with a relatively small Stokes shift, which means that the optimal excitation wavelength is close to the emission peak. Unlike conventional dyes, QDs have absorption spectra that increase dramatically to the blue of the emission, and although the absorption is broad, the emission is narrow, symmetric, and independent of the excitation wavelength. Hence, the optical systems can be simplified if QDs substitute for the conventional dyes as a fluorescent label. Second, QDs are advantageous over fluorescent dyes because of their long-term photostability and high quantum yield. Moreover, the emission color of QDs is tunable by changing the material composition and size of the cores, and * To whom correspondence should be addressed. Phone: (479) 575-2424. Fax: (479) 575-7139. E-mail:
[email protected]. (1) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (2) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016-2018.
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therefore simultaneous multianalyte detection can be realized by using multicolor QDs. Due to their extraordinary properties, QDs have found increasing applications in biological imaging and analyses including cell staining,1,3-5 DNA detection,6,7 cell surface receptor targeting,8 and immunoassays of IgG,2,9 ovalbumin,10 protein toxins such as staphylococcal enterotoxin B (SEB) and cholera toxin,11,12 and TNT.13 Most recently, Goldman et al.14 demonstrated simultaneous detection of cholera toxin, ricin, Shiga-like toxin 1, and SEB using four colors of QDs coated with antibodies. In the other hand, immunomagnetic separation (IMS) is a rapid, technically simple, specific, and efficient method that can be used for isolation of target bacteria directly from original or pre-enriched samples without any need for centrifugation or filtration. Most commonly, after magnetic separation, the bacteria captured by antibody-coated magnetic beads are determined by a conventional plating,15-19 which is reliable, but very time consuming, typically taking 18-24 h. Some methods have been (3) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 41-46. (4) Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Nat. Biotechnol. 2003, 21, 47-51. (5) Watson, A.; Wu, X.; Bruchez, M. Biotechniques 2003, 34, 296-303. (6) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631-635. (7) Parak, W. J.; Gerion, D.; Zanchet, D.; Woerz, A. S.; Pellegrino, T.; Micheel, C.; Williams, S. C.; Seitz, M.; Bruehl, R. E.; Bryant, Z.; Bustamante, C.; Bertozzi, C. R.; Alivisatos, A. P. Chem. Mater. 2002, 14, 2113-2119. (8) Rosenthal, S. J.; Tomlinson, I.; Adkins, E. M.; Schroeter, S.; Adams, S.; Swafford, L.; McBride, J.; Wang, Y.; DeFelice, L. J.; Blakely, R. D. J. Am. Chem. Soc. 2002, 124, 4586-4594. (9) Sun, B.; Xie, W.; Yi, G.; Chen, D.; Zhou, Y.; Cheng, J. J. Immunol. Methods 2001, 249, 85-89. (10) Speckman, D. M.; Jennings, T. L.; LaLumondiere, S. D.; Moss, S. C. Mater. Res. Soc. Symp. Proc. 2001, 676, Y3.6.1-Y3.6.6. (11) Goldman, E. R.; Balighian, E. D.; Mattoussi, H.; Kuno, M. K.; Mauro, J. M.; Tran, P. T.; Anderson, G. P. J. Am. Chem. Soc. 2002, 124, 6378-6382. (12) Lingerfelt, B. M.; Mattoussi, H.; Goldman, E. R.; Mauro, J. M.; Anderson, G. P. Anal. Chem. 2003, 75, 4043-4049. (13) Goldman, E. R.; Anderson, G. P.; Tran, P. T.; Mattoussi, H.; Charles, P. T.; Mauro, J. M. Anal. Chem. 2002, 74, 841-847. (14) Goldman, E. R.; Clapp, A. R.; Anderson, G. P.; Uyeda, H. T.; Mauro, J. M.; Medintz, I. L.; Mattoussi, H. J. Anal. Chem. 2004, 76, 684-688. (15) Wright, D. J.; Chapman, P. A.; Siddons, C. A. Epidemiol. Infect. 1994, 113, 31-39. (16) Chapman, P. A.; Wright, D. J.; Siddons, C. A. J. Med. Microbiol. 1994, 40, 424-427. (17) Karch, H.; Janetzki-Mittmann, C.; Aleksic, S.; Datz, M. J. Clin. Microbiol. 1996, 34, 516-519. (18) Chapman, P. A.; Malo, A. T. C.; Siddons, C. A.; Harkin, M. Appl. Environ. Microbiol. 1997, 63, 2549-2553. (19) Weagant, S. D.; Bound, A. J. Int. J. Food Microbiol. 2001, 71, 87-92. 10.1021/ac049442+ CCC: $27.50
© 2004 American Chemical Society Published on Web 07/10/2004
reported for rapid detection of the magnetic-bead-captured bacteria; they are either based on the use of enzyme-labeled secondary antibodies and an additional enzymatic reaction followed by an optical or electrochemical measurement20-24 or on the use of fluorophore-labeled secondary antibodies followed by epifluorescence microscopy25 or flow cytometry.26 The latter is more favorable for rapid analysis since it does not need the step of enzymatic amplification; however, it requires expensive equipment and must overcome the limitations of the conventional fluorophores that are mentioned above. In this study, an example was demonstrated for rapid detection of immunomagnetic-bead-captured bacteria using QDs as a fluorescent marker and an inexpensive portable spectrometer for the fluorescence measurement. E. coli O157:H7, a common cause of bloody diarrhea and other life-threatening diseases,27,28 was chosen as a model pathogen. The detection limit of the QD-based method was compared with that of the conventional method using fluorescein isothiocyanate (FITC) as a fluorescent label. To our best knowledge, this is the first example to combine IMS with QD biolabeling for bacterial detection. There are very limited literature reports about the application of QDs in bacterial detection. Recently, Zhu et al.29 reported a dual-color image analysis of Cryptosporidium parvum and Giardia lamblia spotted on a glass slide using QDs as labels, but they just tested a high cell concentration (107 cells/mL) without investigating the detection limit. EXPERIMENTAL SECTION Reagents. CdSe-ZnS quantum dot (QD)-streptavidin conjugates (10-15 nm in size) having a maximum emission wavelength of 609 nm (Qdot 605, catalog no. 1000-1) and the incubation buffer were obtained from Quantum Dot Corp. (Hayward, CA). Superparamagnetic, polystyrene microscopic beads covalently coated with affinity-purified polyclonal anti-E. coli O157 antibodies (Dynabeads anti-E. coli O157, diameter 2.8 µm, catalog no. 710.04) were purchased from Dynal Biotech Inc. (Lake Success, NY). Biotin-conjugated anti-E. coli antibodies (catalog no. B65109B) were supplied by Biodesign International (Saco, ME). Fluorescein isothiocyanate (FITC)-labeled affinity-purified anti-E. coli O157: H7 antibodies (catalog no. 02-95-90) were manufactured by Kirkegaard & Perry Laboratories (Gaithersburg, MD). Phosphate buffered saline (PBS, 0.01 M, pH 7.4) and 1% (w/v) bovine serum albumin (BSA)-PBS (pH 7.4) were received from Sigma-Aldrich Chemical Co. (St. Louis, MO). Ultrapure water (18 MΩ cm) produced by a Millipore Milli-Q system (Molsheim, France) was used throughout. Bacteria and Culture Plating Method. Escherichia coli O157: H7 (ATCC 43888), Escherichia coli K12 (ATCC 29425), and (20) Yu, H.; Bruno, J. G. Appl. Environ. Microbiol. 1996, 62, 587-592. (21) Perez, F. G.; Mascini, M.; Tothill, I. E.; Turner, A. P. F. Anal. Chem. 1998, 70, 2380-2386. (22) Tu, S. I.; Uknalis, J.; Gehring, A. J. Rapid Methods Autom. Microbiol. 1999, 7, 69-79. (23) Liu, Y.; Li, Y. J. Microbiol. Methods 2002, 51, 369-377. (24) Ruan, C.; Wang, H.; Li, Y. Trans. ASAE 2002, 45, 249-255. (25) Restanio, L.; Frampton, E. W.; Irbe, R. M.; Allison, D. R. Lett. Appl. Microbiol. 1997, 24, 401-404. (26) Seo, K. H.; Brackett, R. E.; Frank, J. F.; Hilliard, S. J. Food Prot. 1998, 61, 812-816. (27) Rowe, P. C.; Orrbine, E.; Wells, C. A. J. Pediatr. 1991, 119, 218-224. (28) Bunchnan, R. L.; Dolye, M. P. Food Technol. 1997, 51, 69-76. (29) Zhu, L.; Ang, S.; Liu, W. T. Appl. Environ. Microbiol. 2004, 70, 597-598.
Figure 1. Schematic diagram of the fluorescence measurement system.
Salmonella typhimurium (ATCC 14028) were all obtained from ATCC (American Type Culture Collection, Rockville, MD). The pure culture of E. coli O157:H7, E. coli K12, or S. typhimurium was grown in brain heart infusion (BHI) broth (Remel, Lenexa, KS) at 37 °C for 20 h before use. The culture was serially diluted to 10-8 with physiological saline solution, and the viable cell number was determined by conventional plate counting. E. coli O157:H7 and E. coli K12 dilutions were surface plated on sorbitolMacConkey (SMAC) agar (Remel, Lenexa, KS), the plates were incubated at 37 °C for 24 h, and the colonies developed were counted to determine the number of colony-forming units per mL (CFU/mL). S. typhimurium was enumerated in the same way except using xylose lysine tergitol (XLT4) agar (Remel, Lenexa, KS). For safety issues the undiluted cultures were heated in a 100 °C water bath for 15 min to kill all bacteria and then diluted to the desired concentrations with PBS for further use. Fluorescence Measurement System. Fluorescence measurements were performed on a laptop-controlled portable system as shown in Figure 1. The system consisted of a USB2000 miniature fiber-optic spectrometer, a USB-LS-450 LED light source module, an R400-7 UV-vis optical probe, all from Ocean Optics Inc. (Dunedin, FL), and a probe/cuvette holder housed in a plastic dark box. The spectrometer contained a low-cost 2048-element linear CCD-array detector with a working range of 360-900 nm. The LED module contained a blue LED, which had a spectral output peaking at 473 nm with a 27-nm fwhm (full bandwidth at half-maximum emission intensity). The optical probe was composed of a tight bundle of seven optical fibers in a stainless steel ferrule (six illumination fibers around one read fiber; each was 400 µm in diameter). Procedure. The whole analytical procedure could be divided into two steps. The first step was immuno incubation and IMS. An amount of 20 µL of IMBs specific to E. coli O157, 100 µl of 500 µg/mL biotin-conjugated anti-E. coli antibodies, and 1.0 mL of sample solution containing 0∼107 CFU/mL of E. coli O157:H7 were added to 1.7 mL microcentrifuge tubes and vortexed on a VSM-3 mixer (Shelton Scientific Mfg., Shelton, CT) for several seconds. The mixtures were incubated at room temperature for 60 min with gentle mixing on a RKVSD rotating mixer (Appropriate Technical Resources, Laurel, MD) at 10 rpm. Then, the microcentrifuge tubes were loaded into MPC-S magnetic particle concentrators (Dynal Biotech) and allowed 3 min for separation of the IMBs from the liquid matrixes. The liquid part was discarded, and the resulting sandwich immuno complexes consisting of IMBs, target bacteria, and biotin-conjugated anti-E. coli antibodies were rinsed with 1 mL of 1% BSA-PBS followed by IMS, which was repeated 3 times. The second step was QD labeling and fluorescence measurement. A total of 300 µL of 10 nM QD-streptavidin conjugates was added to the above microtubes containing the rinsed sandwich Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
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Figure 2. Principle of bacterial detection based on the combination of quantum dot biolabeling with immunomagnetic separation.
complexes. After vortexing, the mixtures were incubated at room temperature for 30 min with gentle rotation at 10 rpm, followed by IMS. Due to the conjugation of biotin and streptavidin, QDs were attached to the above sandwich complexes. The resulting complexes were washed with 1 mL of PBS followed by IMS 3 times. After the last IMS, the complexes were resuspended in 300 µL of PBS and transferred into 6-mm o.d. × 50-mm long borosilicate glass round cuvettes (VWR, catalog no. 47729-566) to measure the fluorescence emission spectra using an excitation wavelength of 473 nm. The blank (PBS) was subjected to the same treatment as that of the samples. After subtracting the background signal of the blank, the intensities of the fluorescence emission at 609 nm were correlated to the cell concentrations of E. coli O157:H7.
Figure 3. Fluorescence emission spectra obtained with an excitation wavelength of 473 nm and an integration time of 512 ms: (curve a) blank (PBS); (curve b) sample (107 CFU/mL of E. coli O157:H7); (curve c) sample - blank; (curve d) 10-nM quantum dots. Both blank and sample were subject to immunomagnetic separation and quantum dot labeling.
SAFETY CONSIDERATIONS Since E. coli O157:H7 and S. typhimurium are highly pathogenic, experiments directly utilizing these pathogens were conducted in a Biosafety Level 2 laboratory equipped with laminar flow biological hoods by trained personnel. RESULTS AND DISCUSSION Proof of Concept. Figure 2 illustrates the principle for the detection of E. coli O157:H7 with QDs as a marker. For simplification, only one antibody is shown on one immunomagnetic bead (IMB) and one streptavidin on one QD conjugate. In fact, one IMB carries numerous antibodies and one QD conjugate typically carries 12-25 streptavidins.30 Briefly, the detection was based on coupling IMS with QD biolabeling, i.e., the immuno complexes of IMBs, E. coli O157, and biotin-conjugated antibodies obtained with IMS were labeled with QDs via the strong biotin-streptavidin bond. This was followed by a fluorescence measurement using an inexpensive spectrometer consisting of a blue LED and a CCDarray detector. Figure 3 shows some typical fluorescence emission spectra. As can be seen, the blank (PBS), which was subjected to the same treatment as that of the sample (107 CFU/mL), gave no emission peak (curve a), while the sample had a maximum emission at 609 nm (curve b). To eliminate the influence of background reflection/backscattering, the blank spectrum was subtracted from the sample spectrum. The net spectrum of the sample also had a (30) Quantum Dot Corporation. Qdot Streptavidin Conjugates User Manual; PN 90-0003, revision 3.
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Figure 4. Effect of integration time on the fluorescence intensity: (curve a) sample (107 CFU/mL of E. coli O157:H7); (curve b) blank; (curve c) sample - blank.
peak emission at 609 nm (curve c), which was identical with that of the QD solution (curve d). This indicates the successful attachment of the QDs to the IMB-captured E. coli O157 cells. The excitation source used was a blue LED with a maximum emission at 473 nm; it excited the QD conjugates effectively and did not interfere with the fluorescence measurement. Another inherent benefit of blue light is that, unlike UV radiation commonly used as an excitation source for conventional fluorescent dyes, it does not kill cells, and therefore the system used in this study is also suitable for the assay of live cells. Effect of Integration Time. This study aimed to demonstrate a proof of concept of combining QD labeling with IMS for bacterial detection. Hence, no effort was made for extensive parametric optimization. However, the setting of integration time is critical for the fluorescence measurement. As shown in Figure 4, the intensities obtained at 609 nm for the sample (a), blank (b), and sample - blank (c) all increase linearly with integration time. To ensure a constant number of LED pulses during the integration time, the integration time was set to be powers of 2 as recommended by the manufacturer. An integration time of 1024 ms was
Figure 5. Typical fluorescence emission spectra (after subtracting the blank) obtained for detection of E. coli O157:H7; integration time, 1024 ms.
Figure 6. Fluorescence intensity as a function of the concentration of E. coli O157:H7; integration time, 1024 ms. Error bars ) (SDs (n ) 3∼5).
chosen for further experiments to maximize the peak intensity while not saturating the CCD detector. Analytical Characteristics. Typical fluorescence spectra of 101-107 CFU/mL E. coli O157:H7 are presented in Figure 5 after subtracting the blank. As shown in this figure, the spectra of 103107 CFU/mL all have a peak emission around 609 nm, but those of 101 and 102 CFU/mL do not. The peak intensity at 609 nm as a function of the cell concentration is illustrated in Figure 6. It is shown that the peak intensity increases with increasing cell concentration in the range of 103-107 CFU/mL. However, the signals of the blank and the samples of 101 and 102 CFU/mL are indistinguishable, which might be due to the high background reflection/backscattering of MBs. The detection limit obtained in this study was ca. 103 CFU/mL, and the total detection time, from adding a sample solution to obtaining the final result, was less than 2 h. These results are comparable to those of the IMS immunoassay methods20-24 and IMS flow cytometry,26 which have a detection limit of 102-105 CFU/mL with a detection time of 1-2 h. No photobleaching phenomenon was observed after the QDlabeled immuno complexes were exposed to room light for 24 h. The sample-to-sample reproducibility within the same batch was better than 10% RSD. For further comparison, FITC-labeled anti-E. coli O157 antibody solution containing ca. 170 nM FITC was tested for labeling the
Figure 7. Results of the specificity test. The bacterial concentrations were all 106 CFU/mL. Error bars ) SDs.
IMB-captured E. coli O157. Measured on the CCD spectrometer with the blue LED as the excitation source, the FITC-labeled antibody solution showed a peak fluorescence emission at 521 nm, and the relative peak intensity of FITC to QD was 1:230. However, the FITC-labeled IMB complexes did not give any emission peaks and the sample signals could not be discriminated from the blank at a concentration of up to 106 CFU/mL. A FluoroTec fluorometer (St. John Associates, Beltsville, MD) configured specifically for the FITC measurement was thus used for detecting the FITC-labeled IMB complexes. The fluorometer utilizes a 931 B photomultiplier tube (PMT) as a detector, which is more sensitive but also more expensive than the CCD detector. With the use of the PMT-based fluorometer, the FITC-labeled complexes were detectable at a sample concentration of g105 CFU/ mL. Hence, the QD labeling-based method proposed in this work is at least 100 times more sensitive than the FITC method in detecting E. coli O157:H7. The sensitivity of the IMS-QD method could be further improved by optimizing the concentrations of IMBs, biotinconjugated antibodies, and QDs as well as the incubation time to enhance the signal-to-background ratios at lower cell concentrations (
