Anal. Chem. 1998, 70, 2380-2386
Immunomagnetic Separation with Mediated Flow Injection Analysis Amperometric Detection of Viable Escherichia coli O157 Fidel G. Pe´rez* and Marco Mascini†
Dipartimento di Sanita` Pubblica, Epidemiologia e Chimica Analitica Ambientale, Universita` degli Studi di Firenze, via Gino Capponi 9, Firenze, Italy Ibtisam E. Tothill and Anthony P. F. Turner‡
Cranfield Biotechnology Centre, Cranfield University, Cranfield, Bedfordshire, MK43 OAL U.K.
The coupling of an immunological separation (using immunomagnetic beads) with amperometric flow injection analysis detection of viable bacteria is presented. Using a solution containing Escherichia coli O157, the electrochemical response with two different mediators [potassium hexacyanoferrate(III) and 2,6-dichlorophenolindophenol] was evaluated in the FIA system. Antibodyderivatized Dynabeads were used to selectively separate E. coli O157 from a matrix. The kinetics and the capacity parameters regarding the attachment of bacteria to the immunobeads were studied. The immunomagnetic separation was then used in conjunction with electrochemical detection to measure the concentration of viable bacteria. A calibration curve of colony-forming units (cfu) against electrochemical response was obtained. The detection limit for this rapid microbiological method was 105 cfu mL-1, and the complete assay was performed in 2 h. Some advantages over ELISA methods are the direct detection of viable cells (and not total bacterial load) and the need for only one antibody (not enzyme-labeled), thus making the assay faster (only one washing step is necessary) and less expensive. Medical, environmental, and industrial analysts are seeking rapid, inexpensive, and easy-to-use methodologies in order to investigate microbial concentration. Several rapid methods have been proposed (based on different measuring principles, such as biochemical, optical, physical, and electrochemical parameters), and some of them have been commercialized.1,2 Monitoring the electrochemical behavior of redox mediators in solutions containing viable bacterial cells has been studied and proposed as a simple method for detection and quantitation.3-6 †
E-mail:
[email protected]. http://www.igiene.unifi.it/Chimica/sensori. E-mail:
[email protected]. http://www.Cranfield.AC.UK/ibst. (1) Silley, P. Biosens. Bioelectron. 1994, 9, xv-xxi. (2) Hobson, N. S.; Tothill, I. E.; and Turner, A. P. F. Biosens. Bioelectron. 1996, 11, 455-477. (3) Fultz, M. L.; and Durst, R. A. Anal. Chim. Acta 1982, 140, 1-18. (4) Kala´b, T.; and Skla´dal, P. Electroanalysis 1994, 6, 1004-1008. ‡
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Immunological techniques have also been proposed. Antibodyderivatized surface plasmon resonance devices,7 the resonant mirror,8 piezoelectric crystals,9 and enzyme-linked immunosorbent assay (ELISA) techniques have been studied. Detection limits varied from one procedure to another, depending on the sensitivity of the transducing device and, e.g., the use of pre-enrichment cultures or mass-generating or optically active reagents carried by a second antibody. All of them, however, had one principal drawback: they could not discriminate between viable and nonviable cells. In this paper, a study of a model system using immunological selection coupled with electrochemical detection of viable bacteria in solution was performed. For the immunological step, immunomagnetic beads (Dynabeads) were selected as the immunocapture reagent. The bacterial strain selected was Escherichia coli O157. Electrochemical detection was carried out using redox mediators [potassium hexacyanoferrate(III) and 2,6-dichlorophenolindophenol (DCPIP)] (Figure 1). The measurement was performed using an FIA system. Dynabeads are uniform microparticles comprising superparamagnetic material wrapped in a polymer shell. They have an even dispersion of magnetic material (δFe2O3 and Fe3O4) throughout the bead and are coated with a polymer that allows the adsorption or coupling of various molecules. The possibility of maintaining (by shaking or rotating) the beads in suspension ensures a rapid and efficient binding of the target analytes. Shape and size uniformity prevent “clumping” and nonspecific binding due to irregularly shaped particles. Their superparamagnetic properties allow the quantitative magnetic separation of the beads and ensure that they retain no residual magnetism when removed from (5) Nishikawa, S.; Sakai, S.; Karube, I.; Matsunaga, T.; and Suzuki, S. Appl. Environ. Microbiol. 1982, 43, 814-818. (6) Ramsay, G.; and Turner, A. P. F. Anal. Chim. Acta 1988, 215, 61-69. (7) Haines, J.; Patel, P. D.; & Wahlstro ¨m, L. Fifth European BIAsymposium Proceedings, Stockholm, Sweden, 1995. (8) Watts, H. J.; Lowe, C. R.; and Pollard-Knight, D. V. Anal. Chem. 1994, 66, 2465-2470. (9) Minunni, M.; Mascini, M.; Guilbault, G. G.; and Hock, B. Anal. Lett. 1995, 28, 749-764. S0003-2700(97)00715-4 CCC: $15.00
© 1998 American Chemical Society Published on Web 04/24/1998
colitis and hemolytic uremic syndrome (HUS). It can be transmitted via contaminated food (like raw or rare ground beef and unpasteurized milk)20 and from person to person,21 e.g., in nursing homes22 and day care facilities.23 The currently accepted methods for the detection of E. coli O157 strains are based on the direct plating on sorbitol MacConkey agar (SMAC), cefixime-SMAC agar, or SMAC agar supplemented with cefixime and tellurite (CTSMAC) with subsequent serotyping, coupled with the detection of the toxins (directly or at the genomic level).15 Plasmid analysis and phage typing were also used for the identification of E. coli O157.13 This kind of analysis requires highly skilled operators and expensive procedures. A new approach to the rapid detection of viable E. coli O157 is presented in this paper.
Figure 1. Schematic model of the whole method, performed in three separate steps: the selective capture of E. coli O157 using antibodyderivatized magnetic particles (A), the reaction of bacteria with a mediator (B), and the electrochemical measurement of the reduced mediator using an amperometric method (C).
the magnetic field.10 They have been used for the selective separation of bacteria and their quantitation using different methods.11 The inoculation of the beads (with bacteria immobilized on their surfaces) on agar plates,12-15 the staining of the immobilized bacteria with a fluorescent dye,16 the development of ELISA tests,17,18 and the use of electrochemiluminescence19 were all accomplished using immunomagnetic separation. E. coli O157:H7 (or verocytotoxigenic E. coli) has been associated with two important human diseases: hemorrhagic (10) Dynal A. S. Cell Separation and Protein Purification. Technical Handbook, 2nd ed.; Dynal A.S.: Oslo, Norway, 1996. (11) Olsvik, Ø.; Popovic, T.; Skjerve, E.; Cudjoe, K. S.; Hornes, E.; Ugelstad, J.; and Uhle´n, M. Clin. Microbiol. Rev. 1994, 7, 43-54. (12) Wright, D. J.; Chapman, P. A.; and Siddons, C. A. Epidemiol. Infect. 1994, 113, 31-39. (13) Chapman, P. A.; Wright, D. J.; and Siddons, C. A. J. Med. Microbiol. 1994, 40, 424-427. (14) Cubbon, M. D.; Coia, J. E.; Hanson, M. F.; and Thomson-Carter, F. M. J. Med. Microbiol. 1996, 44, 219-222. (15) Karch, H.; Janetzki-Mittmann, C.; Aleksic, S.; Datz, M. J. Clin. Microbiol. 1996, 34, 516-519. (16) Lund, A.; Hellemann, A.; and Vartdal, F. J. Clin. Microbiol. 1988, 26, 25722575. (17) Cudjoe, K. S.; Hagtvedt, T.; and Dainty, R. Int. J. Food Microbiol. 1995, 27, 11-25. (18) Bennett, A. R.; MacPhee, S.; and Betts, R. P. Lett. Appl. Microbiol. 1996, 22, 237-243. (19) Yu, H.; and Bruno, J. G. Appl. Environ. Microbiol. 1996, 62, 587-592.
EXPERIMENTAL SECTION Materials and Reagents. General chemical and biological reagents were obtained from Sigma (Milano, Italy), Merck (Darmstadt, Germany), and Biolife (Milano, Italy). All chemical reagents used were of analytical grade. Deionized water was purified using a Milli-Q system (Millipore) and used for all the procedures. Immunomagnetic beads (Dynabeads M-280, 2.8 µm diameter) against E. coli O157 and a freeze-dried nonpathogenic strain of E. coli O157 were purchased from Dynal (Bromborough, U.K.). All bacterial cultures were incubated at 37 °C. Cultures in nutrient broth were grown on an orbital shaker at 150 rpm. A 50 mM sodium phosphate buffer, pH 6.9, containing 0.1 M KCl was used for the microbiological and electrochemical assays (PBS-KCl). Saline solution was prepared by dissolving 0.9 g of sodium chloride in 100 mL of pure water. Instrumentation. The FIA system comprised a Gilson Minipuls 2 peristaltic pump (Gilson, France), a Rheodyne 7125 model injection valve (Rheodyne Inc., Cotati, CA) with a 20-µL stainless steel loop, and a BAS Unijet MF-1014 (wall jet) electrochemical cell (Bioanalytical Systems Inc., West Lafayette, IN). The electrochemical cell had a platinum disk electrode of 3 mm diameter and a reference electrode of Ag, on which AgCl is chemically generated. The counter electrode was provided by one of the two blocks that constitute the body of the cell (stainless steel). The peristaltic pump was connected to the injection valve and to the Unijet electrochemical cell by Tygon tubes. The carrier solution was PBS-KCl at a flow rate of 100 µL min-1. The working electrode was polarized at +450 mV against the reference electrode. A commercial kit was used for cleaning the electrode (Bioanalytical Systems Inc.), comprising a velvety-textured cloth and alumina powder (0.05-µm particle size). The electrode was polished using alumina on the wet cloth. The electrode was then sonicated using a Vibra cell sonicator (Sonics & Materials, Danbury, CT) for 5 min in distilled water. Finally, it was cleaned with methanol before use. (20) Center of Disease Control 1993. Preliminary report: foodborne outbreak of Escherichia coli O157:H7 infections from hamburgerssWestern United States, 1993; MMWR 42:85-6. (21) Griffin, P. M.; and Tauxe, R. V. Epidemiol. Rev. 1991, 13, 60-98. (22) Carter, A. O.; Borczyk, A. A.; Carlson, J. A.; Harvey, B.; Hockin, J. C.; Karmali, M. A.; Krishnan, C.; Korn, D. A.; and Lior, H. N. Engl. J. Med. 1987, 317, 1496-1500. (23) Belongia, E. A.; Osterholm, M. T.; Soler, J. T.; Ammond, D. A.; Braun, J. E.; and MacDonald, K. L. J. Am. Med. Assoc. 1993, 269, 883-888.
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An Amel 559 potentiostat (Amel, Milano, Italy) was used for amperometric measurements, and the data were recorded on an Amel 868 chart recorder. An Autolab PGSTAT20 (EcoChemie, Utrecht, The Netherlands) was used for voltammetric analysis, and a Unicam 8625 UV/visible spectrometer (Analytical Technology Inc., Unicam, Milano, Italy) was used for the spectrophotometric measurements. The magnets used to concentrate the beads were circular central pole magnets (RS, Milano, Italy), made of sintered anisotropic ferrite (Feroba II). Solutions were not degassed, and positive feed back was not employed for the cyclic voltammetry measurement. Bacterial Cultivation Conditions and Harvesting Procedure. Freeze-dried bacteria were reconstituted by adding 0.5 mL of sterile nutrient broth to the ampule, leaving to rehydrate for a few minutes, and then transferring 0.25 mL to two bottles containing 10 mL of sterile nutrient broth. The culture was grown for 20 h. Bacterial cultures used for all experiments were grown in nutrient broth at 37 °C for 16 h. The cultures obtained in this manner were spun down for 10 min at 3500 rpm. The supernatant was discarded and the pellet resuspended in 10 mL of sterile saline solution. This solution was spun down again and the supernatant discarded. The pellet was resuspended in a final volume of 10 mL with sterile saline solution. This procedure was used for all experiments as the bacterial preparation method. All solutions containing bacteria (including the supernatants of centrifugations and the waste from FIA) were discarded in a 0.5% hypochlorite solution. To know the concentration of colony-forming units (cfu) in the bacterial suspensions, a calibration curve of absorbance against cfu per milliliter was performed. Four serial dilutions of a stock bacterial suspension (prepared as mentioned above) were made in sterile conditions in PBS-KCl. The numbers of colony-forming units per milliliter of each solution were estimated by a conventional serial dilution method with surface spreading of dilutions on nutrient agar 9-cm Petri dishes (with four replicates per treatment).24 The cultures were grown for 24 h at 37 °C. The absorbance of each one of the four solutions was measured. The results were plotted (absorbance versus cfu mL-1) and fitted using the linear regression least-squares method. The turbidity of the bacterial suspensions obtained each day was measured in order to know its cfu concentration. Study of the Binding Kinetics of E. coli O157 to Dynabeads and of the Binding Capacity of Dynabeads. All experiments were performed in 2-mL sterile polypropylene tubes. The general procedure involved the addition of 1.0 mL of bacterial solution and 20 µL (3 × 107 beads) of Dynabeads suspension to each tube, in sterile conditions. All incubation periods elapsed in a rotator at room temperature, to maintain beads in suspension and to facilitate the mixing of the solution. After each respective incubation period, tubes were removed from the rotator, and the beads were separated from the supernatant using a magnet. Beads were washed once with PBS-KCl-0.02% Tween 20 and twice with PBS-KCl. The original supernatant and all the washes from each tube were combined (nonattached bacterial solutions). The cfu mL-1 in each of these solutions and in the original (24) Standards Methods for the Examination of Water and Wastewater, 19th ed.; American Public Health Association: Washington, DC, 1995; Part 9215C.
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solutions were estimated by a conventional serial dilution method with surface spreading of dilutions on nutrient agar 9-cm Petri dishes (the inoculation was performed in triplicate for each solution). The cultures were grown for 24 h. The number of colonies was counted, and the total number of bacteria attached to the beads was calculated. For the determination of the binding kinetics, the concentration of the bacterial solution was constant (∼106 cfu mL-1), and incubation times ranged from 5 to 50 min. To determine the binding capacity of the immunomagnetic beads, Dynabeads were incubated with different solutions ranging from 102 to 108 cfu mL-1 of E. coli O157. The incubation time between beads and bacteria was 30 min. Additionally, beads were resuspended in PBS-KCl and serially diluted. The cfu attached to the beads were estimated by spreading a volume of each dilution on nutrient agar 9-cm Petri dishes (in triplicate for each solution) and cultured in the same conditions of the nonattached bacterial solutions. Determination of the Standard Potentials of Potassium Hexacyanoferrate(III) and 2,6-Dichlorophenolindophenol (DCPIP): Determination of the Working Potential for FIA. To determine the standard potentials of potassium hexacyanoferrate(III) and DCPIP, cyclic voltammetry was used. Cyclic voltammetry was performed in the solutions of mediators at a concentration of 5 mM (in PBS-KCl), at a scan speed of 20 mV s-1, using a working electrode of platinum (disk of 1 mm diameter). To reproduce the experimental conditions, the reference electrode was a Ag wire with electrogenerated AgCl. The counter electrode was a platinum net. The switching potentials were 350 and -50 mV for potassium hexacyanoferrate(III); 100 and -250 mV for DCPIP; and 500 and -250 mV for PBS-KCl. Scans started from the more positive potential. To fix the working potential, a hydrodynamic voltammetry of potassium hexacyanoferrate(II) was performed. The concentration of Potassium hexacyanoferrate(II) was 5 mM. Hydrodynamic voltammetry was performed in a beaker using the stirred-solution method, from 50 to 550 mV, using the same electrochemical cell as for the cyclic voltammetries. All voltammograms were performed without eliminating oxygen. Study of the Response of E. coli O157 in Solution with Potassium Hexacyanoferrate(III) and DCPIP. Study of the Effect of the Addition of Potassium Cyanide to the Mediator Mixture. All amperometric experiments were performed in 15mL glass-stoppered tubes. All solutions were prepared in PBSKCl. The solutions with the mediators (containing 4 mM glucose) were placed in a water-circulating bath at 37 °C. Then, the necessary volume of bacterial stock solution was added to each tube in order to reach the required concentration. After each respective incubation period in the water bath, a 1-mL aliquot was extracted from each tube, and three injections were performed in the FIA system. The current peak heights were recorded, that of the blank was subtracted, and the three measurements for each concentration were averaged. Each experiment had an appropriate blank solution containing 4 mM glucose and the corresponding mediator(s) and excluding E. coli. For the study of the behavior of E. coli O157 with the different mediators, three mediator solutions were used: 30 µM potassium
Table 1. Parameters Regarding the Binding Kinetics of E. coli O157 to Dynabeads M-280 in PBS-KCl time of incubation (min)
average no. of bacteria immobilized on 20 µL of beads
standard deviation (S)
fraction of total E. coli in solution retained on the beads (%)
stock solution 5 10 15 20 30 40 50
97 × 104 a 4 × 105 56 × 104 64 × 104 70 × 104 78 × 104 78 × 104 76 × 104
8 × 104 1 × 105 6 × 104 2 × 104 2 × 104 6 × 104 2 × 104 1 × 104
41 58 66 72 80 80 78
a
Average of cfu of the beads).
mL-1
in the original solution (before the addition
hexacyanoferrate(III), 30 µM DCPIP, and 30 µM potassium hexacyanoferrate(III) + 30 µM DCPIP (mediator mixture). The bacterial concentration was 107 cfu mL-1. Measurement of the amperometric response of the solutions of each tube were performed at different times from 0 to 130 min. Concomitantly, the absorbances of both solutions containing DCPIP were measured at the maximum absorption wavelength of 600 nm (spectrum scan data not shown). For the study of the influence of potassium cyanide in the reaction between bacteria and mediator mixture, harvested bacteria were added in order to reach 105 and 106 cfu mL-1. Two experiments were performed, with and without the addition of 2 mM potassium cyanide. The solutions were incubated for 1 h, and the amperometric responses were measured in the FIA system. Calibration Curve of E. coli O157 Obtained by Coupling the Immunomagnetic Separation with Electrochemical Detection. In each of seven 4-mL stoppered polypropylene tubes was placed 20 µL of Dynabeads against E. coli O157, and then 1.5 mL of bacterial solution was added, ranging from 103 to 6.5 × 108 cfu mL-1. The solutions were incubated at room temperature for 30 min in a rotator. The magnetic beads were then separated with a magnet, and the supernatants were discarded. The magnetic beads were washed once with PBS-KCl with the addition of 0.02% Tween 20 and then twice with PBS-KCl. The blank assay was processed as the samples, but without addition of bacteria. Finally, 0.5 mL of the mediator mixture was added to each tube, and they were left to incubate for 1 h at 37 °C in a circulating water bath. After that, the magnetic beads were separated with a magnet, and each supernatant was injected in triplicate into the FIA system. The current peak heights were recorded, that of the blank was subtracted, and the three measurement for each concentration were averaged. RESULTS AND DISCUSSION Binding Kinetics and Capacity of Dynabeads against E. coli O157. Table 1 shows the relationship between the incubation time of the bacteria with the beads and the percentage (recovery) of bacteria bound on their surface. As can be seen, the reaction is fast, retaining about 80% of the bacteria in 30 min. Thus, 30 min was taken as the incubation time for further experiments. Experiments on binding kinetics using less con-
Table 2. Parameters Regarding the Binding Capacity of Dynabeads M-280 against E. coli O157 in PBS-KCla
E. coli O157 concn (cfu mL-1) 1.6 × 1.6 × 103 1.6 × 104 1.6 × 106 1.6 × 107 1.6 × 108 102
average no. of bacteria immobilized on 20 µL of beads SUP
PART
b 15 × 102 155 × 102 126 × 104 960 × 104 688 × 105
140 14 × 102 157 × 102 110 × 104 752 × 104 192 × 105
standard deviation (S) SUP
PART
b 40 1 × 102 2 × 102 2 8 × 10 7 × 102 1 × 104 2 × 104 7 × 104 3 × 104 6 × 105 7 × 105
fraction of total E. coli in solution, retained on the beads (%) SUP
PART
∼100 94 97 79 60 43
87 87 98 69 47 12
a SUP, data obtained by plating the supernatant and the washings; PART, data obtained by plating the beads. b Bacteria undetected in the cultures.
centrated bacterial suspensions were not performed, because, as can be seen in the experiment on bead capacity, almost all bacteria are retained in this time for concentrations smaller than 106 cfu mL-1. For more concentrated solutions, the amount of bacteria in solution might be high enough to saturate the binding sites of beads in 30 min. Table 2 shows the results of the experiments to determine the capacity of the beads. Using the data obtained from the cultures of the supernatants, at low concentrations the beads captured almost all the bacteria from the solution in a period of 30 min of incubation. At higher concentrations, the percentage of bacteria bound to the surface of the beads decreased, but the absolute number of bound bacteria continued to increase. The recovery (percentage) data obtained in our laboratories differ from those obtained by Fratamico et al.25 for the recovery of E. coli O157 from PBS using immunomagnetic separation. In fact, they found that 60% of bacteria recovered from PBS when the cfu mL-1 concentration was 102, and 39% when the cfu mL-1 concentration was 5 × 104 (for an incubation time of 60 min). Three differences in methodology might explain this. (1) The bead coating procedure. The magnetic beads used in this investigation were coated through a direct procedure with affinity-purified polyclonal anti-E. coli O157 (covalently bound antibodies), while those used by Fratamico et al.25 were coated with sheep anti-rabbit IgG, and then a secondary antibody (rabbit antiserum against E. coli O157:H7) was added. This latter approach effectively introduces a spacer arm, potentially furnishing greater access for large molecules and increased flexibility in the ligand.26 However, the higher capacity of our beads showed that a greater number of antibodies were attached to the surface. (2) The counting method. Fratamico et al.25 performed a direct method (surface spreading of the beads on agar plates), while our method was indirect (surface spreading of the supernatant and the washing solutions). When cfu determination of bacteria attached to beads is performed, the possibility that more than one cell can generate a colony must be taken into consideration: in (25) Fratamico, P. M.; Schultz, F. J.; and Buchanan, R. L. Food Microbiol. 1992, 9, 105-113. (26) Hermanson, G.; Krishna Mallia, A.; Smith, P. A. Immobilized Affinity Ligand Techniques; Academic Press: San Diego, CA, 1992; Chapter 3.1.1, pp 137140.
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Figure 2. Cyclic voltammograms of potassium hexacyanoferrate (PH), DCPIP, and PBS-KCl. Scan rate, 20 mV s-1 (all the experiments); working electrode, platinum disk, 1 mm diameter; reference electrode, Ag/AgCl; counter electrode, platinum net; standard potentials, 0.138 and -0.094 V for potassium hexacyanoferrate and DCPIP, respectively. (b) Hydrodynamic voltammogram of potassium hexacyanoferrate using the same electrochemical cell.
optimal conditions, more than one cell can attach to one bead, giving a false smaller recovery.11 This could be the reason for the results we obtained when beads were plated. As can be seen in Table 2, the results obtained when beads were plated were generally smaller than those obtained when supernatant was plated, especially at high concentrations. The culture of supernatants and washings would give a better result in this sense. Adsorption of bacteria to the tubes could be a reason for our higher recovery results. However, adsorption was not considered to be a problem, because, as demonstrated by Wright et al.,12 E. coli O157 do not attach to polypropylene. Besides, Tween 20 was used in washing steps, and this would detach adsorbed bacteria from the tube walls. (3) The bead diameter. the beads used in this work were M-280 (diameter 2.8 µm), while those used by Fratamico et al.25 were M-450 (diameter 4.5 µm). With an approximately double mass of beads (approximately 4.3 × 10-4 g for M-280 and 1.4 × 10-4 g for M-450), the surface of beads exposed to the solution dramatically increases (approximately 3.4 × 10-3 m2 for M-280 and 5.7 × 10-4 m2 for M-450). Then, a greater surface could impact on the efficiency (kinetics) and the capacity (higher quantities of antibody) of the binding. Determination of the Standard Potentials of Potassium Hexacyanoferrate(III) and 2,6-Dichlorophenolindophenol (DCPIP): Determination of the Working Potential for FIA. Figure 2 shows the voltammograms of potassium hexacyanoferrate, and DCPIP, PBS-KCl and the hydrodynamic voltammogram of potassium hexacyanoferrate. The standard potentials for potassium hexacyanoferrate and DCPIP were respectively 138 and -94 mV vs AgCl, as determined by averaging the peak potentials. The hydrodynamic voltammetry of potassium hexacyanoferrate (the species with higher redox potential) showed a plateau at ∼350 mV. Then, 450 mV was selected as the working potential for further experiments. The PBS-KCl voltammogram shows that 2384 Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
Figure 3. Amperometric FIA response of E. coli O157 (107 cfu mL-1) to potassium hexacyanoferrate(III) 30 µM (b), to DCPIP 30 µM (9), and to the mixture of both (30 µM each) ([) in PBS-KCl. Glucose concentration, 4 mM. Temperature, 37 °C. Each value is the average of three measurements, and the error bars represent the standard deviation of the measurements.
redox species are present neither in the range in which cyclic voltammograms were done nor in the region in which further amperometric FIA measurement were performed. Amperometric Response of E. coli O157 in Solution with Potassium Hexacyanoferrate(III) and DCPIP. Effect of the Addition of Potassium Cyanide to the Mediator Mixture. Figure 3 shows the electrochemical response of E. coli O157 with the mediators assayed. Potassium Hexacyanoferrate(III) showed the smaller response. The response with DCPIP plateaued after about 60 min. The greater response was shown by the mediator mixture. The current values obtained with the mediator mixture cannot be explained simply as the addition of the responses of the bacteria to each mediator separately, because the response of the mediator mixture is higher than the sum of the responses of each mediator alone. It was demonstrated that the electrochemical response of E. coli using potassium hexacyanoferrate(III) is concentration dependent, having a maximum response (with no further increase of the response at higher concentrations of mediator) at 12 mM potassium hexacyanoferrate(III).6 Mediator concentrations higher than 30 µM were tried in this work, to enhance the bacterial electrochemical response, but reproducibility declined with this approach, possibly because of fouling of the electrode.27 Spectrophotometric data showed that in the experiment with DCPIP the response was due to the reduction of DCPIP (Figure 4a). With a mediator mixture, however, the absorbance due to DCPIP initially remained constant, while the amperometric response rose at an enhanced level (Figure 4b). The relative redox potentials of the mediators suggest that hexacyanoferrate(III) can oxidize reduced DCPIP, and this was confirmed experimentally. Thus, the results are consistent with electron transfer from the bacteria to both mediators, but with reduced DCPIP being recycled to the oxidized (27) Kawiak, J.; Jedral, T.; and Galus, Z. J. Electroanal. Chem. 1983, 145, 163171.
Figure 5. Amperometric FIA calibration curve of E. coli O157 performed by coupling the immunomagnetic separation and the further reaction with the mediator mixture (30 µM each mediator in PBSKCl). Glucose concentration, 4 mM; temperature, 37 °C; incubation time, 1 h. Each value is the average of three measurements, and the error bars represent the standard deviation of the measurements.
Figure 4. Comparison of spectrophotometric (9) (at 600 nm, path length 1 cm) and amperometric (b) responses of E. coli O157 using (a) DCPIP 30 µM in PBS-KCl and (b) DCPIP + potassium hexacyanoferrate(III) (30 µM each) in PBS-KCl. Glucose concentration, 4 mM; temperature, 37 °C.
form by hexacyanoferrate(III): E. coli O157
Fe(CN)63- 98 Fe(CN)64E. coli O157
DCPIPox 98 DCPIPred DCPIPred + Fe(CN)63- f DCPIPox + Fe(CN)64-
The transfer of electrons from the respiratory chain of bacteria appears to be associated with both DCPIP28 and potassium hexacyanoferrate(III). Ramsay and Turner6 demonstrated that hexacyanoferrate(III) might be reduced between NADH dehydrogenase and the terminal oxidases of E. coli respiratory chain (placed in the bacterial membrane). As the reaction progresses in the current scheme, hexacyanoferrate(III) becomes fully reduced by the bacteria and, hence, becomes unavailable to recycle reduced DCPIP. At this point DCPIP appears in the reduced form (Figure 4b). Given the synergistic response due (28) Ikeda, T.; Kurosaki, T.; Takayama, K.; Kano, K.; and Miki, K. Anal. Chem. 1996, 68, 192-198.
to the above mechanism, a mixture of mediators was used for all further experiments. In aerobic conditions, oxygen is the final acceptor of electrons from the respiratory chain of aerobic bacteria and could be an interference when mediators (that can be reduced at the cytochromes) are used to measure respiratory activity. Ramsay and Turner6 (for E. coli using potassium hexacyanoferrate(III) as soluble mediator) and Kala´b and Skla´dal4 (for Paracoccus denitrificans using ferrocene, tetrathiafulvalene, and tetracyanoquinodimethane included in a carbon paste electrode) demonstrated some increase in the amperometric response by selectively inhibiting with KCN the transfer of electrons from the terminal oxidases to oxygen (at cytochromes O and d). Responses were 40 and 200 nA (with a standard deviation of 10 nA each) for the experiment without potassium cyanide, for 105 and 106 cfu mL-1 respectively. The experiment performed with the addition of 2 mM potassium cyanide gave the following results: 36 and 190 nA (with standard deviations of 8 and 10 nA, respectively) for 105 and 106 cfu mL-1. There were no significant differences between the experiments with and without KCN, contrary to the results obtained by Ramsay and Turner6 and by Ka´lab and Skla´dal.4 These data were, however, consistent with those obtained by Ikeda and co-workers28 for Pseudomonas fluorescens using DCPIP as mediator. In fact, this group of researchers demonstrated that DCPIP is equally as effective an electron acceptor as oxygen. The addition of KCN to the solution in order to avoid the transfer of electrons to oxygen, in this case, would be unnecessary. KCN was not used in subsequent experiments. Calibration Curve of E. coli O157 Obtained by Coupling Immunomagnetic Separation with Electrochemical Detection. Figure 5 shows a typical result obtained for a calibration curve (curve fitted through a polynomial third-order equation) for E. coli O157 by coupling the immunomagnetic separation with Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
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the FIA amperometric detection. The standard deviation for the injection of the blanks was ∼10 nA. Using three times the noise, the limit of detection is ∼105 cfu mL-1. Above 108 cfu mL-1, the signal tends toward a plateau. Bouvrette and Luong29 proposed an immunoreactor flow system for measuring E. coli. Their system and the system presented here are based on the same principle: the selective immunological separation of a bacterial strain and the generation of a signal by bacterial cells. Even if the time necessary to complete the analysis with our method is slightly longer (2 h vs 27 min), the limit of detection is much lower (105 vs 5 × 107 cfu mL-1) Several ELISA methods were proposed for bacterial detection in the late 1970s,30 using different surfaces for the immobilization of antibodies or antigens. These methods became very popular, and several microbiology laboratories use them as routine detection methods. Microtiter plate-based assays is the format proposed in most commercial kits. However, the main drawback of microtitration plates is the minimal volume of sample they can hold (maximum 300 µL), while immunomagnetic separation could be achieved with sample volumes up to 50 mL. This is of extreme importance in analysis in which only a few cells can be found in some grams of sample. CONCLUSIONS In this paper, we studied a model method that demonstrated the suitability of immunomagnetic separation to rapidly preconcentrate microbial cells over a wide range of concentrations and (29) Bouvrette, P.; and Luong, J. H. T. Int. J. Food Microbiol. 1995, 27, 129137. (30) Krysinski, E. P.; and Heimsch, R. E. Appl. Environ. Microbiol. 1977, 33, 947-954.
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the possibility to use electrochemical FIA detection of viable microbial cells, using E. coli O157 as an example. Interferences and specificity were not studied. In addition, we demonstrated the suitability of a mixture of potassium hexacyanoferrate(III) and DCPIP to measure the respiratory activity of E. coli. This measurement method showed an enhanced response with respect to the experiments performed with each mediator separately. We also demonstrated that the addition of potassium cyanide to the reaction, to prevent oxygen interference, is not necessary. This suggested that DCPIP is (for E. coli) an equally effective electron acceptor as oxygen. We obtained maximum responses without resorting to addition of a respiratory inhibitor or sparging with an inert gas. Some advantages over ELISA methods are the direct detection of viable cells (and not total bacterial load) and the need for only one antibody (not enzyme-labeled), thus making the assay faster (only one washing step is necessary) and less expensive. The detection limit of 105 cfu mL-1, however, would obviously require the inclusion of a fast, nonselective pre-enrichment procedure in order to detect low amounts of bacteria present in food or environmental samples. ACKNOWLEDGMENT F.G.P. gratefully acknowledges the provision of a postgraduate scholarship by the Universidad Nacional del Litoral, Santa Fe, Argentina. Received for review July 7, 1997. Accepted March 7, 1998. AC970715T
