Detection of Escherichia coli O157:H7 Using Interdigitated Array Microelectrode-Based Immunosensor Y. Wang, R. Wang, Y. Li, B. Srinivasan, S. Tung, H. Wang, M. F. Slavik, C. L. Griffis* ABSTRACT. In this study, a label-free interdigitated array (IDA) microelectrode-based immunosensor was designed and tested for rapid and sensitive detection of E. coli O157:H7. The surface of gold IDA microelectrodes (25 pairs of fingers with 700 μm length and 10 μm width and space) was modified with protein A and further immobilized with polyclonal antibodies against E. coli O157:H7. A 20 μL sample containing E. coli O157:H7 in 0.01 M phosphate buffered saline (PBS) at pH 7.4 was dropped on the surface of the modified microelectrodes and incubated for 2 h at room temperature. After the target bacteria were captured by the antibodies immobilized on the microelectrode, a wash with PBS was applied to rinse off all unbound parts of the sample. Finally, the impedance, both magnitude and phase angle, was measured over a broad frequency range from 1 Hz to 1 MHz. The results showed that the impedance in a frequency range of 1 Hz to 1 kHz increased with the increasing number of E. coli O157:H7 in a range from 80 to 2 × 105 cells per 20 μL. A linear relationship between the magnitude of measured impedance and the log number of cells was determined at the frequency of 1 kHz. This biosensing method was able to detect as few as 80 cells of E. coli O157:H7 within 2 h. The specificity of the immunosensor was validated using five non-target bacteria, and none of them generated detectable signals. The application of the immunosensor for food safety was demonstrated with food samples including chicken carcasses, ground beef, and fresh cut broccoli inoculated with E. coli O157:H7. At the same time, the capture of E. coli O157:H7 cells on the microelectrode surface was confirmed using atomic force microscopy. Keywords. E. coli O157:H7, Immunosensor, Impedance measurement, Interdigitated array microelectrode, Rapid detection.
Submitted for review in November 2008 as manuscript number BEJ 7802; approved for publication by the Biological Engineering Editorial Board of ASABE in April 2010. The authors are Yun Wang, Graduate Student, Ronghui Wang, Senior Research Associate, and Yanbin Li, ASABE Fellow, Professor, Department of Biological and Agricultural Engineering; Balaji Srinivasan, Graduate Student, and Steve Tung, Associate Professor, Department of Mechanical Engineering; Hong Wang, Program Associate, and Michael F. Slavik, Professor, Department of Poultry Science; and Carl L. Griffis, Professor, Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, Arkansas. Corresponding author: Yanbin Li, Department of Biological and Agricultural Engineering, 203 Engineering Hall, University of Arkansas, Fayetteville, AR 72701; phone: 479-575-2424; fax: 479-575-7139; e-mail:
[email protected]. Biological Engineering 2(2): 49-62
© 2010 ASABE ISSN 1934-2799
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E
scherichia coli O157:H7, which may cause diarrhea and renal insufficiency, sometimes resulting in death, is one of the major foodborne pathogens. Food products, such as raw or cooked poultry and meat, dairy products, fresh vegetables and fruits, can be easily contaminated with E. coli O157:H7. In recent years, outbreaks of foodborne diseases associated with E. coli O157:H7 include those related to consumption of fresh fruits and vegetables contaminated during cultivation or handling (WHO, 2005). In 2006, a nationwide outbreak of E. coli O157:H7 in spinach from 26 states caused 204 cases of human sickness, 104 hospitalizations, 31 cases involving kidney failure, and three deaths (FDA, 2006). In October 2007, ground beef products contaminated with E. coli O157:H7 caused the illness of 25 people in eight states and the recall of 21.7 million pounds of meat products (USDA, 2007). In order to reduce the risk of E. coli O157:H7 outbreaks, rapid, sensitive and specific methods to identify the target pathogen are needed to control the spread from production to retail of agricultural and food products as well as water supply. The conventional culture plating method for detection of E. coli O157:H7 takes several days to get results (Sanderson et al., 2007). Other detection methods, such as polymerase chain reaction (PCR) and real-time PCR (Oberst et al., 1998; Ibekwe and Grieve, 2003), enzyme-linked immunosorbent assay (ELISA) (Sunwoo et al., 2006), and quartz crystal microbalance (QCM) (Wu et al., 2007; Shen et al., 2007) have been developed based on nucleic acid, enzyme, or antibody-antigen reactions. However, these methods have their own limitations. The PCR method requires complex sample preparation, including pre-enrichment and skillful manipulations. ELISA takes a relatively long analysis time due to multistage procedures (Park et al., 2008), and QCM lacks sensitivity. Development of biosensor technology in the last decade has provided rapid, specific, and sensitive detection methods for various substances such as chemicals, bacteria, and other biomolecules in agricultural production, food processing, and environmental monitoring. Various sensing materials and transducing technologies have been employed in biosensors. Among the sensing materials, antibodies with properties such as stability and specificity are an attractive sensing material for the purpose of constructing a high-affinity interface between an analyte and a transducer, and immunosensors based on antibody-antigen interaction have been well developed. The interaction can be measured with different transducers. Electrochemical biosensors measure the change in electrical properties of electrode structures as bacterial cells are entrapped or immobilized on or near the electrode. Impedimetric analyte detection, where the binding event results in a change in the electrical surface properties, is frequently applied for immunosensing (Lisdat and Schaefer, 2008). Measuring the electrical impedance of an interface in AC steady state with constant DC bias conditions, impedance biosensors are powerful tools to probe the features of surface-modified electrodes and the chemical processes associated with conductive support (Bard and Faulkner, 1980; Daniels and Pourmand, 2007). Electrochemical impedance spectroscopy (EIS) measurement with immunosensing material is considered an effective way to detect bacteria and viruses, as reported in recent studies of interdigitated array (IDA) microelectrode-based EIS measurement for bacteria detection (Yang et al., 2004a, 2004b; Elsholz et al., 2006; Varshney and Li, 2007). Advantages of IDA microelectrodes include maximizing impedance changes at the surface, reducing detection time, minimizing interfering effects of non-target analytes in solution, requiring small sample volume, and presenting a high signal-to-noise
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ratio (Thomas et al., 2004; Radke and Alocilja, 2005). Because distances between electrodes are in the range of microns or nanometers, IDA microelectrodes can overcome the obstacle of detecting particles with micro- or nano- sizes, such as bacteria, DNA, and viruses whose sizes cause difficulties for conventional electrode systems (usually 1 cm distance between electrodes). For most E. coli detection systems based on antibody immobilization and impedance measurement, redox probes such as Fe(CN)63-/4- are included in the detection as Faradaic impedance measurements for obtaining detectable signals and quantifying bacterial cells (Ruan et al., 2002; Yang et al., 2004b). The influence of the electrical field on the biological recognition event can also be investigated by capacitance measurements made in the absence of Faradaic currents (Lisdat and Schaefer, 2008), which is more suitable for rapid and in-field detection. The current work describes the development of a gold IDA microelectrode-based immunosensor with non-Faradaic impedance measurement and demonstrates that the immunosensor could be used for rapid and sensitive detection of E. coli O157:H7. In this study, anti-E. coli antibodies were immobilized onto the surface of a gold microelectrode via the linkage by protein A, which can help control the amount and orientation of immobilized antibodies on a gold surface (Briand et al., 2006). E. coli suspensions at different concentrations in PBS were incubated, followed by the non-Faradaic impedance measurement. Both immobilization of antibodies and binding between the immobilized antibodies and target bacteria were characterized by impedance analysis. The measurement in absence of a redox probe in PBS may maintain the activities of live bacterial cells for further study. To better understand the impedance immunosensor, an equivalent circuit was generated to interpret changes in the impedance and to investigate contributions of each component in the circuit to the impedance signal. Effects of E. coli concentration on the impedance signal are discussed, and the detection limit and sensitivity of the immunosensor were determined. The specificity of the immunosensor was tested using five non-target bacteria, and the application of the immunosensor was demonstrated with three types of food samples inoculated with E. coli O157:H7. Atomic force microscopy (AFM) was used to characterize cells of E. coli O157:H7 captured by the antibodies immobilized on the IDA microelectrode surface.
Experimental Section Chemicals and Reagents Protein A from Staphylococcus aureus was purchased from Sigma-Aldrich (St. Louis, Mo.) and stored at 4°C. It was dissolved in 0.01 M phosphate buffered saline (PBS) to 2 mg mL-1 and stored in a refrigerator. Affinity purified goat anti-E. coli O157:H7 antibody (1.0 mL, 3.2 mg mL-1) was obtained from Biodesign International (Saco, Maine). A 1:3 dilution, which gave an antibody concentration of ~1 mg mL-1, i.e., the optimized concentration for immobilization used in our previous studies (Ruan et al., 2002; Yang and Li, 2005), was prepared with 0.01 M PBS before use. Phosphate buffered saline (PBS; 0.01 M, pH 7.4) was purchased from Sigma-Aldrich (St. Louis, Mo.). A 1% (w/v) solution of bovine serum albumin (BSA) in PBS was prepared by dissolving 1 g bovine serum albumin (BSA; EM Science, Gibbstown, N.J.) in 0.01 M PBS buffer. Wash solution from KPL (Gaithersburg, Md.) was used to clean the IDA 2(2): 49-62
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microelectrodes before and after each test. All other solutions were prepared with deionized (DI) water (Milli-Q, 18.2 MΩ·cm, Millipore, Bedford, Mass.). Bacterial Cultures E. coli O157:H7 (ATCC 43888), Listeria monocytogenes (ATCC 43251), Salmonella Typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 25923), E. coli K12 (ATCC 29425) and Campylobacter jejuni (ATCC 33291) were obtained from American Type Culture Collection (Rockville, Md.). Stock cultures of E. coli O157:H7, L. monocytogenes, S. Typhimurium, S. aureus, and E. coli K12 were grown for 18 to 20 h at 37 °C in brain heart infusion (BHI) broth (Remel, Lenexa, Kans.). C. jejuni was inoculated into Bolton enrichment broth and incubated at 42°C for 20 h under microaerobic conditions. Serial 10-fold dilutions were made in 0.01 M PBS (pH 7.4). The viable cell numbers of E. coli O157:H7, L. monocytogenes, S. Typhimurium, S. aureus, E. coli K12, and C. jejuni were determined by surface plating 0.1 mL of the appropriate dilutions onto MacConkey sorbitol agar (Remel), modified Oxford Listeria agar (EMD Chemicals, Inc., Gibbstown, N.J.), XLT4 agar (Remel), MacConkey sorbitol agar (Remel), Mannitol salt agar (BD Diagnostics, Franklin Lakes, N.J.), and Campy-Cefex agar (Hardy Diagnostics, Santa Maria, Cal.), respectively, and colonies were counted after incubation at 37°C for 24 h (for E. coli O157:H7, S. Typhimurium, S. aureus, and E. coli K12) or 48 h (for L. monocytogenes). C. jejuni plates were placed in a CO2 incubator filled with a mixture of 5% O2, 10% CO2, and 85% N2 gasses to provide a microaerobic atmosphere and incubated for 48 h at 42°C before the colonies were counted. The cultures were thermally killed by boiling them in glass tubes placed in a water bath for 15 min for further use. Food Sample Preparation and Inoculation Chicken carcasses, ground beef, and fresh cut broccoli were purchased from a local grocery store and kept in a cooler at 4°C. Each of five chicken carcasses was individually washed by shaking it in a sterile plastic bag filled with 100 mL of 0.1% PBS. Ten mL of chicken carcass wash solution was collected from each chicken sample and inoculated with 4 × 106 cfu mL-1 E. coli O157:H7 bacteria culture for use in the tests. Each of five 25 g samples of ground beef or five 25 g samples of fresh cut broccoli was mixed with 225 mL of 0.1% PBS in a stomacher and stomached for 1 min. Ten mL of stomaching solution were collected from each ground beef or fresh cut broccoli sample and inoculated with 4 × 106 cfu mL-1 E. coli O157:H7 bacterial culture for use in the tests. IDA Microelectrodes and Surface Modification A gold IDA microelectrode chip was designed and fabricated in the High-Density Electronics Center (HiDEC) at the University of Arkansas. Briefly, the chips were fabricated on a glass wafer with a 500 nm gold layer on 25 nm Cr as the adhesion layer. The wafer was pin coated with AZ 4330 photoresist in the thickness of 4 μm. Photolithography was used to pattern the photoresist, and the resist was developed. The electrodes were obtained by etching the Au and Cr on the wafer with the patterned photoresist as a masking layer. The length of each electrode was 0.7 mm. Both the widths of the digits and the spaces between them were 10 μm. The area of 25-pair electrodes was 0.703 mm2, and the perimeter of this area was 3.4 mm. Before use and
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after each test, each chip was gently wiped with lens paper wetted with wash solution to remove the reactants binding on the surface of the electrodes and glass substrate. Antibodies were immobilized onto the surface of the electrodes through protein A linkage. First, the surface area of the electrodes was covered with a drop (20 μL) of protein A and incubated for 2 h at room temperature. After incubation, the electrodes were rinsed with DI water to remove unbound protein A and dried with a nitrogen stream. Then, 20 μL of 1.07 mg mL-1 anti-E. coli O157:H7 antibody was incubated at 4°C overnight. Finally, 1% (w/v) BSA solution in the same volume was incubated for 30 min at room temperature after the antibody immobilization to block non-specific binding. The chips were ready for testing after rinsing with DI water for 20 s and drying in a nitrogen stream for 2 min. After each test, the surface of the electrodes was cleaned, and the above procedure beginning with protein A incubation was performed. Detection of E. coli O157:H7 Twenty μL of E. coli O157:H7 suspension in 0.01 M PBS (pH 7.4) was dropped on the surface of the microelectrode coated with the antibody and incubated for 2 h at room temperature. The volume of the suspension (20 μL) was employed to cover the whole surface area of the microelectrode. In this case, the sample volume was minimized, and the coverage of the area by the sample enabled the contact between the microelectrode and the sample. To remove unbound bacteria and non-specifically bound proteins and cells, the chip was rinsed with DI water and dried in a nitrogen stream after incubation. Impedance measurement in the frequency range of 1 Hz to 1 MHz immediately followed the washing process. Suspensions with different cell numbers of E. coli O157:H7, from 80 to 2 × 105 cells per 20 μL, were tested. The procedures from the surface modification to the capture of bacteria are shown in figure 1.
(a)
(d) (b)
(e) (c) Protein A
Antibody
BSA
E. coli
Gold digit
Figure 1. Schematic diagram of the testing steps: (a) the bare microelectrode ready, (b) modifying the microelectrode with protein A, (c) immobilizing antibodies onto the microelectrode, (d) applying BSA blocking, and (e) capturing E. coli O157:H7.
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Impedance Analyzer and Impedance Measurement The impedance measurement was performed using a BAS IM-6 impedance analyzer (BAS, West Lafayette, Ind.) with IM-6/THALES software. A sine-modulated AC potential of 100 mV was applied, and the magnitude of impedance and phase angle were measured for the frequency range from 1 Hz to 1 MHz to study electrochemical phenomena over this wide frequency range. The measurements were performed in the presence of 0.01 M PBS buffer. The two poles of the IDA microelectrode chip were connected to the test-sense probe and counter-reference probe, respectively. Non-Faradaic impedance was recorded, and both the resistive and capacitive properties of the biomaterials were analyzed by electrochemical impedance spectroscopy (EIS). Each chip was rinsed gently with DI water and dried in nitrogen before measurement. At the end of each test, all chips were wiped with lens paper wetted with wash solution, rinsed with water and then dried with nitrogen. The impedance was recorded and presented in a Bode diagram that contained information on the values of impedance (Ω), phase angle (°) and frequency (Hz). The impedance change (IC) was calculated as the difference between the magnitude of the impedance recorded after and before the incubation of one sample of E. coli suspension. A calibration curve for impedance change (IC) versus the number of bacteria cells was plotted, and the detection limit was determined from the curve. Specificity and Food Sample Tests To determine the specificity of the immunosensor, non-target foodborne bacteria, including L. monocytogenes, S. Typhimurium, S. aureus, E. coli K12, and C. jejuni, were tested at a concentration of 107 cfu mL-1 (105 cfu per 20 μL). The same protocol as described earlier for detection of E. coli O157:H7 was followed in the test, except only non-target bacteria were used. Three replicates for each non-target bacterium were used, and the means and standard deviations of their impedance values were calculated. The impedance change caused by these non-target bacteria was compared with that caused by target E. coli O157:H7. To demonstrate the applications of the immunosensor for food safety, food sample tests were conducted using chicken carcasses, ground beef, and fresh cut broccoli, which were inoculated with target bacteria of E. coli O157:H7 at a concentration of 4 × 106 cfu mL-1 (2 × 104 cfu per 20 μL). The same protocol as described earlier for detection of E. coli O157:H7 was followed in the test, except food samples were used instead of pure bacterial culture of E. coli O157:H7. Five replicates for each type of food samples were used, and the means and standard deviations of their impedance values were calculated for data analysis. Atomic Force Microscopy Since atomic force microscopy (AFM) has the advantages of having high resolution and providing three-dimensional images of even nanoscale samples, it has been widely applied to bioimaging in biomedical and biotechnological fields (Cidade et al., 2003; Maalouf et al., 2007). AFM images were taken to characterize the electrode surface before and after incubation with bacteria. Images were recorded in air in tapping mode using a Nanoscope III AFM (Digital Instruments, Santa Barbara, Cal.). Digital Instruments Nanoscope Software V5.30r3.sr3 was used to analyze the AFM images. E. coli K12, a nonpathogenic strain very similar to E. coli O157:H7, was used for AFM images to prevent any biological contamination by pathogenic bacteria since the poly54
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clonal antibodies could capture live E. coli K12 cells as well O157:H7 cells. The procedure to prepare samples for AFM was the same as shown in figure 1. Bacterial stock was used, with the cell number at 108 cells mL-1 level. The main operation parameters are presented on the AFM images. The section analysis was conducted using the software.
Results and Discussion Typical Sensorgram Figure 2 presents responses of the IDA immunosensor to the surface modification, antibody immobilization, blocking, and binding of E. coli O157:H7 (800 cells per 20 μL) during sensor preparation and bacteria detection. Since protein A has a tendency to bind non-specifically to microorganisms, 1% (w/v) BSA solution was used in this study to block non-specific binding sites and minimize non-specific binding. The non-Faradaic impedance amplitude and phase angle of the bare electrode, protein A, antibodies, BSA, and E. coli cells are shown in a Bode diagram (impedance and phase vs. frequency) in figure 2. With the step-by-step addition of biomaterials, obvious differences in the Bode diagram were observed, especially for the amplitude of the impedance at low frequencies from 1 Hz to 10 kHz and for the phase angle of the impedance in a frequency range from 1 kHz to 500 kHz. Equivalent Circuit The data from EIS can be simulated using the IM-6/THALES software with an equivalent circuit that consists of several elements, as shown in figure 3. The spectrum indicated that kinetic and diffusion processes might account for polarization.
Figure 2. Bode diagram of the electrochemical impedance spectra for the bare gold microelectrode, protein A modification, antibody immobilization, BSA blocking, and E. coli O157:H7 (800 cells per 20 μL) binding in the frequency range from 1 Hz to 1000 kHz. The data points from left to right correspond to increasing frequency. Voltage amplitude is 100 mV.
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Cdl
Cdl
RS
W Zw
Rp
W Zw
Rp
Figure 3. Equivalent circuit of the impedance measurement system based on the IDA microelectrode with immobilized antibodies for the detection of E. coli O157:H7.
The circuit consists of four elements: Rs, the ohmic resistance of the electrolyte; ZW, the Warburg impedance resulting from the oxygen diffusion; Rp, the polarization resistance of dissolved oxygen; and Cdl, the double-layer capacitance. Based on the equivalent circuit, 45 data points on the spectrum for bacteria detection recorded in the presence of 0.01 M PBS were selected by the software for simulation, and a fitting spectrum was generated. It is noted that the spectrum used for fitting was obtained after the capture of bacteria. Both the measured spectrum and the fitting spectrum for impedance amplitude and phase angle of E. coli O157:H7 in PBS are presented in figure 4, and the agreement between the measured and fitting spectra with an acceptable mean error of 0.4% indicates the excellent fit of the equivalent circuit model shown in figure 3. The simulated values of Rs, ZW, Rp, and Cdl were 64.59 Ω, 48.5 kΩ/s1/2, 254.5 Ω, and 5.396 nF, respectively. Simulations were also presented with spectra obtained before and after surface modification (protein A, antibody, and BSA), and the same circuit fitted well with all the data. This indicated that the addition of biolayers (protein A, antibody, BSA, and bacteria) changed the impedance of the system. According to the equivalent circuit in figure 3 and the Bode diagram in figure 2, there are three regions that behave differently depending on the frequency. At a high frequency from approximately 500 kHz to 1 MHz, the current passed through the branches containing the capacitances, so that the resistance of the electrolyte was re-
Figure 4. Impedance spectra of E. coli O157:H7 cells in PBS with the fitting curves. 56
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sponsible for the value of the impedance. This explains why there were nearly no differences of impedance in this range when different layers of materials were immobilized on the surface, because the resistance of the PBS was almost constant. When the frequency was in the range of 100 Hz to 10 kHz, both branches including ZW and Rp in each of the two identical parallel cells in the circuit were activated, and the impedance was mainly attributed to the resistive components. At low frequencies, the doublelayer capacitance dominated the impedance. With the addition of the biomaterial to the electrode surface, the impedance changed mostly in the frequency range below 10 kHz. Therefore, the impedance measured at 1 kHz was chosen to determine the relationship between the impedance change and the captured E. coli O157:H7. At a frequency of 1 kHz, changes in dielectric and insulating features at the electrode/electrolyte interface contributed to the change in the impedance magnitude. Detection of E. coli O157:H7 at Different Concentrations Detection of E. coli O157:H7 at different concentrations was performed using 0.01M PBS as a dilution buffer. The same buffer without bacteria served as a control, and a negligible signal (only several ohms) was obtained for the control. A linear relationship, with a slope of 300.3 and coefficient of determination (R2) of 0.97, was found between the change in the amplitude of impedance at the frequency of 1 kHz and the logarithmic value of E. coli O157:H7 concentration in a range of 80 to 2 × 105 cells per 20 μL (fig. 5). The detection limit could have been affected by several factors. The resistance dominated the impedance in the higher frequency range, while the double-layer capacitance existed at low frequencies where the impedance change was caused by the bacteria captured. The double-layer capacitance might affect the sensitivity of the sensor. Because the double-layer capacitance resulted in large impedance as the background, a relatively large number of bacteria attached to the microelectrode surface was needed to alter the impedance at low frequencies (Radke and Alocilja, 2005). The diffusion and adsorption of E. coli O157:H7 cells to the microelectrode surface should be considered as well. The bacteria were incubated for 2 h at room temperature and captured onto the microelectrode surface via the antibody-antigen reaction. Therefore, the affinity of antibodies to antigens had a critical impact on the detection of target bacteria. At the same time, the diffusion of target bacteria in the sample solution determined whether all E. coli O157:H7 cells had a chance to contact the immobilized antibodies during the short incubation. To improve the biosensor’s performance, the conditions for antibody immobilization, including surface modification materials, antibody concentration, and incubation time and temperature, should be optimized to improve the capture efficiency of E. coli O157:H7. Higher incubation temperature may increase the diffusion rate and the binding kinetics of target bacteria, which would reduce the total detection time, as reported in other research when a temperature of 37°C was applied (Moll et al., 2007). The detection limit of this impedance immunosensor is comparable with other reported research based on a similar principle for detection of E. coli. Meanwhile, this method, with impedance measurement in the absence of a redox probe and protein A facilitating antibody immobilization, provided high sensitivity without further amplification of signals, which revealed the potential for this method to be developed for practical use, even for detection of live bacteria. Radke and Alocilja (2005) developed
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1400 y = 300.3x - 439.26
Impedance change (Ohm)
1200
2
R = 0.97
1000 800 600 400 200 0 -200
0
1
2
3
4
5
6
-400 Number of E. coli O157:H7 (log cells/20 µL)
Figure 5. Linear relationship between the change in the amplitude of the impedance and the logarithmic cell number of E. coli O157:H7 at the frequency of 1 kHz.
an impedance biosensor based on antibody immobilization that could directly detect in the range from 104 to 107 cfu mL-1. Boehm et al. (2007) utilized a microfluidic labchip immunosensor for the detection and identification of 104 cfu mL-1 of E. coli. Varshney and Li (2007) developed an impedance biosensor based on an IDA microelectrode combined with the separation of the target bacteria using magnetic nanoparticle-antibody conjugates (MNAC), resulting in the lowest detection limit of 7.4 × 104 cfu mL-1 for E. coli in the pure culture. When compared with conventional methods, this sensing method needs much less detection time and has a lower detection limit. As described by Fratamico et al. (1992), 10 cfu mL-1 of E. coli O157:H7 in meat products could be detected using a surface plating method, but 24 h enrichment and plating time was required. Characterization of Bacteria Captured on the Microelectrode by AFM Figure 6a presents the simultaneously obtained topographic and phase AFM images of the surface of one digit of the IDA microelectrode after incubation with E. coli cells in PBS suspension. As can be seen in the images, there were no obvious particles on the gold digit (the band in the middle of the image) in figure 6a, except for two particles that are similar in size. The horizontal section analysis of the topograph, shown in figure 6b, measured dimensions of the particles. The measured length and width were as expected for a live E. coli cell (2 μm in length and 0.5 μm in width), but the height of 200 nm was less than a typical live cell. The loss of cell contents during the drying process for AFM sample preparation may have caused the reduced height. Because the scanning area of the AFM was 15.9 μm × 15.9 μm, only part of one gold digit could be seen in one scan. Several different areas of the IDA microelectrode were scanned, and E. coli cells were found in each area only on the gold digit, which demonstrated that E. coli cells were specifically captured on the gold digits but not on the glass substrate, and the attachment of the bacteria might cause the change in impedance.
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600nm
0nm
(a)
(b)
Figure 6. AFM images (15.9 μm × 15.9 μm) of E. coli O157:H7 cells on the microelectrode with immobilized antibodies: (a) top view, and (b) horizontal cross-section review.
The actual number of cells attached to the electrodes was calculated based on the AFM images, and for the total area of electrodes, there were approximately 4300 cells. Since 20 μL of bacterial stock (~108 cells mL-1, by cell counting) was used for imaging and our calibration curve was built based on cell counts, there should be about 2 × 106 cells introduced to the electrode surface. These cells could generate a signal of impedance magnitude at about 1500 Ω, calculated using the calibration curve presented in figure 5. However, it should be noted that since a nonpathogenic strain, E. coli K12, was used in the AFM images for biosafety reasons, the variation in antibody affinity to E. coli K12 and E. coli O157:H7 may result in a different number of cells attached to the electrode surface. Immunosensor Specificity and Detection of Food Samples As shown in figure 7, the impedance signals resulting from the non-target bacteria L. monocytogenes, S. Typhimurium, S. aureus, E. coli K12, and C. jejuni at a concentration of 107 cfu mL-1 (105 cfu per 20 μL) were negligible, indicating that they did not interfere with the detection of the target bacteria, E. coli O157:H7.
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Figure 7. Specificity tests with five non-target bacteria (L. monocytogenes, S. Typhimurium, S. aureus, E. coli K12, and C. jejuni) and food sample tests (chicken carcasses, ground beef, and fresh cut broccoli).
The results of the food sample tests are also shown in figure 7. The chicken carcass wash solution, ground beef, and fresh cut broccoli, which were inoculated with target bacteria of E. coli O157:H7 at a concentration of 4 × 106 cfu mL-1 (2 × 104 cfu per 20 μL), generated detectable signals with impedance values of 678 ±137 Ω, 628 ±222 Ω, and 491 ±111 Ω, respectively.
Conclusions In this study, an IDA microelectrode-based, label-free impedance immunosensor was developed for rapid and sensitive detection of E. coli O157:H7. The surface of a gold IDA microelectrode was modified with protein A and then immobilized with the antibody against E. coli O157:H7. The impedance change caused by E. coli O157:H7 captured on the modified microelectrode could be measured without a redox probe, and an equivalent circuit could be generated to explain the electrochemical impedance spectrum. It was found that the impedance measured in the absence of a redox probe in PBS in a certain frequency range increased with increasing number of E. coli O157:H7 in a sample. The linear relationship of the impedance magnitude and the cell number of E. coli O157:H7 was found in the range of 80 to 200,000 cells per 20 μL with the detection limit of 80 cells per 20 μL within 2 h. Atomic force microscopy (AFM) images confirmed the capture of E. coli O157:H7 by the antibody immobilized on the microelectrode. None of the five non-target bacteria generated any detectable signals, clearly indicating that the immunosensor could be very specific to the target bacteria. The tests on three types of food samples inoculated with E. coli O157:H7 successfully demonstrated the applications of this immunosensor for food safety. This study indicated that the IDA microelectrode-based immunosensor may provide a simple, rapid, sensitive, and specific method for quantitative detection of E. coli 60
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O157:H7 in foods, which should have the potential for applications to the rapid detection of different pathogens in various foods. For biosafety reasons, the E. coli O157:H7 used in the study were thermally killed before tests. The on-going research will continue to improve the IDA immunosensor (e.g., further reduce the finger size of the IDA microelectrode) and evaluate the IDA immunosensor for rapid detection of viable E. coli O157:H7 cells from ready-to-eat food samples. Furthermore, a flowthrough microfluidic cartridge may be designed and fabricated, and the method described herein can then be modified for simultaneous detection of multiple foodborne pathogens using multi-antibody-modified IDA microelectrodes. Acknowledgements This research was supported by the Food Safety Consortium. The authors thank Lisa Cooney for her help in the microbial tests and Husein Rokadia for his help in AFM tests.
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