Novel 3-D Biosensor for Rapid Detection and Accurate Identification of E. coli 0157:H7 in Beef

NON-TECHNICAL SUMMARY: Escherichia coli O157:H7 is clearly one of the deadliest food borne pathogenic bacteria. It causes an estimated 73,000 cases of infection and 61 human deaths in the United States each year (Centers for Disease Control and Prevention, 2006). This bacterium has been linked to hemolytic uremic syndrome and hemorrhagic colitis. These illnesses may cause diarrhea, seizure, stroke, kidney failure and even death (Food and Drug Administration, 2008). They are often misdiagnosed, resulting in expensive medical testing before they are correctly diagnosed. In addition, E- coli has the potential to cause enormous national and international economical devastation due to medical costs and product recalls, as recently occurred with the recall of tomatoes due to E. coli O157:H7 contamination. It can also be found in vegetables, unpasteurized milk, juice and unchlorinated water. Contamination can have a significant impact on businesses such as the beef -industry. E. coli O157:H7 can be found on most cattle farms and can live in the intestines of healthy cattle. Thus, the meat can become contaminated with E. coli O157:H7 during slaughter. It is important to note that the infectious doses of E-coli O157:H7 is as low as 10 cells (Federal Register, 1990, 1991). Therefore, effective detection techniques are crucial to monitor and control E-coli O157:H7 in food products. Testing for the bacteria requires extensive analysis which has to meet certain challenging criteria. Sensitivity and response time for the analysis are imperative factors related to the usefulness of microbiological testing. An extremely selective detection methodology is also required because low numbers of pathogenic bacteria are often present in a complex biological environment along with many other nonpathogenic organisms. Traditional methods for the detection of bacteria are not available in the time scale desired in a clinical laboratory. In response to this problem, a number of instruments have been developed using various principles of detection, such as flow cytometry polymerase chain reaction, immunomagnetic separations, bioluminescence and mass spectrometry. These methods, however, are still time consuming and expensive. The proposed project will develop a novel 3-dimensional (3-D) interdigitated microelectrode array (IDE) based impedance biosensor. This biosensor will be capable of rapid detection and selective for accurate identification of E. coli O157:H7. This design is unique in the use of a 3-D IDE which increases the surface area compared to a single (2-D) IDE sensor. The increased surface area will enhance the sensitivity of impedance detection. Each IDE biosensor consists of 100 pairs of gold electrode "fingers" with a length of 0.5 mm. The IDE array will be designed with spaces between the interdigitated electrodes nearly the size of the bacteria in order to detect a single or a few bacteria cells.

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APPROACH: <BR> Biosensor Design: <BR>The proposed E-Coli O157:H7 biosensor will be designed with a 3-D high density cylindrical interdigitated electrode array (IDE). The IDE surface is modified to immobilize antibodies and is used as the sensing surface to detect bacteria concentration in solution. The design is unique in the use of 3-D IDE with a microchannel to increase the surface to volume ratio and decrease the sample volume, resulting in a rapid and high sensitivity impedance biosensor. The biosensor will be exposed to bacteria via a flow channel. When bacteria bind to antibodies, only a region of 2-4 micrometer above the sensor surface is modified. The impedance measurement results will change as the concentration of bacteria bound to the sensor surface changes. The advantages of using 3-D IDE impedance system include a reduction in the sample volume, a more rapid detection time (within several minutes), low resistance, and a high signal to noise ratio. <BR><BR>Culture and Coating of Bacteria: <BR>The next stage is immobilization of the antibodies on to the IDE by forming a Self-Assembled Multilayer (SAM). Between and after these treatments, the electrodes will be rinsed thoroughly with ultra pure water, then rinsed with absolute ethanol and dried in a flow of pure nitrogen. After cleaning, the substrates will be immersed immediately into a mixed solution of biotin thiol and spacer alcohol thiol in 50/50 ethanol/chloroform solution. The electrodes functionalized with mixed SAMs will be removed, rinsed with the corresponding solvent, dried under N2 flow/ and fixed to the supporting prism of the Surface Plasmon Resonance (SPR) instrument (n) 1.61. The mixed SAMs will be stabilized by injecting Phosphate-Buffered Saline (PBS) (pH 7.0). Subsequently, Bovine Serum Albumin (BSA) solution will be added to prevent any further nonspecific adsorption on the mixed SAMs. Next, the same protocol will be used to inject neutravidin followed by adding the biotinylated polyclonal anti-E. coli antibodies. After each step, the electrode will be rinsed by injecting PBS solution (pH 7.0) to remove the unbound biological compounds. <BR><BR>Testing and Characterization:<BR> The magnitude and phase of the impedance across the interdigitated electrode array will be measured using an impedance analyzer. A modulated AC voltage (sine wave) will be applied to the microelectrode array at frequency range from 10 - 10 MHz. The impedance will be measured using two beef samples, one with bacteria and the other without bacteria as described above. The measurement will be performed prior to immobilizing the antibodies on to the surface of the IDE array, after immobilizing the antibodies, and after the exposure of bacteria. This technique will establish the baseline impedance and enable the extraction of the impedance of the bacteria effect alone. In addition, the effect of frequency on impedance measurement will be monitored and analyzed. The total detection time will be measured. The detection time will include immunoreactions, washing, and measurement. A calibration curve for E-coli O157:H7 concentration will be obtained using standard techniques adopted by FDA.