Detection of E. Coli O157-H7 Using a Nanoneedle Probe Biosensor

NON-TECHNICAL SUMMARY: The working principle of nanoneedles as bacterial detectors is that when an AC electric field is applied to a needle in contact with a biological solution, bacterial cells are attracted to the needle by electrical forces, i.e. dielectrophoresis. As the needle is withdrawn from the solution, the cells stick to the needle surface by capillary action. By tuning the size of nanoneedles and frequency of electric field, selective capture based on size and electric properties will be achievable. Discrimination of live and dead cells is a good example since they have different permittivities. This study will follow two tracks in parallel, (1) development of an immunofluorescence detector for single step bacterial detection and isolation, and (2) design of nanoneedle conductance biosensors with antibody binding. The first track will focus on demonstrating the ability to selectively capture E. coli O157:H7 onto a nanoneedle network by detection with fluorescence-labeled antibodies. The second track will focus on developing a simple conductance sensor to detect selective capture and antibody labeling of E. coli O157:H7 on the nanoneedle probe surface. The small size and capability of these semiconductor nanowires for sensitive, label-free, real-time detection of a wide range of biological species could be exploited in array-based screening, in vivo diagnostics, and separation and concentration of targeted microbes. <P>

APPROACH: Task 1: Design and fabrication of the nanoneedle device. To fabricate a platform for immuno-fluorescence sensor, an oxide layer is grown on Si wafer (orientation: 100) and a Si3N4 is deposited by low pressure chemical vapor deposition (LPCVD) on the oxide layer. The backside of the Si substrate is patterned and opened by KOH etching. Nanowires are fabricated on the substrate by using the shadow edge lithography (SEL) method followed by suspension in HF solution. The device will consist of three main components: a polydimethylsiloxane (PDMS) well, a patterned wafer, and XY stage. A thin layer of PDMS (approx. 1mm thick) will be prepared and a 3 mm diameter well is then punched out of the PDMS strip. The PDMS well is rinsed and dried using deionized (DI) water and N2 gas. Then it will be placed over the gold electrode. Light pressure will be applied to ensure good contact between the PDMS well and the electrode. Rinsing with water immediately after microbial capture will make it possible to recycle the electrode and PDMS well. <P>
Task 2: Investigate and optimize the concentration and capturing mechanism of the nanoneedle device for E. coli O157:H7. The Immersed Electrokinetic Finite Element Method (IEFEM) will be employed for this simulation. The critical particle dimension at the crossing point will be investigated to optimize the diameter of nanoneedles to selectively capture bacterial pathogens from the sample solution. <P>
Task 3: Evaluate the performance of the nanoneedle device coupled with immunofluorescence sensing. After capturing E. coli O157:H7 cells onto the nanoneedle device, an immunofluorescence measurement is performed using a FITC-labeled monoclonal antibody against the pathogen (ViroStat, Inc., Portland, ME). The number of E. coli cells is varied between 103 to 106 /mL. Dr. Jun will lead the effort on the evaluation of immunofluorescence nanoneedle devices, along with technical and microbial supports from Drs. Li and Jenkins. <P>
Task 4: Evaluate the functionality of the nanoneedle conductance biosensor with antibody binding. We propose to study ligand-receptor binding of a nanoneedle device with captured E. coli O157:H7. The measurements show the conductance of biotin-modified silicon nanowires (SiNWs) increases rapidly to a constant value upon addition of a 250 nM streptavidin solution and that this conductance value is maintained after addition of pure buffer solution. The observed conductance changes are due to the specific bindings of streptavidin to the biotin ligand. After capturing E. coli O157:H7 cells onto the nanoneedle device, the device will be placed in the anti- E. coli O157:H7 solution. The antibody binding to E. coli O157:H7 cells on the nanoneedle tip results in a change in electric charge on the surface of the nanoneedle, thereby changing the electrical conductance of the nanoneedle. To confirm the increased conductance due to the antibody-antigen reactions, we will add anti- E. coli O157:H7 to the reference nanoneedle probe to see the conductance profile. To confirm the sensing selectivity, captured cells with antibodies selective to non-select bacteria including Salmonella Enteritidis anti E. coli O55 will be used.