Impedance Biosensing to detect food allergens, endocrine disrupting chemicals, and food pathogens

Electrochemical impedance biosensors can be viewed as an AC electroanalytical method for the analyte detection in the fields of biomedicine, environmental monitoring, and food and agriculture, amongst others. The most common format for AC impedance biosensing involves surface immobilization of an antibody, receptor protein, DNA strand, or other species capable of bio-recognition, and AC impedance detection of the binding event. Technological application of AC impedance biosensors has been hindered by several obstacles, including the more complex circuitry required for AC relative to DC electrochemistry, chemical and physical interference arising from non-specific adsorption, and the stability and reproducibility of protein immobilization. One focus of these PhD studies is on methods to reduce or compensate for non-specific adsorption, including sample dilution, site blocking with BSA, and the use of control electrodes onto which reference antibodies are immobilized. Examples that will be presented include impedance detection of food pathogens, such as Listeria monocytogenes, using a mouse monoclonal antibody immobilized onto an Au electrode. This yields detection limits of 5 CFU/ml and 4 CFU/ml for ideal solutions and filtered tomato extract, respectively. Control experiments with an Au electrode onto which a mouse monoclonal antibody to GAPDH is immobilized demonstrate that non-specific adsorption is insignificant for the system and methodology studied here. Control experiments with Salmonella enterica demonstrate no cross-reactivity to this food pathogen. In addition, Detection of two endocrine-disrupting chemicals (EDC), norfluoxetine and BDE-47, is reported here by impedance biosensing, with a detection limit of 8.5 and 1.3 ng/ml for norfluoxetine and BDE-47, respectively. Additional research has focused on alternative substrates and linker chemistries for protein immobilization, including the use of degenerate (highly doped) Si and bidendate thiol monolayer onto Au. Advantages of degenerate Si include a simpler equivalent circuit, simple and reproducible surface preparation, easy incorporation into ULSI devices, and the greater strength of Si-C bonds (~520 kJ/mole) relative to Au-S bonds (125-150 kJ/mole). New results demonstrating antibody regeneration atop degenerate (highly doped) Si are also reported. Using 0.2 M KSCN and 10 mM HF for antibody regeneration, peanut protein Ara h 1 is detected daily during a thirty-day trial. An impedance biosensor is reported that employs the bidentate thiol 16-[3,5-bis(mercaptomethyl)phenoxy]-hexadecanoic acid (BMPHA) to immobilize the mouse monoclonal antibody to peanut protein Ara h 1. The detection limit for Ara h 1 is approximately 0.71 ng/mL (0.01 nM), which is about one order of magnitude lower than that obtained for antibody immobilization atop the monodendate thiol, 16-mercaptohexadecanoic acid (16 MHA). Antibody regeneration was studied daily using a gentle denaturing agent, 0.2 M KSCN at pH 7.3. The antibody-coated on Au electrodes retained activity towards Ara h1 for 10 and 20 days of regeneration of the monodendate- and BMPHA-coated Au electrodes, respectively. This prolonged activity illustrates the superior stability of protein films atop the BMPHA bidentate thiol- coated Au electrode relative to the 16-MHA monodendate thiol-coated Au electrode.