The main objective of this Phase I proposal is to develop and test a strip biosensor electrode which can detect two different antigens (LH and FSH) in urine. The project is based on several technologies already established by this group (Enzyme Technology Research Group, Inc. or ETRG). First, this group has pioneered the technique of using colloidal gold to immobilize antibody to an electrode surfaced without loss of biological activity. Second, this group has established that an antibody coupled to horse radish peroxidase when exposed to the appropriate substrate will generate an electron flow that can be measured by a sensing electrode. Third, this group will test the concept that with the use of two different antibody-enzyme conjugates each with different working potentials, it may be feasible to measure two analytes in solution by altering the redox potential without additional analyte separation.Thus, in a typical reaction, the antigen will bind to the immobilized antibody-electrode surface, the second antibody coupled to an enzyme will sandwich the antigen, and it the presence of a fixed substrate concentration, a current will be generated. The activity of the second antibody coupled enzyme will be dependent on the redox potential of the system. The prototype biosensor is an electrochemical cell consisting of a working electrode coated with an electrical conducting layer containing colloidal gold adsorbed antibodies to LH and FSH, FSH antibody coupled to horse radish peroxidase, LH antibody coupled to glucose oxidase, substrates for both enzymes, a reference electrode, and a counter electrode. The commercial biosensor will consist of a disposable test strip having an electrical conducting layer containing absorbed immunocomponents, and the electronic components used for the test strip signal readout.
