STUDIES OF THE PIEZOELECTRIC IMMUNOSENSOR FOR THE DETECTION OF FIBRINUSING PROTEIN-A ORIENTED IMMOBILIZATION OF ANTIBODY

A highly sensitive piezoelectric crystal immunosensor has been develop ed for the detection of human fibrin. The crystal used was 9 MHz quart z AT-cut with gold coated electrodes. Three methods for immobilization of anti-fibrin antibody on the gold electrodes were employed. The imm obilization method with a thin protein A layer gave the best results i n terms of sensitivity, stability and reproducibility. The crystal was precoated with either polyethyleneimine adhesion and glutaraldehyde c ross-linking, or bovine serum albumin adsorption and glutaraldehyde cr oss-linking, gave inferior results. Protein A molecular membrane could be developed as a matrix for oriented immobilization of antibody mole cule because of both specific binding to the Fc fragment of IgG and ad sorption strongly to the gold electrode surface. In this paper, quartz crystal was dipped into 0.1 g/L protein A solution. Then a thin prote in A membrane was performed on the crystal surface so that fibrin anti body could be effectively self-assembled. Then an orderly and closely arranged self-assembled antibody molecular membrane was immobilized or ientationally on the protein A layer. The coated crystal using this im mobilization method was stable for 6 weeks when stored in silica gel d ry over at room temperature. The influence factors related to the sens itivity of the system such as the concentration of protein A coated, t he titer of antibody, the absorption time of protein A, the immobiliza tion time of antibody, the reaction time of immunoreaction etc., were discussed. Under the optimized experimental conditions chosen, the coa ted crystal had a good response to fibrin in the concentration range o f 1 X 10(-4)similar to 1 X 10(-2) g/L. This sensor had a good selectiv ity, other materials in human serum did not interfere the detection. T he reusability of the immunosensor was also studied. We eluted the bou nd materials on the crystal surface with strong acid and alkali soluti ons, then the crystal was washed with ultrasonic bath. The crystal cou ld be regenerated nearly 10 times without detectable-loss of activity.