Abstract: Ahighly sensitive piezoelectric crystal immunosensor has been developed for the detection of human fibrin. The crystal used was 9 MHz quartz AT-cut with gold coated electrodes. Three methods for immobilization of anti-fibrin antibody on the gold electrodes were employed. The immobilization method with a thin protein A layer gave the best results in terms of sensitivity, stability and reproducibility. The crystal was precoated with either polyethyleneimine adhesion and glutaraldehyde cross-linking, or bovine serum albumin adsorption and glutaraldehyde cross linking, gave inferior results. Protein A molecular membrane could be developed as a matrix for oriented immobilization of antibody molecule because of both specific binding to the Fc fragment of IgG and adsorption strongly to the gold electrode surface. In this paper, quartz crystal was dipped into 0.1 g/L protein A solution. Then a thin protein A membrane was performed on the crystal surface so that fibrin antibody could be effectively self-assembled. Then an orderly and closely arranged self-assembled antibody molecular membrane was immobilized orientationally on the protein A layer. The coated crystal using this immobilization method was stable for 6 weeks when stored in silica gel dry over at room temperature. The influence factors related to the sensitivity of the system such as the concentration of protein A coated, the titer of antibody, the absorption time of protein A, the immobilization time of antibody, the reaction time of immunoreaction etc., were discussed. U nder the optimized experimental conditions chosen, the coated crystal had a good response to fibrin in the concentration range of 1×10^-4～1×10^-2 g/L. This sensor had a good selectivity, other materials in human serum did not interfere the detection. The reusability of the immunosensor was also studied. We eluted the bound materials on the crystal surface with strong acid and alkali solutions, then the crystal was washed with ultrasonic bath. The crystal could be regenerated nearly 10 times without detectable loss of activity.

Key words: Protein A, Oriented immobilization of antibody, Piezoelectric immunosensor, Fibrin