蛋白A定向固定抗体的纤维蛋白压电免疫传感器的研究

Studies of the Piezoelectric Immunosensor for the Detection of Fibrin Using Protein A Oriented Immobilization of Antibody

将９ＭＨｚ双面镀金石英晶体浸入蛋白Ａ溶液中，在晶体电极表面形成一层均匀的蛋白Ａ薄层，用于定向固定人体纤维蛋白抗体．在蛋白Ａ层上形成一层有序致密的自组装抗体分子膜，研制成一种新型的用于人体纤维蛋白检测的压电免疫传感器．比较了３种固定抗体方法的效果，从传感器的灵敏度、稳定性、重现性等考虑，蛋白Ａ吸附法优于聚乙烯亚胺及牛血清白蛋白固定抗体的方法．研究了蛋白Ａ浓度、抗体效价以及抗原抗体反应时间等对传感器灵敏度的影响，考察了电极的选择性和再生能力．纤维蛋白在１×１０－４～１×１０－２ｇ／Ｌ浓度范围内有良好响应．

A highly 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. Under 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.