基于蛋白A定向固定的Love波免疫传感技术研究

A Protein A-based Immobilization Stategy for Love Wave Immunosensor

报道了一种基于蛋白A固定技术的新型Love波免疫传感器,可用于检测B型葡萄球菌肠毒素(SEB)。本传感器由压电石英基片、叉指换能器和SiO2声波导层构成。为提高传感器对SEB分子的响应能力和检测灵敏度,设计了抗体分子的蛋白A定向固定路线,通过APTES和戊二醛层交联蛋白A,随即用100mmol/L NaBH4还原蛋白A与戊二醛层之间形成的Schiff′s base结构,实现抗体分子在Love波器件表面高效率的定向固定。该固定化方案使传感器的再生能力和检测灵敏度都得到显著提高,对1·0mg/L SEB进行连续5次检测后,其响应能力没有明显下降;而对SEB的浓度检出限度达到了1·0μg/L。原子力显微镜对传感器表面形态的成像结果表明,与戊二醛共价交联法相比,蛋白A结合法使抗体分子探针在传感器表面拥有更高的覆盖度和活性,对SEB具有更强的结合能力。

A Love wave immunosensor based on protein A immobilization strategy for staphylococcal enterotoxins B （SEB） is presented. The Love wave devices with the substrate of ST-cut quartz, IDTs and the guiding layers of SiO2 were fabricated successfully. In the immobilization of antibodies to the sensor by protein A-based immobilization procedure, NaBH4, a selective reducing agent was used to reduce the Schiff＇s bases resulting from the reaction of glutaraldehyde-activated surface with protein amino groups to improve the regeneration of the immunosensor. The active area of sensor was precoated with y-aminopropylethoxysilane （APTES） and glutaraldehyde, protein A were coupled to the surface by formation of Schiff＇s base and reduction with 100 mmol/L NaBH4 to form stable - CH2 - NH - bonds, and antibody molecule probes were immobilized in a highly oriented manner by the protein A on SiO2 surface. Results indicated that such a preparation can greatly increase regeneration and sensibility of the immunosensor, up to 5 successive assay repetitions to 1.0 mg/L SEB were achieved without significant loss of sensitivity using the new immunosensor, moreover, SEB serum concentration as low as 1.0 μg/L could be detected by the same system. The surface morphology of the active area characterized by using atomic force microscopy revealed that in contrast to the glutaraldehyde immobilization, protein A-NaBH4 procedure could allow antibody probes bound with higher bioactivity and loading amount, achieving better SEB-binding capabilities.