材料之表面經過修飾後可與蛋白質、胜肽或小分子藥物以共價鍵結合，形成之耦合物可廣泛應用於生醫檢測方法和治療藥物之開發。本研究推算奈米粒子表面之羧基量，以及蛋白質耦合量，其目的為優化合成方法，降低活化試劑與蛋白質使用量，並應用於耦合抗體之磁性奈米粒子及耦合抗體與葡萄糖氧化酶之膠珠的製備暨電化學反應分析以期建立偵測免疫反應之電化學分析法。實驗流程首先分析奈米粒子與蛋白質耦合之優化條件，接著應用耦合反應之最佳條件製備抗體-奈米粒子或抗體-奈米粒子-蛋白質之耦合物。實驗方法包括: (1) 應用Ninhydrin 方法推算奈米粒子之羧基含量，以降低活化劑與蛋白質之用量；(2) 應用 Bradford 定量未反應之蛋白質，推算耦合反應所需蛋白質之用量，建立耦合反應優化的條件；(3) 應用耦合反應之優化條件，製備葡萄糖氧化酶-奈米粒子之耦合物，並應用電化學分析法之電流訊號，判別耦合物的製備是否成功; (4) 應用優化之條件，將捕捉抗體耦合至奈米粒子，專一性地辨識其抗原；(5) 應用優化之條件，將偵測抗體與葡萄糖氧化酶耦合至奈米粒子；(6) 應用電化學分析其完整免疫反應。本研究電化學方法之建立，應用於偵測特定抗體與抗原之間的專一性反應，研究成果有助於疾病檢測方法之開發。 The surface of the material can be modified to form covalent bonds with proteins, peptides or small molecular drugs, and the coupling products can be widely used in the development of biomedical testing methods and therapeutic drugs. This study estimated the amount of carboxyl groups and protein coupling on the surface of nanoparticles. The purposes are to optimize the synthesis method and reduce the amount of activation reagents and protein. The preparation and electrochemical analysis of magnetic nanoparticles conjugated with antibody and silica nanoparticles conjugated with antibody and glucose oxidase, are expected to establish an electrochemical analysis method for detecting immunoreaction. The first of experimental procedure analyzed the optimal conditions for the protein-conjugated nanoparticles, and prepared antibody-nanoparticles or antibody-nanoparticle-protein coupling products with the optimal conditions of the coupling reaction. The experimental methods include: (1) Apply Ninhydrin method to estimate the carboxyl group of the nanoparticles to reduce the amount of activation reagent and protein; (2) Apply Bradford assay to quantify the unreacted protein, estimating the amount of protein required for the coupling reaction, and establishing the optimal condition; (3) Glucose oxidase was conjugated with nanoparticles by the optimal conditions and detected whether the conjugation was successful by the electrochemical analysis; (4) The capture antibody was conjugated with nanoparticles by the optimal conditions, and the antibody-conjugated nanoparticles specifically identified the antigen; (5) Antibody and glucose oxidase were conjugated with nanoparticles by the optimal conditions; (6) Apply electrochemical method to analyze immunoreaction. In this study, the established electrochemical analysis method can be applied to detect the specific reaction between antibody and antigen, and the results contribute to the development of disease detection.
