多重苯硼酸衍生物之設計、合成和生物感測之應用

Abstract

抗體的非位向性固定化可能導致生物辨識力下降，而影響生物感測之靈敏度。由於苯硼酸在 pH 7-9 環境下能與雙醇有效鍵結。根據此特性，本實驗將合成一系列含有單、雙和三苯硼酸基團之連接子 ( linker )，供生物分子(抗體)之固定化使用，其一端是利用硼酸與抗體的 Fc 端的寡醣區進行鍵結，以達到位向控制的效果，另一端則含有氫硫基(-SH)供金硫自組裝(self-assembly)修飾用或苯胺基(aminophenyl group)供電沉析修飾用。我首先將連接子修飾於金晶片表面，再以循環伏安法和電阻抗分析法評估晶片表面修飾之緻密度，我們證實，相較於金硫鍵之自組反應，電沉析修飾法能大幅縮短修飾所需的時間，且能提高連接子於金表面之覆蓋率。 接著，我們以石英晶體微天平 ( Quartz crystal microbalance, QCM ) 為分析平台，利用 QCM 的壓電效應分析並比較上述兩種方法修飾所得的晶片(含有不同苯硼酸基團)對抗體修飾量的差異，並比較在酸性條件下，晶片上之抗體脫附量，藉以研究此一系列連接子對抗體作用力的強弱差異。實驗結果顯示，三種苯硼酸連接子皆可快速修飾上抗體，其中雙、三苯硼酸連接子之修飾速率相當(約1.1 ng/s)，而單苯硼酸之速率則較低，約為前兩者之70%；抗體之修飾量以雙苯硼酸連接子最佳，約較單苯硼酸高36%，而三苯硼酸則僅較單苯硼酸高25%。可能因苯硼酸間之立體障礙的關係致使三苯硼酸連接子之修飾量不如雙苯硼酸者，但對抗體之作用力則如預測為三苯硼酸>雙苯硼酸>單苯硼酸。結論，依所設計之結構，雙苯硼酸連接子為最佳的選擇，以電沉析法可將表面修飾時間縮短至20分鐘，遠比金硫鍵之自組裝修飾法(約需24小時)快速且修飾之緻密度大幅提升!此連接子和修飾法對生醫感測之晶片製作將有重大的助益!

This study is aimed to overcome the weakness of low recognition power of antibody that resulted from its random immobilization on biosensor. Phenylboronic acid has been shown to effectively form complex with diols within pH7-9. Based on this feature, we designed and synthesized a series of bi-functional linkers with one end containing 1-3 phenylboronate moieties and the other end containing either sulfhydryl group (-SH) or an aminophenyl group. The phenylboronates were employed for specifically forming complex with the glycosyl portion at the Fc region of antibody and consequently to control the orientation of antibody immobilization. The -SH and aminophenyl group were used for conjugation of the linker to gold surface through self-assembly and electrical deposition process, respectively. The density of linker (or coverage ratio) were characterized and evaluated by cyclic voltammetry and electric impedance assessments. Our studies confirm the electrical deposition method (require <20 min) is much more efficient than the self-assembly process (require more than 24 hr). The coverage ratio derived from electrical deposition is much higher than that of self-assembly process.Quartz crystal microbalance (QCM) was also used as the detection platform to compare the binding capacity of antibody on chip labeled with linker containing single, double, or triple phenylboronate moiety. All of them successfully demonstrated the effective function of antibody immobilization. The rate of antibody immobilization on chip with double and triple phenylboronate moiety as linker are similar. The rate was estimated to be ~1.1 ng/s. Yet, the rate for single phenylboronate moiety was only 70 % of that with double or triple phenylboronate linker. By Comparison of the total quantities of immobilized antibody, the double and triple phenylboronate linkers showed 36 % and 25% higher capacity than that of single phenylboronate, respectively. The unexpected low capacity and density of triple phenylboronate linker may due to the steric hindrance of the adjacent phenylboronate moiety itself. Nevertheless, the binding affinity of antibody toward the three phenylboronate linkers are in the sequence of triple > double > single phenylboronate. This argument was confirmed by the study of antibody desorption from QCM chip in the acidic condition.