利用苯硼酸衍生物作為抗體專一位向固定化並應用於前降鈣素檢測

Abstract

敗血症主要是由全身性細菌感染所引起，傳統上的檢測方法是酵素連結免疫吸附法(ELISA)或是以血液培養，ELISA大約需要六個小時，而血液培養更是需要兩到三天的時間，耗費時間並延誤治療時程，檢測血液中CRP的濃度也是常被使用的檢測法，但對於鑑別細菌感染與病毒感染仍有部分誤差，很容易造成醫生誤判，延誤病情。近年來，用來判別敗血症的新生物指標前降鈣素 (Procalcitonin)已被研究出來，根據不同的嚴重程度，血液中的前降鈣素濃度也會不同。因此如何檢測前降鈣素就成為重要的課題。

本研究是以石英晶體微天平為平台感測器，以進行生物感測，此感測器的固體材料為金電極，在嘗試過許多清洗金電極的方式之後，發現使用化學方法搭配電化學方法的清洗方式對於金電極有最佳的清洗效果。

目前也有很多文獻指出，金的材料與烷基硫醇反應，其表面會形成排列緻密的自組裝單層薄膜，因此實驗設計一端為硫醇一端為苯硼酸的長鏈烷基化合物，利用此化合物的硫醇與金反應，固定化在金表面上，接著利用另外一端的苯硼酸對前降鈣素單株抗體Fc端寡醣區，作有位向性的鍵結，以增加與前降鈣素鍵結的位置提高實驗的再現性，並利用噬菌體與降鈣素原鍵結位置不同，達到增強訊號，使偵測前降鈣素靈敏度達到10-11 g/ml的程度。

Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state (called systemic inflammatory response syndrome or SIRS) and the presence of a known or suspected infection. Traditional detection methods used ELISA (Enzyme-linked immunosorbent assay) or blood incubation, it takes 6 hours to screen the blood by ELISA and 2-3 days to incubate bacteria. The method was time consuming, and delay for medical treatment . Detecting CRP concentration in blood is also a method to diagnosis sepsis. Unfortunately, this kind of method also hard to seperate inflammatory and virus infection. In recent years, Procalcitonin (PCT) becomes an innovative and highly specific marker for the diagnosis of clinically relevant bacterial infections and sepsis. Procalcitonin supports early diagnosis and clinical decision making which could direct an effective therapy immediately and save unnecessary spending for critically ill patients.

This study is based on Quartz Crystal Microbalance as a biosensor, sensor chip is composed of gold electrode. During different experiments, we found cleaning the gold surface using standard wash (for example: piranha, or standard cleaning method) was inefficient. The result was proved by the fluorescence labeling antibody.

Previous study shows that gold substrate and thiol can form densely packed self-assembly monolayer. Base on this idea, the design for our synthesis strategy was that composed of thiol and phenylboronic acid bi-funtional probe immobilized on the gold, and coupling with antibodies via carbohydrate moiety, located on the Fc region. This method offer a gentle oriented immobilization of proteins, maintaining protein’s activity, and increase reproducibility. Furthermore, in order to increase the sensitivity during detection of low concentration, we use the Fab-phage particle to enhance the signal successfully which has different binding site between the procalcitonin monoclonal antibody and Fab-phage.