以鍵擊化學製備可專一性結合二氧化矽之功能性蛋白質A及其應用於中孔洞二氧化矽三維蛋白質晶片之研究

本研究首先利用鍵擊化學，將有二氧化矽結合能力之功能性胜肽(Silica Binding Peptide, SBP)與蛋白質A(protein A)結合，形成具有二氧化矽專一性親和力之蛋白質A (SBP-proteinA)，做為生物檢測之探針。利用其可與二氧化矽結合之能力，將其固定於具中孔洞結構的二氧化矽氣凝膠基材表面，製備出具有三維立體結構之蛋白質晶片。藉由蛋白質A與IgG結合之特性，進行三明治免疫分析，可應用於疾病之檢測。本研究選用兩種生物分子進行目標檢測：1.可作為發炎感染指標之抗原分子-白血球間質素-6(IL-6)、2.前列腺癌之生物指標抗原- METCAM/MUC18，並比較所製備之蛋白質晶片與其他方式之蛋白質晶片檢測之能力。本研究經由蛋白質膠體電泳、蛋白質濃度檢測 (BCA)，證實成功合成出與二氧化矽具專一親和力之蛋白質A(SBP-proteinA)，基材所使用之中孔洞二氧化矽，利用SEM和BET鑑定證實其為具有高比表面積之孔洞性材料。生物晶片螢光部分則利用3D 定量活體螢光冷光影像系統 (In Vivo Imaging System，IVIS)和生物晶片掃描儀(Microarray scanner)進行檢測，以螢光訊號強度探討蛋白質晶片之背景值，以及抗體在具備SBP-proteinA之蛋白質晶片的固定化效果，且已證實本研究成功合成出SBP-proteinA。經由本研究結果得知，SBP-proteinA可以成功捕捉抗體，並成功進行後續對IL-6和METCAM/MUC18之三明治免疫分析，其中對於IL-6之偵測極限可達到1pg/ml；對METCAM/MUC18之偵測極限也可達到1.35ng/ml，顯示此方法具有良好的敏感性和穩定性。對各時期前列腺癌患者之血清檢驗結果可知：抗原METCAM/MUC18具有區分癌症和非癌患者之能力，且通過和PSA的共同檢驗，可以有效改善前列腺癌診斷之準確性，減少誤判之情形發生。

關鍵字

生物晶片；鍵擊化學；免疫螢光分析

並列摘要

In this study, a functional protein A (SBP-proA) containing a functional peptide with silica binding ability (SBP) was prepared to use as a probe for the biological detection. This SBP-proA was synthesized by Click Reaction. The SBP-proA was, then, immobilized on the surface of the mesoporous silica aerogel (named A60) substrate, which was synthesized by a sol-gel reaction using an ionic liquid as the template, to prepare a three-dimensional protein chip. The A60 chip with SBP-proA has the ability to capture biological active protein molecules by the sandwich immunoassays method and can be applied on the detection of some diseases. In this study, it was used to detect two antigens, interleukin-6 (IL-6) and METCAM/MUC18. IL-6 is an antigen molecule used as an indicator of inflammatory infection, and the METCAM/MUC18 was used as a diagnostic biomarker for the detection of prostate cancer. The sensitivity of the as-prepared A60 chip was also compared with that of the traditional chemically modified chips. The SBP-proA was characterized with a combination of protein concentration detection (BCA) and gel electrophoresis. The results showed that the probe was successfully prepared by the Click Reaction. The prepared A60 material was characterized with FTIR, SEM and BET. The results confirmed its three-dimensional network structure and also demonstrated that it is a mesoporous material with a large internal surface area. The fluorescence analysis showed that the A60 chip is a low background substrate. In this study, the In Vivo Imaging System (IVIS) and Microarray Scanner were used to investigate the effect of immobilization of antibodies on A60 by the fluorescence signal intensity measurements. It confirmed that SBP-proteinA was successfully synthesized. The results of this study showed that SBP-proteinA successfully captured the antigens, IL-6 and METCAM/MUC18, and were analyzed with sandwich immunoassay method. The detection limit of IL-6 reached 1pg/ml and that of METCAM/MUC18 1.35ng/ml. This revealed that the method provided high sensitivity and stability for detection of the antigens selected. In addition, the method was used to measure the METCAM/MUC18 concentration in the human serum samples of several prostate cancer patients, indicating that the detected METCAM/MUC18 concentration has the ability of distinguishing the cancerous and the non-cancerous patients. Furthermore, it also confirmed that METCAM/MUC18 can be used as a biomarker and the concentrations of the patients as the supporting data for PSA index to improve the accuracy of prostate cancer diagnosis, reducing the occurrence of misjudgment.