應用免標記光纖式粒子電漿共振生化感測平台檢測退化性膝關節炎之基質金屬蛋白酵素第十三型

Detection of Matrix Metalloproteinase-13 in Synovial Fluid Using Fiber-Optic Particle Plasmon Resonance Biosensors

林玉潔(Yu-Jie LIN)

指導教授 ： 周禮君

國立中正大學/理學院/化學暨生物化學系/碩士(2013年)

摘要

退化性膝關節炎(Osteoarthritis, 簡稱OA)，常見於五十歲以上的人，是老年人最常見的關節炎。關節在長期承受重力及運動下，關節軟骨會退化導致病變，連帶引起軟骨下硬骨增厚、關節變形、滑液膜和關節周圍結構相關變化的疾病。
基質金屬蛋白酵素第十三型(Matrix Metalloproteinase-13, 簡稱MMP-13)，分子量約為52kDa，為基質金屬蛋白酵素(Matrix Metalloproteinases, 簡稱MMPs)系列蛋白之一，過去有研究顯示，在患有關節炎病人軟骨組織的表面，發現其MMP-1、MMP-3、MMP-13的表現量有增加的情形 [1,2,4,5]，也有結果發現在OA病人的膝關節軟骨組織比一般正常人的軟骨組織含有較多的MMP-13 [3]。
本研究首先使用水相金奈米粒子取代有機相金奈米粒子應用於本實驗室所開發之免標記光纖式粒子電漿共振生化感測平台(fiber-optic particle plasmon resonance sensor, 簡稱FO-PPR)進行感測能力的評估，即以不同濃度的蔗糖溶液測試感測器對折射率的感測解析度，測試結果感測解析度可達到3.78 × 10-6 RIU，感測靈敏度亦可達到4.87 AU / RIU，對於基礎生化樣品Anti-OVA的偵測極限則可達到9.36 × 10-7g / ml，結果皆與有機相金奈米粒子偵測能力相當，但使用水相金奈米粒子其製程更為健康環保。
藉由FO-PPR系統的感測能力，我們利用混合自組裝單層膜(Mixed self-assembled monolayer) MUA (11-Mercaptoundecanoic acid)與MCH (Mercaptohexanol)，以及AUT (11-Aminoundecanethiol hydrochloride)與MCH (Mercaptohexanol)當作架橋分子修飾於金奈米粒子表面，並與Anti-MMP-13的抗體做鍵結，對MMP-13進行多濃度的標準品偵測，初步實驗結果對於MMP-13偵測極限可達1.35 × 10-10g / ml (MUA/MCH)以及2.7 × 10-9g / ml (AUT/MCH)。兩者訊號對濃度有相當良好的線性關係R = 0.9987 (MUA/MCH)、0.9979 (AUT/MCH)。同時我們也使用FO-PPR系統和酵素連結免疫吸附分析方法(Enzyme-linked immunosorbent assay, ELISA)進行真實樣品之定量檢測及結果比對。

關鍵字

免標記光纖式粒子電漿共振生化感測平台 ； 基質金屬蛋白酵素-13 ； 混合自組裝單層膜 ； 退化性膝關節炎 ； 金奈米粒子

並列摘要

Knee osteoarthritis (Osteoarthritis, OA), common to people over fifty years old, is the most common form of arthritis in the elderly. Many factors can lead to the degeneration of the knee cartilage, including overuse, injury and hereditary. Joints suffering long periods of gravity and overuse often lead to the cartilage degradation and joint space narrowing.
MMP-13 (Matrix metalloproteinase-13, MMP-13), with a molecular weight of about 52 kDa, is one member of the series of MMPs (matrix metalloproteinases, MMPs). Past studies have shown that the MMP-1, MMP-3 and MMP-13 concentrations on the cartilage in OA patients, are higher than normal.
In this study, we modified the aqueous phase gold nanoparticles on optical fiber probes for analysis of real biological samples by the fiber-optic particle plasmon resonance (FO-PPR) sensor.
The FO-PPR sensor has many advantages such as label-free, high sensitivity, real-time, and low-cost. It offers a potential to monitor biomarkers of various diseases. In this work, we focuses on osteoarthritis concerned biomarker, MMP-13.
First, we tested the sensor with different concentrations of sucrose solutions to determine the sensor resolution and sensor sensitivity. Experimental results show that the sensor resolution can reach 3.78 × 10-6 RIU and the senor sensitivity can reach 4.87 AU / RIU. For conjugation of a biorecognition molecule on the gold nanoparticle surface, we modified a mixed self-assembled monolayer (Mixed SAM) on the surface of the gold nanoparticles, then further conjugate OVA or anti-MMP-13 on the optical fiber probe. For anti-OVA standards, a limit of detection (LOD) of 9.36× 10-7 g/ml was obtained. For MMP-13 standards, the sensor response versus log concentration of MMP-13 is linear (R = 0.9978) and a LOD of 1.35× 10-10g / ml was obtained, which is similar to that by ELISA (Enzyme-linked immunosorbent assay, ELISA).

並列關鍵字

mixed self-assembled monolayer ； osteoarthritis ； fiber-optic particle plasmon resonance sensor ； Matrix Metalloproteinase-13 ； gold nanoparticles

參考文獻

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