酵素顯色偵測法之發展與特性探討及其在生醫科學上的應用

Abstract

本論文以酵素顯色法在靈敏度及多重分析上的發展為研究目的。酵素顯色法利用酵素與特定受質反應產生色素沉澱而達到偵測效果。論文中分別針對酵素顯色法與活化標記佈放(CARD)顯色增益法之動態範圍與偵測極限作探討，同時比較兩種方法在分析上訊號的一致性，並以實際篩選基因驗證結果。在CARD顯色增益法的實例應用上，則研究人類鼻咽癌細胞在抗癌藥物Camptothecin(CPT)處理前後基因表現的情形。根據實驗結果，利用活化標記佈放(CARD)顯色增益法，可將酵素顯色法的偵測靈敏度提高至原來的六十倍，並得到與酵素顯色法一致的結果。 在多色酵素顯色系統的發展方面，利用在標記探針的過程中加入不同比例混合的半抗原，理論上可標記出無限多種顯色後呈現不同呈色的探針。論文中比較以聚合脢連鎖反應及隨機引子法標記多色探針的效率，及顯色結果與加入之半抗原比例的相對應關係﹔實驗最後並以隨機引子法成功的標記出二十一種顯色結果經數值化分析後可解析之探針，若運用在實際分析上，將可達到同時分析多種樣品的目的，使分析的效率大大提昇。

The goals of the research project are on the development of novel methods for enhancing the sensitivity of enzyme colorimetric detection and multiplex detection based on multicolor enzyme colorimetry. Enzyme colorimetric detection is achieved by the enzymatic reaction to create a colour precipitate on the immediate area. The thesis focuses on the dynamic range and limit of detection for both the enzyme colorimetric detection and the signal amplification method by Catalyzed Reporter Deposition(CARD). With the amplification of CARD, studies have been done on the effect of camptothecin(CPT) to the expression profile of human nasopharyngeal cell line(KB cell). The experimental results show that CARD amplification can raise the sensitivity of the technique by sixty folds comparing with enzyme colorimetric detection. Data consistency is achieved at the same time. On the development of multicolor enzyme colorimetric detection, combining different ratios of different haptens in probe labeling can create many probes with different colors. Two different labeling methods, polymerase chain reaction and random-primed labeling, were compared on the labeling efficiency and the correlation of hapten ratios with color . Using random-primed method, 21 probes with distinct colors were successfully created. They can be used to analyze up to 21 samples simultaneously, which greatly increases the efficiency of analysis.