設計並合成分子轉子螢光蛋白質探針

Design and Synthesis of Protein Probe Based on the Fluorescent Molecular Rotor

賴修平(Lai, Hsiu Ping)

指導教授 ： 陳貴通

國立清華大學/理學院/化學系/碩士(2015年)

摘要

蛋白質在細胞中的位置、含量及交互作用對生物體有著重要的資訊，與大部分生物進程如生長、代謝、繁殖等息息相關，因此蛋白質偵測極其重要，然而傳統偵測方法需經過一系列複雜的處理步驟，費時且不方便，具有快速偵測、具專一性、高靈敏度、優化簡易、高信號雜訊比等優點的小分子型蛋白質螢光探針便在近年來越來越受到重視，雖然目前有許多小分子型螢光探針證實可用來偵測特定蛋白質，但其多為酶活性型探針，亦即利用蛋白質本身之催化活性，進而達到其偵測目的，但若是針對非酶活性蛋白質進行偵測將受種種限制，由於非酶活性蛋白質在人體中亦佔有重要的地位，開發非酶活性蛋白質探針相當重要。
在此我們以環境敏感螢光分子與對蛋白質之親和性配體結合，並形成一對目標蛋白質具專一性偵測之螢光探針，利用蛋白質結合後其巨大立體障礙限制分子本身旋轉的淬熄機制，使環境敏感螢光分子產生劇烈的螢光變化。我們成功建立一套TO型螢光探針模型，並成功應用於人類碳酸酐酶(hCAII)、凝血酶(thrombin)及抗生物素蛋白(avidin)之專一性檢測，其螢光增益最高可達90倍，並延伸應用於細胞膜上hCAII表達之成像及數種磺胺藥物偵測。

關鍵字

螢光探針

並列摘要

Protein location and interaction in the cells are important, which involve many biological processes, such as growth, metabolism and reproduction. Traditional protein detection methods have to undergo operation complex steps, therefore small molecule fluorescent turn-on probes are getting more attention due to their high sensitive, simple operation, and high specific detection with high signal-to-background ratios. Until now, the design of fluorescence probes for non-enzymatic proteins remains a challenging task.
Here we introduce a general design to construct florescence probes by using conjugates of a fluorescent molecular rotor and protein specific ligands, for the selective fluorescence turn-on detection of proteins through non-enzymatic process.
When the probe bind to the target proten, because of stupendous steric effect caused by the large protein volume, the fluorescence quench mechanism will be restricted, and highly fluorescence can be observed. We successfully established a TO type model for specific detection of hCAII, thrombin and avidin with fluorescent turn-on ratios up to 90-fold. We also demonstrated that one of the fluorescent probes can be employed to visualize carbonic anhydrase expressed on the cell surface, as well as sulfa drugs detection.

並列關鍵字

probe

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