合成電化學探針並應用在代謝物偵測

Synthesis and Application of Electrochemical Probe for the Metabolites Detection

陳泉佑(Chuan-Yu Chen)

指導教授 ： 陳貴通

國立清華大學/理學院/化學系/碩士(2013年)

https://doi.org/10.6843/NTHU.2013.00386

摘要

代謝物是任何在代謝過程中的反應物、中間體或是產物。目前已知的代謝物在生物體中有約有7900種，由於代謝物涉及生長、發育與生殖或能量產生及移轉，亦或是在生物上具有重要生物學功能，因此對代謝物的濃度變化進行分析，有助於了解生物的生理概況。
在代謝物中磺胺類藥物是最早拿來用在制菌的藥劑，目前大多數雖然已被抗生素所取代，但對於某些疾病、動植物用藥仍具有不可忽視的價值，故有其研究的重要性存在。
由於磺胺類藥物並無相對應的氧化還原酵素來偵測電流變化，所以我們運用結構轉換型電化學感測器的概念，設計出不需氧化還原酵素亦可偵測代謝物之模型。
電化學生物感測器之設計是利用雙硫化物鏈（Lip-BG），藉由自組裝單分子層在電極表面做修飾，BG負責標記SNAP-hCAⅡ蛋白中的SNAP-tag，則將蛋白修飾在電極表面。當Fc-Linker-SFA探針加入後，探針的SFA會與hCAⅡ蛋白的抑制區結合，使得探針距離電極表面較近，而產生電流訊號；當其他磺胺類分析物加入後，與hCAⅡ蛋白抑制區的探針做競爭，而被競爭游離走之探針因為距離電極表面較遠，造成電流訊號下降，借此達到分析磺胺類藥物。

關鍵字

代謝物 ； 生物感測器 ； 電化學 ； 磺胺類藥物 ； 二茂鐵 ； 探針 ； 循環伏安法 ； 方波伏安法

並列摘要

Metabolites are the reactants, intermediates, and products of metabolism. It is estimated that metabolism of a biological cell comprises 7900 metabolites. Metabolites are important in many biological functions and involve in the normal cell growth, development, reproduction, energy generation and transfer. Hence, development of a selective and sensitive analytical method for the detection of metabolites can provide important insights for many cellular processes.
For the metabolites detection using electrochemical method, one of the fundamental limitations is the requirement of redox enzymes to generate electrochemical signals. However, there are only limited numbers of redox enzymes in nature which are applicable for metabolites detection by using electrochemical method. Many metabolites and drug molecules which do not have the corresponding redox enzymes are important in medical diagnosis and pharmaceutical industry. For example, sulfa drugs which are important for the treatment of many diseases and killing bacteria, have no corresponding redox enzymes to catalyze redox reaction to deliver the electrochemical signals.
The objective of this thesis is to develop an electrochemical sensor which does not require a redox enzyme to generate electrochemical signal to overcome the fundamental limitation of electrochemical method for the selective detection of metabolites and drug molecules.
The electrochemical sensor is constructed by using a recombinant protein, SNAP-hCAII and a dialkyldisulfide linker (Lip-BG) which consists of a disulfide moiety for the self-assembly on gold electrode and a benzylguanine (BG) for SNAP-tag protein labeling. The addition of electrochemical probe Ferrocene-Linker-Sulfonamide (Fc-Linker-SFA) will result in the binding of SFA to the hCAII protein in which the ferrocene will be in the close proximity to the gold electrode to enhance the electrochemical current. Upon addition of sulfonamide drug, the drug will displace the Fc-Linker-SFA from the hCAII binding and as a result, the electrochemical signals will decrease.

並列關鍵字

metabolite ； biosensor ； electrochemistry ； sulfa drug ； ferrocene ； probe ； Cyclic Voltammetry ； Square-wave Voltammetry

參考文獻

50. 施柏銘 利用半合成感測器檢測蛋白酶之活性, 國立清華大學碩士論文, 2012

12. Pijanowska, D.G.; Sprenkels, A.J.; van der Linden, H.; W. Olthuis, P. Bergveld,; van den Berg A., Sens. Actuators B, Chem. 2004, 103 (1-2), 350-355

38. Lai, H. T.; Wang, T. S.; Chou, C. C. Bioresour. Technol. 2011, 102, 5017-5023

9. Bard, A. J.; Faulkner L. R. Electrochemical merhods - fundamentals and applications, 2001, John Wiley & Sons, Inc.

16. van den Berg, C.M.G.; Khan, S. H.; Riley, J. P. Anal. Chem. Acta 1989, 222 (1), 43-54