A study of the practicability of using disposable electrochemical biosensor to detect α-methylacyl-CoA racemase

α-甲醯基輔酶A消旋酶(α-methylacyl-CoA racemase, AMACR)是近年來被發現針對男性前列腺癌的生物標記，本研究的目的為利用本研究室所開發的單次使用、可拋棄式含Ir奈米粒子的生物感測器為基礎，開發偵測AMACR濃度的生物感測器。利用具有空間異構物的降植烷酸(pristanic acid)為前驅物，與輔酶A(Coenzyme A)、鎂離子(Mg2+)及三磷酸腺苷(Adenosine 5’-triphosphate,ATP)混合均勻產生反應物pristanoyl-CoA，經與AMACR反應後將2R型異構物轉化成2S型，再與過氧化物酶醯基輔酶A氧化酶3(Peroxisomal acyl-coenzyme A oxidase 3, Acox3)反應產生過氧化氫(H2O2)，固定降植烷酸的濃度以及與兩種酵素的反應的時間即可為AMACR定量。利用安培電流檢測法，將電壓固定在+0.5V(相對於銀/氯化銀參考電極)，對過氧化氫進行一系列的檢測。發現將降植烷酸轉化為pristanoyl-CoA的最佳反應時間約為3天。由電化學檢測可知此生物感測器檢測過氧化氫氧化電流的效果良好，且在緩衝溶液中電流大小與AMACR濃度呈現良好的線性關係，其中又以pH 7.0的緩衝溶液效果最佳。α-methylacyl-CoA racemase (AMACR) was recently found the newest biomarker for prostate cancer. The purpose of this study is to develop an AMACR biosensor on the basis of the iridium nano-particle based single-use, dioposable biosensor made in our lab. Using pristanic acids, a chemical naturally has stereoisomers, mixed with CoA (Coenzyme A), Mg2+, ATP (Adenosine 5'-triphosphate,ATP) to generate the substrate, pristanoyl-CoA. AMACR could transfer (2R)-isomer to (2S)-isomer, which is further react with peroxisomal acyl-coenzyme A oxidase 3 (Acox3) and produce the end product H2O2. In a fixed concentration of pristanic acids and fixed reaction time with enzymes could quantitative the concentration of AMACR. The H2O2 is monitored by amperometric tests at an operating potential of +0.45V versus Ag/AgCl reference electrode. The result comes out that the best incubation time for the transformation from pristanic acid to pristanoyl-CoA is three days. Electrochemical measurement shows this disposable biosensor is capable of detecting the oxidizing current of H2O2, and has good linear relationship between response current and AMACR concentration in phosphate buffer solution. The best pH value for operating this sensor is pH7.0.Table of Content Chinese Abstract………………………………………………………………………I English Abstract………………………………………………………………………II Acknowledgement……………………………………………………………………III Table of Contents………………………………………………………………… IV List of Tables and Figures ………………………………………………………… VI Chapter 1 Introduction……………………………………………………………… 1 1.1 Motivation……………………………………………………………………1 1.2 Objective…………………………………………………………………… 5 Chapter 2 Background and theory…………………………………………………… 6 2.1 The Background of Alpha-Methylacyl-CoA Racemase (AMACR) ……… 6 2.1.1 The Relationship with Prostate Cancer…………………………………6 2.1.2 The Role and Physiological Importance……………………………… 8 2.2 Biosensor……………………………………………………………………11 2.3 Electrochemical Biosensor…………………………………………………13 2.3.1 Enzyme-Based Biosensor………………………………………………13 2.3.2 Theoretical Considerations of Enzyme Electrode…………………… 14 2.4 Electrochemical Methods………………………………………………… 17 2.4.1 Cyclic Voltammetry……………………………………………………18 2.4.2 Chronoamperometry……………………………………………………19 Chapter 3 Experimental………………………………………………………………22 3.1 Chemicals and Materials Used in the Test of AMACR Concentration…… 22 3.2 Preparation of Chemical solution………………………………………… 24 3.2.1 Phosphate Buffer solution (PBS) …………………………………… 24 3.2.2 Pristanic Acid solution (Test media) …………………………………24 3.3 The construction of disposable, mini biosensor……………………………25 3.4 Procedures………………………………………………………………… 27 Chapter 4 Results and Discussion……………………………………………………30 4.1 Cyclic Voltammogrametric Studies of AMACR in Phosphate Buffer Solution……………………………………………………………………30 4.2 The Effect of pH Value on the Performance of Detecting AMACR……… 32 4.3 The Influence of Incubation time for Pristanic Acid solution………………36 Chapter 5 Conclusion and Future Perspective…………………………………… 40 References………………………………………………………………………… 41 List of Tables and Figures Fig. 2-1 The metabolism pathway from phytol to pristanic acid. Phytanyl-CoA first undergoes alpha-oxidation, produce pristanic acid. Then (2R)-pristanoyl-CoA is converted to (2S)- pristanoyl-CoA as the substrate of beta-oxidation……10 Fig. 2-2 Schematic diagram of the construction of biosensor……………………… 12 Fig. 2-3 Enzyme electrode contains a biocatalytic layer attached on electrode…… 15 Fig. 2-4 The relationship between reaction rate of an enzymatic-catalyzed reaction and the concentration of substrate (in a fixed enzyme activity) ………… 17 Fig. 2-5 The schematic diagram of a cyclic voltamoogram…………………………19 Fig. 2-6 Scheme of chronoamperometric experiment: (a) waveform of applied potential with time; (b) the profile of reactant concentration which changes with time; (c) a typical chronoamperometric diagram…………………… 21 Fig. 3-1 Structure of Coenzyme A-sodium salt hydrate…………………………… 23 Fig. 3-2 Structure of ATP……………………………………………………………23 Fig. 3-3 Structure of Pristanic Acid…………………………………………………24 Fig. 3-4 The construction and real object of our lab-made disposable biosensor… 26 Fig. 3-5 The flowchart of experimental procedures in our study……………………28 Fig. 3-6 Scheme of operating electrochemical working station…………………… 29 Fig. 4-1 Cyclic Voltammogram of the disposable biosensor showing the response current of pristanic acid solution, with 1ul Acox3 and with both 1ul AMACR and 1ul Acox3………………………………………………………………31 Fig. 4-2 The i-t curve plots shows the response current of different amount of AMACR in (a) pH 6.55 (b) pH7.0 and (c) pH7.5 test media…………… 34 Fig. 4-3 The calibration lines of pH 7.0 and pH 7.5 in (a) 100 sec. (b) 400 sec……35 Fig. 4-4 The i-t curve plots shows the response currents of different AMACR amounts when the incubation time was (a) 1 day (b) 3 days (c) 5 days…………… 38 Fig. 4-5 The calibration lines made from 3 days and 5 days incubation time in (a) 100 seconds (b) 400 seconds……………………………………………………3

THE PREPARATION METHOD OF WELL-DISPERSED ELECTROCATALYST WITH SUPERIOR PERFORMANCE

本發明一種高效能與高分散性之電極觸媒製備方法,係以分散劑將載體顆粒分散,使載體顆粒表面形成一立體阻障層,再將奈米貴金屬觸媒之前趨物結合於該立體阻障層上,經熱處理方式除去該立體阻障層,並以還原劑將前趨物還原成奈米貴金屬觸媒顆粒,並以面心立方晶體結構(Face-Centered CubicCrystal Structure,簡稱F.C.C)均勻的分佈於載體顆粒上,使奈米貴金屬觸媒顆粒於載體上的分散程度及披覆狀況達到最佳合成條件,從而實現提昇貴金屬觸媒顆粒的利用率,增加電化學催化活性面積,提昇電極觸媒效率及燃料電池整體反應效率