本實驗利用黃嘌呤氧化酶 (Xanthine oxidase, EC 1.17.3.2) 氧化次黃嘌呤(Hypoxanthine) 和酪氨酸酶 (Tyrosinase, EC 1.14.18.1)氧化兒茶酚(catechol)這兩個平行的競爭氧氣酵素反應來建立新型異嘌呤醇(Allopurinol)生化感測器。在本方法中，抑制劑異嘌呤醇的濃度上升會抑制黃嘌呤氧化酶的活性而降低氧氣的消耗，同時酪氨酸酶可以得到較多的氧氣催化更多的兒茶酚氧化物，並能在電極上以0.0V(相對於 Ag/AgCl參考電極)偵測到更多來自兒茶酚氧化物的還原電流。有別於傳統抑制型的測量方式，異嘌呤醇的濃度和訊號成反比，但是本實驗中，可以得到與異嘌呤醇的濃度成正比的訊號。另外，此異嘌呤醇生化感測器採用單層酵素修飾電極以0.0V作為偵測電位有效的防止常見氧化物的干擾且得到快速的響應時間( t90%-10%)為2.9秒。線性範圍可達5μM－100μM (R=0.998)可適用藥物體內濃度5mg-15mg。靈敏度為8.79 nA/μM，偵測極限為1.4μM，連續重複20次的注射異嘌呤醇樣品，所得到的標準偏差(RSD)為4.2%。
異嘌呤醇是治療高尿酸血症(Hyperuricemia)及其併發症的一級用藥，但是在某些病患會發生嚴重的過敏反應例如史蒂芬斯－強森症候群(Stevens–Johnson syndrome)和毒性表皮溶解症(Toxic epidermal necrolysis)，致死率可達20-30%，因此不管是在臨床檢驗或是製藥品管方面，異嘌呤醇的定量都是有必要的。

Two parallel enzymatic oxygen-consumed reaction including oxidation of hypoxanthine by xanthine oxidase (EC 1.17.3.2) and oxidation of catechol by tyrosinase (EC 1.14.18.1) were utilized to constructed novel allopurinol biosensor. In this project, the concentration increase of allopurinol (inhibitor) would inhibit the activity of enzymatic oxygen-consumption by xanthine oxidase. Subsequently, tyrosinase could divvy more oxygen to produce catechol quinone, and it could be observed that more current response was recorded from electrochemical reduction of catechol quinone at 0.0V (vs. Ag/AgCl). In contrast to the determination of traditional inhibition type which the signal was inverse proportional to the concentration of inhibitor, the signal is proportional to the concentration of allopurinol. Moreover, in this biosensor, the monolayer of enzyme modifier and 0.0V detection potential were adapted to obtains fast response (t90%-10% = 2.9 second) and efficiently avoids common interference of substances that co-exist in serum. This allopurinol biosensor possesses linear range 5μM－100μM (R=0.998) that satisfy with therapeutic range (5-15mg/L), sensitivity is 8.79 nA/μM, detection limit is 1.4μM, the relative standard deviation (RSD) is 4.2%.
Allopurinol was used to primary treatment of hyperuricemia and its complication. However, it potentially causes serious hypersensitivity such as Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TENS) on several patients after allopurinol treating. Therefore, the determination of allopurinol is important and necessary for clinical monitoring and quality assurance of pharmacy.

TABLE OF CONTENTS

Abstract
LIST OF FIGURES	VII
LIST OF TABLES	IX
CHAPTER 1 INTRODUCTION	1
1-1 Definition and composition of biosensors	1
1-1-1 Recognition element	1
1-1-2 Types of transducers	3
1-1-3 Electrochemical sensors	7
1-2 Enzyme immobilizations	14
1-2-1 Adsorption method	14
1-2-2 Entrapment method	15
1-2-3 Heterogeneous Blending method	16
1-2-4 Covalent attachment	16
1-3 Research of allopurinol	22
1-3-1 Introduction of uric acid and relative disease	22
1-3-2 Clinical significant of allopurinol	22
1-3-3 Determination of allopurinol	24
1-4 Research goals	31
CHAPTER 2 EXPERIMENTAL SECTION	32
2-1 Materials and reagents	32
2-2 Instrumentations and measurements	32
2-3 Enzyme-modified electrode preparation	33
2-3-1 Graphite modified electrode preparation	33
2-3-2 Enzyme immobilized electrode preparation	34
2-3-3 Enzyme-modified RGDE preparation	34
2-4 Experimental design	34
2-4-1 Preparation of modified electrode	35
2-4-2 Optimization of operation parameter	36
2-4-3 Estimation of analytical performace	39
CHAPTER 3 RESULTS AND DISCUSSION	40
3-1 Mechanism of detection	40
3-2 Electrode pretreatment investigation	42
3-2-1 Effect of period pre-anodization	46
3-2-2 Graphite coating ratio study	47
3-2-3 Effect of enzyme loading amount	50
3-3 Determination of allopurinol optimization	52
3-3-1 Relationship between working potential and sensitivity	52
3-3-2 Effect of acidity	53
3-3-3 Influence of electrolyte	56
3-3-4 Influence of buffer ionic strength	57
3-3-4 Investigation of hypoxanthine and catechol in running buffer	58
3-3-5 Flow rate optimization	60
3-3-6 Sample loop optimization	61
3-4 Evaluation of analytical features for allopurinol biosensor	62
3-4-1 Evaluation of analytical performance	62
3-4-2 Correlation with other method	66
3-4-3 Evaluation of kinetic parameter after enzyme immobilization	67
3-4-4 Interference study	71
CHAPTER 4. CONCLUSION	72


 
List of Figures

Figure 1-1 Relation between potential and time in single-step chronoamperometry	12
Figure 1-2 Typical current response of single-step chronoamperometry	13
Figure 1-3 Series process activates cellulose to carboxymethyl azide and coupling with enzyme.	17
Figure 1-4 Coupling reaction is an alkylation reaction involving the primary amino groups of the enzyme.	18
Figure 1-5 Primary amine of poly-p-styrene are activated to give diazonium, isocyanato and isothiocyanoto group and further coupling with enzyme.	20
Figure 1-6 Reactions involving EDC, including activation as an NHS ester.	21
Figure 1-7 Metabolic pathway of allopurinol.	24
Figure 3-1 Mechanism of allopurinol sensing.	41
Figure 3-2 IR spectra of modified electrode step by step	44
Figure 3-3 Impedance test of electrode modification step by step	45
Figure 3-4 Time of pre-anodization study.	48
Figure 3-5 Weight percent of graphite and ink mixture.	49
Figure 3-6 Effect of enzyme loading amount.	51
Figure 3-7 Potential study.	53
Figure 3-8 Effect of pH for catechol solution storage.	54
Figure 3-9 Acdity study.	59
Figure 3-10 Type of buffer solution study	56
Figure 3-11 Amperometric current response in various buffer concentrations.	58
Figure 3-12 Investigation of hypoxanthine and catechol and in running buffer.	59
Figure 3-13 The relationship between flow rate and peak resolution.	60
Figure 3-14 Influence of sample loop for peak resolution.	61
Figure 3-15 Typical amperometric calibration curve of allopurinol biosensor.	64
Figure 3-16 Reproducibility of continuously measurement by twenty successful injections.	65
Figure 3-17 Correlation plot with other method.    66
Figure 3-18 Effect of different rotating speed to mass transfer in RGDE system.	68
Figure 3-19 “Lineweaver-Burk like” reciprocal plots in steady-state current	70
 








List of Tables
Table 3-1 List of optimal operation parameter.	65
Table 3-2 Analytical performance of the allopurinol biosensor after optimizing.	68
Table 4-1 Comparison analytical performance with published literature for determination for allopurinol.	75

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