臺灣博碩士論文加值系統

本實驗利用四氧化三鈷(Co3O4)在鹼性下催化過氧化氫還原的特性研究，利用交聯法將對尿酸具有專一性辨識力的尿酸酶(Uricase, EC 1.7.3.3)固定於四氧化三鈷的修飾旋轉電極上，發展成尿酸電化學生化感測器。分別利用高解析度 X光繞射儀(HRXRD)與場發式掃描電子顯微鏡(FE-SEM)，針對由水熱法所製備的鈷氧化物定性與表面分析的量測，結果顯示自製鈷氧化物為四氧化三鈷且為矩形奈米片堆積結構。而此尿酸生化感測器之最佳化製備條件為70% (w/w%) Co3O4修飾於RGDE電極表面，置於80℃烘箱1小時乾燥，隨後在電極上修飾5 μL 0.5%小牛血清蛋白，靜置4℃乾燥後再加入5 μL 0.1%戊二醛靜置4℃乾燥，最後再滴上0.5 units尿酸酶靜置4℃乾燥即完成電極的製備。而該生物傳感器最佳化偵測條件為0.05 M pH 9.5 Clark & Lubs 緩衝液，偵測電位為0.05V (vs. Ag/AgCl)，電極旋轉速度為400rpm環境下進行尿酸偵測。根據上述操作條件，此感測器分析特性如下:線性範圍可達2 μM - 102 μM(R=0.999)，靈敏度為18.24 μA/mM，偵測極限為0.6μM，在精確度(precision)方面連續重複偵測25 μM尿酸20次操作下，所得到的標準偏差(RSD)為1.38%，反應時間(t90%/10%)為27.34秒。根據干擾物測量結果顯示，在此電位下常見的干擾物，例如acetaminophen、creatin以及dopamine不會有明顯干擾外，其他的干擾比率界於-556.56 % ~5.8% 之間。而針對抗壞血酸(Ascorbic acid)的干擾，若在偵測前1小時先加入抗壞血酸酶(Ascorbate oxidase)進行預處理，即可避免抗壞血酸之干擾。最後，此感測器在不使用時保存於4℃冰箱緩衝溶液中，其活性維持至少83 天不變，結果顯示該電極具有良好的穩定性，其83 天間的精確度為4.12 %。

In current study, a cobalt oxide based uric acid biosensor is fabricated through a drop coating of glutaraldehyde, and uricase onto a BSA coated Co3O4 based rotating disc graphite electrode (RGDE). Uric acid is measured via an uricase based Co3O4 modified biosensor cathodicly. The amperometric signal of this sensor is measured based on the uricase converts uric acid into hydrogen peroxide and reduces by Co3O4. The homemade cobalt oxide fabricated by hydrothermal synthesis is better than the commercial one. Qualitative analysis and morphology of cobalt oxide was characterized by High resolution X-ray diffractmeter (HRXRD) and scanning electron microscopic (SEM). The results show that homemade cobalt oxide is Co3O4 and exhibits rectangular sheets with pore and piled the nanosheets of each other. The uric acid biosensor was simply fabricated with mixing carbon ink and 70% Co3O4 then placed on RDGE and dried in the oven at 80℃ for an hour. Followed drop coated 5 μL 0.5 % BSA, 5 μL 0.1% glutaraldehyde, and 5 μL 0.5 units uricase onto electrode till dried in 4℃ refrigerator. The biosensor showed optimum conditions for the analysis of uric acid are in 0.05 M pH 9.5 Clark & Lubs buffer, when applied at 0.05 V (vs. Ag/AgCl) with rotating rate of 400rpm. The linear range of this scheme covers from 2 μM to 102 μM (R=0.999), with sensitivity is 18.24 μA/mM and a detection limit of 0.6 μM. The relative standard deviation (RSD) for 20 times successive measurement of 25 μM UA is 1.38%, and the response time (t90%/10%) is 27.34 s. These metabolites have interference except acetaminophen, creatine, and dopamine. Others ratio of interference were between -556.56 % and 5.8%. Among these metabolites, ascorbic acid has -556.56 % interference. Therefore, the removal of AA interference could be eliminated by pretreatment of 10 units ascorbate oxidase with sample for one hour in advance. This sensor has good stability during 83 days study with 4.12% of RSD.

Contents
Chapter 1 Introduction 1
1-1 Definition and Composition of Biosensor 1
1-2 Development of Biosensors 2
1-3 Modified Electrode 3
1-3-1 Scheme of Modification 4
1-3-2 Functionality of Modified Electrode 9
1-4 Medical Researches of Uric Acid 11
1-4-1 Purine Metabolism and Formation of Uric Acid 11
1-4-2 Clinical Significance of Uric Acid 12
1-5 Uric Acid Sensors 17
1-5-1 Spectrophotometry 18
1-5-2 Chemiluminescence 21
1-5-3 Chromatography 24
1-5-4 Electrophoresis ‒ Capillary Electrophoresis 26
1-5-5 Electrochemical Schemes 27
1-6 Brief Introduction of Cobalt (II, III) Oxide (Co3O4) 36
1-6-1 Methods of Preparation Co3O4 37
1-7 Research Goals 41
Chapter 2 Experimental Section 42
2-1 Instrumentations and Measurements 42
2-2 Reagents 42
2-3 Synthesis of Co3O4 Nanosheet 43
2-4 Electrodes Preparation of Co3O4 Based Electrode for Hydrogen Peroxide 44
2-4-1 Pretreatment of Electrodes 44
2-4-2 Co3O4 Based Modified Electrode 44
2-4-3 Immobilization of Uricase onto Co3O4 Based Electrode 45
2-5 Experimental Design 45
2-5-1 Primary Investigation of Cobalt Oxide 46
2-5-2 Calcined Temperature Study 46
2-5-3 Mechanism of Co3O4 Based Uric Acid Biosensor 46
2-5-4 Consideration of Hydrogen Peroxide Detect 46
2-5-5 Optimized Operating Parameters of Uric Acid Biosensor 47
2-6 Analytical Performance of Uric Acid Biosensor 50
Chapter 3 Results and Discussion 51
3-1 Primary Investigation of Cobalt Oxide 51
3-2 Mechanism of Co3O4 Based Uric Acid Biosensor 56
3-3 Optimized Operating Parameter of Uric Acid Biosensor 59
3-3-1 Consideration of Hydrogen Peroxide Detect 59
3-3-2 Composition Study ‒ Co3O4 and Ink Ratio 62
3-3-3 Optimization of the Immobilization Scheme 64
3-3-4 Relationship between Apply Voltage and Sensitivity 66
3-3-5 Effect of pH on the Response 67
3-3-6 Influence of Electrolyte solution 68
3-3-7 Influence of Buffer Ionic Strength 69
3-3-8 Effect of Temperature 70
3-3-9 Effect of Revolution Rate 72
3-4 Evaluation Analysis Features of Uric Acid Biosensor 73
3-4-1 Analytical Performance of Uric Acid Biosensor 73
3-4-2 Interference Study 77
3-4-3 Storage Stability of Storage Stability of Co3O4 Based Uric Acid Biosensor 79
Chapter 4 Conclusion 81
Reference 83
Figure and Table Caption
Fig. 1 Purine metabolism 12
Fig. 2 HRXRD patterns of cobalt oxide 52
Fig. 3 FE-SEM micrographs of Co3O4 53
Fig. 4 Calcined temperature study 54
Fig. 5 Typical reductive current response 55
Fig. 6 Sensing mechanism of Co3O4 based uric acid biosensor 57
Fig. 7 Dependence of Co3O4 toward hydrogen peroxide 58
Fig. 8 Sensitivity of hydrogen peroxide and ambient oxygen 61
Fig. 9 Composition study 63
Fig. 10 Composition study 63
Fig. 11 Composition study of BSA, glutaraldehyde, and uricase 65
Fig. 12 Potential effect study 67
Fig. 13 Effect of pH on the response 68
Fig. 14 Influence of electrolyte solution 70
Fig. 15 Influence of buffer concentration. 71
Fig. 16 Effect of temperature 72
Fig. 17 Effect of rotation speed 73
Fig. 18 Typical steady-state amperometric calibration plot 76
Fig. 19 Investigate the reproducibility of uric acid biosensor 76
Fig. 20 Lineweaver-Burk like reciprocal plot 77
Fig. 21 Lifetime of the uric acid sensor. 80


Table 1 Comparison of the Co3O4 nanoparticles shape and size with different surfactant and fabricate methods 40
Table 2 Optimization of conditions to detect uric acid 74
Table 3 Analytical performances of the biosensor 77
Table 4 Effect of interferents on the amperometric response of uric acid biosensor 79
Table 5 Comparison analytical performance of the literature proposed methods with other electrochemical methods for the determination of uric acid 82

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