臺灣博碩士論文加值系統

人的體內會因為L-半胱氨酸(L-cys)濃度異常而引發各種疾病，因此本研究以簡單、準確、快速檢測的電化學方式結合玻璃碳電極(GCE)及可拋棄式金電極(AuE)測定L-cys的濃度。然而，直接使用GCE和AuE會使感測的靈敏度不足，故本研究使用兩種不同的系統分別修飾GCE及AuE以達到高感測靈敏度。
第一個系統為製備普魯士藍-殼聚醣混和物(PB-Chi)與銅-銀纖維複合材料(500-Ag-Cu/PVP)並將兩者修飾於GCE表層(Cu-Ag-PB-Chi/GCE)，第二個系統以電鍍方式將銅沉積於金電極表面(Cu/AuE)。
本論文第一部分利用電紡絲製備PVP纖維，並且將PVP纖維硫醇官能化 (thiol-functionalization)，在500 oC下將銀還原於PVP纖維上，再以化學鍍銅方式將500-Ag-Cu/PVP製備而成。而GCE的修飾層是將已製備出的500-Ag/PVP 、500-Ag-Cu/PVP、化學合成PB，及殼聚醣溶液，用於製備三種不同的修飾GCE ( PB-Chi，Ag-PB-Chi，以及Cu-Ag-PB-Chi/ GCE)。
利用SEM觀察熱燒結含有銀離子之PVP纖維(Ag+-RSH-CL-PVP fibers)，依然存在絲狀結構。同時可以觀察到，經過銅化學鍍層法製備出的500-Ag-Cu/PVP複合纖維較500-Ag/PVP纖維平均直徑大。透過FTIR分析可觀察到Si-O-Si以及Si-O-CH3官能基訊號，由此可說明硫醇官能化反應成功。更利用XRD觀察500-Ag/PVP纖維與500-Ag-Cu/PVP複合纖維的結晶型態，由訊號結果可得到Ag和Cu良好的結晶性。
將三種不同的修飾GCE分別利用循環伏安法去檢測L-cys，從循環伏安法結果得知，L-cys的感測能力分別為Cu-Ag-PB-Chi/GCE > Ag-PB-Chi/GCE > PB-Chi/GCE。有這樣的改善結果是由於，500-Ag-Cu/PVP複合纖維中的Ag-Cu協同效應和對於L-cys有好的氧化催化效果。在電化學測試結果中可以找到兩線性範圍及相對靈敏度，分別為40-1800 µM : 0.1501 µA.µM-1cm-2以及1800-2500 M : 0.0707µA.µM-1cm-2，並可計算出最低偵測極限為1.42 M。此外，在抗干擾測試中發現Cu-Ag-PB-Chi/GCE對L-cys有很高的專一性，且不受sucrose、 glucose、 citric acid、 oxalic acid、 urea、 EDTA以及uric acid的影響。
本論文第二部分為利用Cu/AuE感測L-cys，Cu/AuE主要將金電極浸泡於含銅之水溶液，並利用恆電位沉積法使銅離子沉積在金電極上面，以製備出Cu/AuE。透過XRD與SEM做表面形態以及結晶性分析。從SEM表面特性分析中可以證明，銅電鍍層會受到電化學條件(如: 供應電流、沉積時間)以及前驅物組成溶液(如: CuSO4以及Na2SO4濃度比例不同)的影響，進而發現在沉積時間大於480秒會有較均勻的銅層覆蓋；此外，在較高的供應電流下銅會有樹突狀結構形成。在XRD的分析中可以確認，利用電化學沉積方法成功地將銅沉積在金電極表面。透過最適化測試，可以找出在供應電流為-0.4 V下沉積480秒有最高的電流響應。而製備出的Cu/AuE也利用循環伏安法做電化學特性的測試。根據循環伏安法的測試結果可以看出，Cu/AuE在0.1 M NaOH鹼性溶液中，對L-cys有較好的電催化活性。而最適化參數下Cu/AuE有最小的偵測極限約為0.21 M，並可以找到兩段線性範圍及其相對靈敏度，分別為1-400 µM : 1.0493 µA.µM-1.cm-2以及 400-1800 µM : 0.5090 µA.µM-1.cm-2。此外，在抗干擾測試中發現Cu/AuE對L-cys有很高的專一性，且不受sucrose、 glucose、 citric acid、 oxalic acid、 urea、 EDTA，以及uric acid的影響。

The development of effective strategy to perform electrochemical determination of l-cysteine (L-cys) is of great importance for physiological and clinical diagnosis of various diseases owing to the abnormal level of L-cys. In this study, the application of electrochemical method aim to perform simple, accurate, and fast detection of L-cys using glassy carbon (GCE) and gold electrodes (AuE).
Since direct use of bare GCE and AuE possessed problems of insufficient sensitivity and specificity, two designs of sensors for electrochemical determination of L-cys which are copper-silver fibers composite (500-Ag-Cu/PVP fibers) incorporated in Prussian blue-chitosan modified on GCE (Cu-Ag-PB-Chi/GCE) and copper electrodeposited on AuE (Cu/AuE) were prepared in order to address those problems. The modified layer of GCE was made under three steps: (1) preparation of 500-Ag-Cu/PVP fibers involving electrospinning, crosslinking, thiol-functionalization, silver-ions loading, heat reduction and copper electroless plating, (2) chemical synthesis of PB, and (3) preparation of chitosan solution (Chi). The as-prepared 500-Ag/PVP fibers, 500-Ag-Cu/PVP fibers, chemically synthesized PB, and Chi were used to fabricate: PB-Chi, Ag-PB–Chi, and Cu-Ag-PB–Chi/ GCE.
The surface modification on the electrodes were evaluated by different analytical methods. Surface morphology observation by SEM revealed that after heat reduction allow to load silver-ions on PVP fibers (Ag+-RSH-CL-PVP fibers), where the structure of fibers was maintained. Meanwhile, by means of copper electroless plating, the 500-Ag-Cu/PVP fibers which possessed thicker average diameter than 500-Ag/PVP fibers was generated. The thiol-functionalization process was confirmed by FTIR that the peaks corresponded to the functional group of Si-O-Si and Si-O-CH3 were found. Moreover, XRD pattern results declared that the as-prepared 500-Ag/PVP and 500-Ag-Cu/PVP fibers reveal the crystalline phases of Ag and both Cu and Ag, respectively.
The three modified GCE were characterized by cyclic voltammetry to investigate the current responses toward L-cys. The results of CV response of L-cys revealed the following findings: (1) PB-Chi/GCE was the least sensitive to L-cys, (2) Ag-PB-Chi/GCE exhibited higher current response than that of PB-Chi/GCE (1.2 times fold higher), and (3) Cu-Ag-PB-Chi/GCE owned a significant increase of sensitivity toward L-cys, as compared to that of PB-Chi (1.8 times fold increase) and Ag-PB-Chi/GCE (1.5 times fold increase). This improvement implied the contribution of synergetic effects of catalytic behavior of 500-Ag-Cu/PVP fibers in electrooxidation of L-cys. The sensing performance of Cu-Ag-PB-Chi/GCE was examined by amperometric test under optimized conditions. The Cu-Ag-PB-Chi/GCE showed two linear ranges over conentrations of 40-1800 and 1800-2500 µM with corresponding sensitivities of 0.1501 and 0.0707µA.µM-1cm-2, respectively, with the detection limit of 1.42 µM. According to interference tests, Cu-Ag-PB-Chi/GCE was selective toward L-cys and showed negligible response to sucrose, glucose, citric acid, oxalic acid, urea (concentration ratio L-cys to interferent= 1:1), uric acid, and EDTA (concentration ratio L-cys to interferent= 10:1).
The second objective of this thesis is to prepare L-cys sensor based on Cu/AuE. The Cu/AuE was developed by potentiostatic deposition of metallic Cu from a precursor solution onto AuE. The surface morphology and crystallinity of the Cu/AuE were studied by SEM and XRD, respectively. According to SEM observation, the homogeneous coverage of copper layer on AuE was obtained for longer deposition time (≥ 480 s). In addition, dendrites structure of copper can be produced when high overpotential (≤ -0.7 V) was applied. XRD pattern of Cu/AuE confirmed that copper was successfully electro-deposited on the surface without the presence of its corresponding oxide forms. For further assessments, AuE was electrodeposited at -0.4 V for 480 s from solution containing 0.005 M of CuSO4 and 0.3 M Na2SO4, concerning the highest amperometric response resulted from Cu/AuE farbricated under these parameters. The electrochemical characteristics of Cu/AuE were investigated using cyclic voltammetry tests. Based on CV results, the Cu/AuE displayed a prominent anodic peak ascribed for electrooxidation of L-cys, indicating greater electrooxidation activity toward L-cys than bare AuE. The amperometric test run under optimized conditions showed that the Cu/AuE had lowest detection limit of 0.21 µM and two linear ranges between 1-400 and 400-1800 µM with corresponding sensitivities of 1.0493 and 0.5090 µA.µM-1.cm-2, respectively. Additionally, the Cu/AuE also exhibited high specificity to L-cys, as minor influence from sucrose, glucose, citric acid, oxalic acid, urea, EDTA (concentration ratio L-cys to interferent= 1:1), and uric acid (concentration ratio L-cys to interferent= 40:1) to the L-cys signal.

Abstract i
Acknowledgement vi
Contents viii
List of figures x
List of tables xiv
Abbreviations xv
Chapter 1. Introduction
1. Fabrication of a non-enzymatic l-cysteine sensor based on copper-silver fibers incorporated with Prussian blue (Cu-Ag-PB-Chi/GCE) 1
2. Disposable l-cysteine amperometric sensor based on electrodeposited copper on gold electrode (Cu/AuE) 3
Chapter 2. Literature review
1. Importance of L-cysteine detection 6
1-1. L-cysteine 6
1-2. Common methods for l-cysteine detection 7
2. Biosensor 15
2-1. Chemically modified electrodes 18
2-2. Prussian Blue (PB) 19
2-3. Silver nanomaterials-modified electrode 23
2-4. Copper nanomaterial-modified electrode 24
3. Electrospinning 31
3-1. Basic setup and mechanism of electrospinning 32
3-2. Fabrication of fibers decorated metals or metal oxides based on electrospinning and surface functionalization techniques 34
Chapter 3. Experimental
1. Chemicals 37
1-1. Fabrication of composite of copper-silver fibers (500-Ag-Cu/PVP fibers) 37
1-2. Fabrication of Cu-Ag-PB-Chi/GCE 37
1-3. Electrodeposition of copper on AuE 38
1-4. Electrochemical measurements 38
2. Equipment and instruments 38
3. Experimental procedures 39
3-1. Fabrication of composite of copper-silver fibers (500-Ag-Cu/PVP fibers) 39
3-2. Fabrication of Ag-Cu-PB-Chi/GCE 42
3-2-1. Preparation of Prussian Blue (PB) 42
3-2-2. Preparation of Chitosan (Chi) 43
3-2-3. Incorporation of PB-Chi, Ag-PB-Chi, and Cu-Ag-PB-Chi modified layers on glassy carbon electrode (GCE) 43
3-2-4. Preparation of Cu/AuE 44
3-3. Electrochemical Analyses 44
3-3-1. Electrochemical experiments 44
3-3-2. Cyclic voltammetry 46
3-3-3. Amperometric i-t test 49
4. Material Characterizations 50
4-1. Fourier Transform Infrared Spectroscopy (FTIR) 50
4-2. Field Emission Scanning Electron Microscopy (FE-SEM) 51
4-3. X-Ray Diffraction (XRD) 51
4-4. Thermogravimetry Analysis (TGA) 51
5. Experimental flow chart 51
5-1. Preparation of Cu-Ag-PB-Chi/GCE for l-cysteine determination 51
5-2. Electrodeposition of copper on gold electrode for l-cysteine sensor 53
Chapter 4. Results and Discussion
1. Material characterizations and electrochemical sensing behavior of Cu-Ag-PB-Chi/GCE 54
1-1. Thermal characteristics of cross-linked PVP (CL-PVP) fibers 54
1-2. Surface morphology by FE-SEM 55
1-3. Surface functionalities by FTIR 58
1-4. Surface crystallinity by XRD 59
1-5. Electrochemical characterization of the modified electrode 60
1-6. Electrocatalytic oxidation of l-cysteine at Cu-Ag-PB-Chi/GCE 62
1-7. The pH effects on electrochemical performance of Cu-Ag-PB-Chi/GCE toward l-cysteine 65
1-8. The effects of copper-silver fibers (500-Ag-Cu/PVP fibers) concentration 69
1-9. The effects of volume ratio of Prussian blue (PB) to chitosan (Chi) 70
1-10. Amperometric response of Cu-Ag-PB-Chi/GCE toward l-cysteine 71
1-11. Anti-interference evaluation of Cu-Ag-PB-Chi/GCE 73
1-12. Comparison of various electrodes for l-cysteine determination 74
2. Material characterizations and electrochemical sensing behavior of Cu/AuE 76
2-1. Surface morphology by FE-SEM 76
2-2. Surface crystallinity by XRD 84
2-3. Electrochemical characterization of the modified electrode 86
2-4. Electrocatalytic oxidation of l-cysteine at Cu/AuE 87
2-5. Optimization of electrodeposition parameters of Cu/SPE 91
2-5-1. The effects of applied potential during electrodeposition 91
2-5-2. The effects of electrodeposition period 92
2-5-3. The effects of CuSO4 concentration 93
2-5-4. The effects of NaSO4 concentration 94
2-6. The amperometric response of Cu/AuE toward l-cysteine 95
2-7. Anti-interference evaluation of Cu/AuE 98
2-8. Comparison of electrode performance 102
Chapter 5. Conclusions 104
References 107

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