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研究生： 楊振
Yang, Jhen
論文名稱： 開發馬來醯亞胺修飾電極用於尿液白蛋白之阻抗式感測
Development of maleimide-modified biosensor for the impedimetric detection of albumin in urine
指導教授： 許梅娟
Syu, Mei-Jwyan
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2021
畢業學年度： 109
語文別： 英文
論文頁數： 68
中文關鍵詞： 生物感測器 、尿液白蛋白 、馬來醯亞胺 、電化學阻抗頻譜
外文關鍵詞： biosensor, urine albumin, maleimides, impedimetric detection
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    • 隨著高齡化社會的來臨，定點照護檢驗技術 (point-of-care testing) 的發展也日益重要，若能定時檢測潛在病患的各項健康指標，便可及早發現病因、及早治療，並降低後續併發症的發生機率，而具有快速檢測、高靈敏度、攜帶便利性等優點的生物感測器，在商業應用上已為各界的重點開發項目之一。對於健康的成人而言，尿液白蛋白 (albumin) 應小於3 mg/dL。然而，在慢性腎臟病人的尿液中，白蛋白濃度可達3~30 mg/dL，稱為微白蛋白尿 (microalbuminuria) 現象。本研究目的為開發尿液白蛋白之生物感測器，並應用於尿液檢體之臨床檢測。於此使用半胱胺酸 (cysteine) 修飾於金電極上並藉由EDC/NHS (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide) 與11-MA (11-maleimidoundecanoic acid) 反應形成馬來醯亞胺修飾電極，白蛋白分子會吸附結合於修飾電極表面，進而產生電化學阻抗訊號，以此校正並定量其濃度。
    本研究先以UV-Vis吸收光譜與FTIR分析白蛋白與11-MA的反應性與結構，接著以拉曼光譜、XPS與SEM對各階段電極表面之鍵結機制與表面形貌進行定性分析。同時以循環伏安法 (cyclic voltammetry, CV) 與 Nyqiust 分析探討電極之電化學性質，且尋求較佳的實驗參數。最終以電化學阻抗變化對白蛋白濃度進行校正，可得具有高線性關係之檢量線，亦將此修飾電極應用於成大醫院提供之尿液檢體，以此修飾電極進行對白蛋白的濃度檢測，具有80% 的檢測準確率，可確認此電化學阻抗式感測器於臨床尿液白蛋白之檢測具有初步可行性。

    To develop health-care devices for POCT (point-of-care testing) is an essential issue owing to the aging society. Biosensor or biochip, a device for quantification of disease biomarkers, plays an important role in this field. The normal concentration range of albumin in urine should be less than 3 mg/dL. Nevertheless, if for renal dysfunction, the albumin concentration in urine could be increased to 3~30 mg/dL, where it is defined as microalbuminuria. Thus, to develop a low-cost biosensor for the determination of urinary albumin concentration is the aim of this work. In this work, the Au electrode was modified by 11-MA (11-maleimidoundecanoic acid) for the preparation of an impedimetric biosensor.
    First of all, the reactivity between albumin and 11-MA and the product structure were analyzed by UV-Vis spectrum and FTIR. The characterization of the as-prepared albumin sensor was carried out by Raman spectroscopy, XPS and SEM. The electrochemical behavior was confirmed by CV (cyclic voltammetry) and Nyquist analysis. With the optimized experimental conditions, the calibration curve for the impedimetric detection of albumin were thus established. Besides, the specimens provided from NCKU Hospital were tested to be 80% accuracy. Consequently, the developed albumin sensor could be feasible for the clinical application.

    中文摘要 I Abstract II Acknowledgement III Contents IV List of Figures VII List of Tables XI Chapter 1. Introduction 1 1.1 Preface 1 1.2 Microalbuminuria 1 1.3 Motivation and Purpose 2 Chapter 2. Literature Review 3 2.1 Albumin 3 2.1.1 Human Serum Albumin (HSA) 3 2.1.2 Clinical Evaluation 3 2.1.3 Hydrophobic Cavity 4 2.1.4 Post-translational Modification of Cys 5 2.2 Biosensors 7 2.2.1 Introduction 7 2.2.2 Recognition Elements (Bioreceptor) 7 2.2.3 Immobilization Techniques 10 2.3 Albumin Sensing Platform 12 2.3.1 Introduction 12 2.3.2 Dye-Binding Methods 12 2.3.3 Fluorescent Sensing Techniques 13 2.3.4 Electrochemical Biosensors 13 Chapter 3. Materials and Methods 16 3.1 Fabrication of Working Electrodes 16 3.1.1 Gold Electrode 16 3.1.2 Zinc protoporphyrin (ZnPP)/Cysteine (Cys) Modified Electrode 16 3.1.3 Maleimides/Cys Modified Electrode 16 3.1.4 Maleimides/2-aminoethanethiol (MEA) Modified Electrode 17 3.1.5 Three-Electrode System 17 3.2 Optimization of 11-MA/Cys Modified Electrode 17 3.2.1 Cys Concentration 17 3.2.2 11-MA Concentration 17 3.2.3 NHS/EDC Molar Ratio 18 3.2.4 Rinse Time 18 3.2.5 Cys Reaction Time 18 3.2.6 11-MA/EDC/NHS Reaction Time 18 3.3 Electrochemical Behavior Analysis 18 3.3.1 CV (Cyclic Voltammetry) 18 3.3.2 Nyquist Plot 18 3.4 Material Characteristics Analysis 19 3.4.1 UV-Vis Spectroscopy 19 3.4.2 FTIR (Fourier-Transform Infrared Spectroscopy) 19 3.4.3 Raman Spectroscopy 19 3.4.4 XPS (X-ray Photoelectron Spectroscopy) 19 3.4.5 SEM (Scanning Electron Microscope) 20 3.5 Clinical Examination 20 3.5.1 Selectivity 20 3.5.2 Reproducibility 20 3.5.3 Stability 20 3.5.4 Clinical Sample Test 20 3.6 Chemicals 21 3.7 Apparatus 22 Chapter 4. Results and Discussion 23 4.1 Biorecognition Materials: Fabrication & Comparison 23 4.1.1 ZnPP/Cys 23 4.1.2 Maleimides/Cys 24 4.1.3 Maleimides/MEA 25 4.1.4 Closure 26 4.2 Reactivity: 11-MA & Albumin 27 4.2.1 Absorbance Variation by UV-Vis Spectrophotometer 27 4.2.2 Structure Analysis by FTIR 30 4.3 Surface Characterization 32 4.3.1 Electrode Characterization by Raman Spectroscopy 32 4.3.2 Surface Element Analysis by XPS 34 4.3.3 Surface Morphology of the Electrodes by SEM 36 4.4 Electrochemical Behavior 36 4.4.1 Modification Characteristics 36 4.4.2 Adsorption of Albumin 39 4.5 Optimization of Instrumental Conditions 40 4.5.1 Frequency 40 4.5.2 Stirring Rate 42 4.5.3 Optimization of pH 44 4.5.4 Calibration Time 45 4.6 Optimization of Electrode Fabrication 46 4.6.1 Concentration 46 4.6.2 Rinse Time 48 4.6.3 Reaction Time 49 4.7 Quantification of Albumin 51 4.7.1 Charge Transfer Resistance 51 4.7.2 Impedimetric Parameters 52 4.7.3 Injection Methods 53 4.8 Clinical Examination 54 4.8.1 Selectivity 54 4.8.2 Reproducibility 56 4.8.3 Stability 57 4.8.4 Standard Urine Calibration 58 4.8.5 Clinical Sample Test 59 4.9 Comparison with the Previous Literature 61 Chapter 5. Conclusions 63 References 64

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