近年來，衛生保健對人類的生活日趨重要，如果能夠即時診斷並預測疾病的發展，在疾病的治療與預防上便得以及早應對；或是在食品安全與環境監測等領域，提供人們保障自身健康的途徑。因此，以具有快速檢驗、高靈敏度、微量樣本、非侵入、可攜式且使用簡便為開發目標，如今已有許多生物感測器商業化並應用於臨床醫學與居家照護。本研究的重點在於開發交流阻抗式生物感測器，對人類血清白蛋白 (human serum albumin, HSA) 進行濃度校正，並著墨於各項參數對HSA檢測的影響、電極材料分析、電極的電化學特性分析以及實際應用於人體血清中HSA的檢測。
本文選用pyrrole與其衍生物P3CA，利用循環伏安法 (cyclic voltammetry, CV) 以電聚合的方式製備導電性高分子薄膜於金工作電極上，並將6-MA (6-maleimidohexanoic acid) 吸附於修飾後的電極表面。6-MA可與HSA發生高選擇性反應，造成電極阻抗大幅提升以及顯著的電容效應，故能藉由測量阻抗變化以及相位角變化，來達到定量HSA的目的。
本研究先以UV-Vis吸收光譜與HPLC-UV/Vis對HSA與6-MA之間的反應進行驗證；接著各階段之修飾電極皆以Raman光譜鑑定表面官能基，驗證P3CA、6-MA及HSA特徵峰的存在；以SEM和AFM分析各階段修飾電極的表面型態；以CV探討電極修飾前後電化學活性面積的變化，並以EIS數據模擬各階段電極的等效電路；以交流阻抗對HSA進行濃度校正，針對感測的靈敏度來調整各項實驗參數，HSA濃度校正線分別在自製電極與市售網印電極均具高度線性，分別為：y=0.472x+1.709, R2=0.9995以及y=0.192x+7.920, R2=0.998；最後針對醫院的人血清檢體進行HSA濃度檢測，以阻抗變化率為訊號時，感測得以在15分鐘內完成，並具有71% 的檢測準確率，由實驗結果判斷，此交流阻抗式感測器是高效且可行的。

Human serum albumin (HSA) is an important biomarker for renal or liver diseases. This study focuses on the development of electrochemical impedance biosensor for the quantification of HSA. Handmade gold electrodes deposited with a conducting copolymer layer, poly(Pyr-P3CA), by electropolymerization were futher modified with 6-maleimidohexanoic acid (6-MA). 6-MA could bind with HSA and thus lead to impedance change as well as phase angle change. Conjugation reaction between maleimide group of 6-MA and Cys34 residue of HSA can be comfirmed by HPLC-UV/Vis spectrophotometer. The as-prepared electrodes were characterized by Raman spectroscopy, SEM, and AFM. Equivalent circuit of HSA electrochemical sensing system can be perfectly simulated from EIS data. Consequently, the signal corresponding to HSA concentration is confirmed to be calibrated with excellent linearity and also measured for clinical applications in this work.

[1] OECD Health Statistics. Health expenditure and financing, http://stats.oecd.org/Index.aspx?DataSetCode=SHA.
[2] AN Sekretaryova, M Eriksson, AP Turner. Bioelectrocatalytic systems for health applications. Biotechnology Advances 34, 177−197, 2016.
[3] G Inzelt. Conducting polymers: a new era in electrochemistry. Springer-Verlag, Inc., Berlin, 2008.
[4] G Kaur, R Adhikari, P Cass, M Bown, P Gunatillake. Electrically conductive polymers and composites for biomedical applications. RSC Advances 5, 37553−37567, 2015.
[5] R Balint, N Cassidy, S Cartmell. Conductive polymers: towards a smart biomaterial for tissue engineering. Acta Biomater 10, 2341−2353, 2014.
[6] JY Wang, KC Ho, LC Chen. Preparation of redox polymer nanocomposites for application in biosensors and biofuel cells. PhD thesis, 1−169, 2014.
[7] E Rivard. Inorganic and organometallic polymers. Annual Reports Section "A" (Inorganic Chemistry) 108, 315−329, 2012.
[8] R Gracia, D Mecerreyes. Polymers with redox properties: materials for batteries, biosensors and more. Polymer Chemistry 4, 1−2206, 2013.
[9] EJ Calvo, C Danilowicz. Amperometric enzyme electrodes. Journal of the Brazilian Chemical Society 8, 563−574, 1997.
[10] A Patra, YH Wijsboom, SS Zade, M Li, Y Sheynin, G Leitus, M Bendikov. Poly(3,4-ethylenedioxyselenophene). Journal of the American Chemical Society 130, 6734–6736, 2008.
[11] TVA Dam, D Pijanowska, W Olthuis, P Bergveld. Highly sensitive glucose sensor based on work function changes measured by an EMOSFET. The Analyst 128, 1062−1066, 2003.
[12] R Kumar, S Singh, BC Yadav. Conducting polymers: synthesis, properties and applications. International Advanced Research Journal in Science, Engineering and Technology 2, 110−124, 2015.
[13] HF Mark. Oxidative polymerization. Encyclopedia of Polymer Science and Technology 10, 740−764, 2013.
[14] IY Sapurina, MA Shishov. Oxidative polymerization of aniline: molecular synthesis of polyaniline and the formation of supramolecular structures. New Polymers for Special Applications, 252−312, 2012.
[15] Y Wei. Nonclassical or reactivation chain polymerization: a general scheme of polymerization. Journal of Chemical Education 78, 551−553, 2001.
[16] NY Abu-Thabit. Chemical oxidative polymerization of polyaniline: a practical approach for preparation of smart conductive textiles. Journal of Chemical Education 93, 1606−1611, 2016.
[17] OY Posudievsky, OA Goncharuk, VD Pokhodenko. Mechanochemical preparation of conducting polymers and oligomers. Synthetic Metals 160, 47−51, 2010.
[18] D Braga, L Maini, F Grepioni. Mechanochemical preparation of co-crystals. Chemical Society Reviews 42, 7638−7648, 2013.
[19] H Segawa, T Shimidzu, K Honda. A novel photo-sensitized polymerization of pyrrole. Journal of the Chemical Society, Chemical Communications, 132−133, 1989.
[20] JM Kern, JP Sauvage. Photochemical deposition of electrically conducting polypyrrole. Journal of the Chemical Society, Chemical Communications, 657−658, 1989.
[21] HB Brom, Y Tonikiewicz, A Aviram, A Broers, B Sunners. On a new conducting polymer-pyrolyzed Kapton. Solid State Conmiunications 35, 135−139, 1980.
[22] CS Park, DH Kim, BJ Shin, DY Kim, HK Lee, HS Tae. Conductive polymer synthesis with single-crystallinity via a novel plasma polymerization technique for gas sensor applications. Materials (Basel) 9, 812−822, 2016.
[23] CS Park, DH Kim, BJ Shin, HS Tae. Synthesis and characterization of nanofibrous polyaniline thin film prepared by novel atmospheric pressure plasma polymerization Technique. Materials (Basel) 9, 39−50, 2016.
[24] M Gvozdenovic, B Jugovic, J Stevanovic, B Grgur. Electrochemical synthesis of electroconducting polymers. Hemijska industrija 68, 673−684, 2014.
[25] D Kumar, RC Sharma. Advances in conducting polymers. European Polymer Journal 34, 1053−1060, 1998.
[26] S Zhou, T Wu, J Kan. Effect of methanol on morphology of polyaniline. European Polymer Journal 43, 395−402, 2007.
[27] H Goto, H Yoneyama, F Togashi, R Ohta, A Tsujimoto, E Kita, K Ohshima. Preparation of conducting polymers by electrochemical methods and demonstration of a polymer battery. Journal of Chemical Education 85, 1067−1070, 2008.
[28] DD Ateh, HA Navsaria, P Vadgama. Polypyrrole-based conducting polymers and interactions with biological tissues. Journal of The Royal Society Interface 3, 741−752, 2006.
[29] P Camurlu. Polypyrrole derivatives for electrochromic applications. RSC Advances 4, 55832−55845, 2014.
[30] R Brooke, P Cottis, P Talemi, M Fabretto, P Murphy, D Evans. Recent advances in the synthesis of conducting polymers from the vapour phase. Progress in Materials Science 86, 127−146, 2017.
[31] DP Dubal, SH Lee, JG Kim, WB Kim, CD Lokhande. Porous polypyrrole clusters prepared by electropolymerization for a high performance supercapacitor. Journal of Materials Chemistry 22, 3044−3052, 2012.
[32] JY Kim, K Lee, NE Coates, D Mose, TQ Nguyen, M Dante, AJ Heeger. Efficient tandem polymer solar cells fabricated by all-solution processing. Science 317, 222−225, 2007.
[33] WSP Carvalho, M Wei, N Ikpo, Y Gao, MJ Serpe. Polymer-based technologies for sensing applications. Analytical Chemistry 90, 459−479, 2018.
[34] AJ Heeger. Semiconducting and metallic polymers: the fourth generation of polymeric materials. The Journal of Physical Chemistry B 105, 8476−8479, 2001.
[35] PS Sharma, A Pietrzyk-Le, F D'Souza, W Kutner. Electrochemically synthesized polymers in molecular imprinting for chemical sensing. Analytical and Bioanalytical Chemistry 402, 3177−3204, 2012.
[36] N Aydemir, J Malmstrom, J Travas-Sejdic. Conducting polymer based electrochemical biosensors. Physical Chemistry Chemical Physics 18, 8264−8277, 2016.
[37] M Umaña, J Waller. Protein-modified electrodes. The glucose oxidase/polypyrrole system. Analytical Chemistry 58, 2979−2983, 1986.
[38] NC Foulds, CR Lowe. Enzyme entrapment in electrically conducting polymers: immobilisation of glucose oxidase in polypyrrole and its application in amperometric glucose sensors. Journal of the Chemical Society, Faraday Transactions 1 82, 1259−1264, 1986.
[39] A Ramanavicius, K Habermüller, E Csöregi, V Laurinavicius, W Schuhmann. Polypyrrole-entrapped quinohemoprotein alcohol dehydrogenase. evidence for direct electron transfer via conducting-polymer chains. Analytical Chemistry 71, 3581−3586, 1999.
[40] JN Barisci, D Hughes, A Minett, GG Wallace. Characterisation and analytical use of a polypyrrole electrode containing anti-human serum albumin. Analytica Chimica Acta 371, 39−48, 1998.
[41] SK Jha, M Kanungo, A Nath, SF D'Souza. Entrapment of live microbial cells in electropolymerized polyaniline and their use as urea biosensor. Biosensors and Bioelectronics 24, 2637−2642, 2009.
[42] Rajesh, V Bisht, W Takashima, K Kaneto. An amperometric urea biosensor based on covalent immobilization of urease onto an electrochemically prepared copolymer poly (N-3-aminopropyl pyrrole-co-pyrrole) film. Biomaterials 26, 3683−3690, 2005.
[43] F Darain, SU Park, YB Shim. Disposable amperometric immunosensor system for rabbit IgG using a conducting polymer modified screen-printed electrode. Biosensors and Bioelectronics 18, 773−780, 2003.
[44] PT Kissinger, WR Heineman. Cyclic voltammetry. Journal of Chemical Education 60, 702−706, 1983.
[45] F Scholz, AM Bond, RG Compton, DA Fiedler, G Inzelt, H Kahlert, etc. Electroanalytical methods: guide to experiments and applications. Second Ed., Springer, Inc., Berlin, 2010.
[46] Z Taleat, C Cristea, G Marrazza, M Mazloum-Ardakani, R Săndulescu. Electrochemical immunoassay based on aptamer-protein interaction and functionalized polymer for cancer biomarker detection. Journal of Electroanalytical Chemistry 717−718, 119−124, 2014.
[47] DR Crow. Principles and applications of electrochemistry. Fourth Ed., CRC Press, 1994.
[48] T. Cassagneau, F. Caruso. Conjugated polymer inverse opals for potentiometric biosensing. Advanced Materials 14, 1837-1841, 2002.
[49] G Yang, KL Kampstra, MR Abidian. High performance conducting polymer nanofiber biosensors for detection of biomolecules. Advanced Materials 26, 4954−4960, 2014.
[50] E Barsoukov, JR Macdonald. Impedance spectroscopy: theory, experiment, and applications. Third Ed., John Wiley & Sons, Inc., USA, 2018.
[51] M David, MM Barsan, CMA Brett, M Florescu. Improved glucose label-free biosensor with layer-by-layer architecture and conducting polymer poly(3,4-ethylenedioxythiophene). Sensors and Actuators B: Chemical 255, 3227−3234, 2018.
[52] M Fasano, S Curry, E Terreno, M Galliano, G Fanali, P Narciso, S Notari, P Ascenzi. The extraordinary ligand binding properties of human serum albumin. IUBMB Life 57, 787−796, 2005.
[53] V Arroyo, R Garcia-Martinez, X Salvatella. Human serum albumin, systemic inflammation, and cirrhosis. Journal of Hepatology 61, 396−407, 2014.
[54] XM He, DC Carter. Atomic structure and chemistry of HSA. Nature 358, 209−215, 1992.
[55] M Amiri, K Jankeje, JR Albani. Characterization of human serum albumin forms with pH. Fluorescence lifetime studies. Journal of Pharmaceutical and Biomedical Analysis 51, 1097−1102, 2010.
[56] DG Levitt, MD Levitt. Human serum albumin homeostasis: a new look at the roles of synthesis, catabolism, renal and gastrointestinal excretion, and the clinical value of serum albumin measurements. International Journal of General Medicine 9, 229−255, 2016.
[57] JK Sun, F Sun, X Wang, ST Yuan, SY Zheng, XW Mu. Risk factors and prognosis of hypoalbuminemia in surgical septic patients. PeerJ 3, e1267, 2015.
[58] OA Aguayo-Becerra, C Torres-Garibay, MD Macías-Amezcua, C Fuentes-Orozco, MG Chávez-Tostado, E Andalón-Dueñas, etc. Serum albumin level as a risk factor for mortality in burn patients. Clinics 68, 940−945, 2013.
[59] ME Jellinge, DP Henriksen, P Hallas, M Brabrand. Hypoalbuminemia is a strong predictor of 30-day all-cause mortality in acutely admitted medical patients: a prospective, observational, cohort study. PLoS One 9, e105983, 2014.
[60] G Fanali, A di Masi, V Trezza, M Marino, M Fasano, P Ascenzi. Human serum albumin: From bench to bedside. Molecular Aspects of Medicine 33, 209−290, 2012.
[61] F Kratz, A Warnecke, K Scheuermann, C Stockmar, J Schwab, P Lazar, P Drückes, N Esser, J Drevs, D Rognan, C Bissantz, C Hinderling, G Folkers, I Fichtner, C Unger. Probing the Cysteine-34 position of endogenous serum albumin with thiol-binding doxorubicin derivatives. Improved efficacy of an acid-sensitive doxorubicin derivative with specific albumin-binding properties compared to that of the parent compound. Journal of Medicinal Chemistry 45, 5523−5533, 2002.
[62] L Turell, R Radi, B Alvarez. The thiol pool in human plasma: the central contribution of albumin to redox processes. Free Radical Biology & Medicine 65, 244−253, 2013.
[63] BH Northrop, SH Frayne, U Choudhary. Thiol-maleimide “click” chemistry: evaluating the influence of solvent, initiator, and thiol on the reaction mechanism, kinetics, and selectivity. Polymer Chemistry 6, 3415−3430, 2015.
[64] CF Brewer, JP Riehm. Evidence for possible nonspecific reactions between N-ethylmaleimide and proteins. Analytical Biochemistry 18, 248−255, 1967.
[65] G Hermanson. Bioconjugate techniques. Third Ed. Elsevier, Inc., USA, 2013.
[66] AD Baldwin, KL Kiick. Tunable degradation of maleimide-thiol adducts in reducing environments. Bioconjugate Chemistry 22, 1946−1953, 2011.
[67] T Kantner. Bioconjugation strategies through thiol-alkylation of peptides and proteins. PhD thesis, 1−295, 2015.
[68] DE Chung, F Kratz. Development of a novel albumin-binding prodrug that is cleaved by urokinase-type-plasminogen activator (uPA). Bioorganic & Medicinal Chemistry Letters 16, 5157–5163, 2006.
[69] J Stejskal, M Trchová, P Bober, Z Morávková, D Kopeckỳ, M Vrňata, J Prokeš, etc. Polypyrrole salts and bases: superior conductivity of nanotubes and their stability towards the loss of conductivity by deprotonation. RSC Advances 6, 88382–88391, 2016.
[70] Y Su, H Zhu, H Dong. A novel electrochemical immunosensor incorporating a pyrrole/4-(3-pyrrolyl) butyric acid conducting polymer. Analytical Letters 48, 477–488, 2015.
[71] J Jehlička, HGM Edwards, A Culda. Using portable Raman spectrometers for the identification of organic compounds at low temperatures and high altitudes: exobiological applications. Philosophical Transactions of The Society A 368, 3109–3125, 2010.
[72] R Aroca, M Scraba. SERS and normal coordinate analysis of maleimide chemisorbed on colloidal silver. SpccnochimicAacta 47A, 263–269, 1991.
[73] NK Howell, G Arteaga, S Nakai, ECY Li-Chan. Raman spectral analysis in the C-H stretching region of proteins and amino acids for investigation of hydrophobic interactions. Journal of Agricultural and Food Chemistry 47, 924–933, 1999.
[74] P Zanello. Inorganic Electrochemistry: Theory, Practice and Application. First Ed. RSC, UK, 2003.
[75] MC Rodriguez, AN Kawde, J Wang. Aptamer biosensor for label-free impedance spectroscopy detection of proteins based on recognition-induced switching of the surface charge. Chemical Communications, 4267–4269, 2005.
[76] I Cseresnyés, K Rajkai1, E. Vozáry. Role of phase angle measurement in electrical impedance spectroscopy. International Agrophysics 27, 377–383, 2013.
[77] KF Wang, CC Zhou, YL Hong, XD Zhang. A review of protein adsorption on bioceramics. Interface Focus 2, 259–277, 2012
[78] JZ Tsai, CJ Chen, K Settu, YF Lin, CL Chen, JT Liu. Screen-printed carbon electrode-based electrochemical immunosensor for rapid detection of microalbuminuria. Biosensors and Bioelectronics 77, 1175–1182, 2016.
[79] A Fatoni, A Numnuam, P Kanatharana, W Limbut, Panote Thavarungkul. A novel molecularly imprinted chitosan-acrylamide, graphene, ferrocene composite cryogel biosensor used to detect microalbumin. Analyst 139, 6160–6167, 2014.
[80] YH Chuang, YT Chang, KL Liu, HY Chang, TR Yew. Electrical impedimetric biosensors for liver function detection. Biosensors and Bioelectronics 28, 368–372, 2011.
[81] D Caballero, E Martinez, J Bausells, A Errachid, J Samitier. Impedimetric immunosensor for human serum albumin detection on a direct aldehyde-functionalized silicon nitride surface. Analytica Chimica Acta 720, 43–48, 2012.
[82] AF Ogata, JM Edgar, S Majumdar, JS Briggs, SV Patterson, MX Tan. Virus-enabled biosensor for human serum albumin. Analytical chemistry 89, 1373−1381, 2017.