在臨床的腎臟疾病診斷時，需要檢測人體血清或尿液中肌酸酐的濃度。肌酸酐的濃度可以協助醫療人員判斷病人腎功能的狀態，若能在腎病變的初期發現症狀，患者在積極治療後有較多復原的希望，反之病情若在後期發現，治癒的機會較少。故體內肌酸酐濃度的即時監測可作為預防疾病的早期訊號，有助於人們對自我健康的管理。
本實驗成功開發出一個非酵素型光學感測電極，使用經電化學氧化之2 ,2-聯氮-二(3-乙基-苯并噻唑啉-6-磺酸) (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), ABTS) 作為感測元件，與肌酸酐進行化學反應。從比色動力研究結果得知氧化態ABTS與肌酸酐的反應機制，得知氧化態ABTS與肌酸酐在反應中之反應級數皆為1。
本研究感測電極是將甲殼素膜或甲殼素-二氧化矽複合膜滴塗於電極上，再將ABTS吸附並固定其上。在相同吸附條件下，甲殼素-二氧化矽複合膜比單純甲殼素膜穩定，可長時間浸泡於溶液中。在相同浸泡條件下，ABTS比氧化態ABTS易吸附於電極上。此感測機制受尿液中抗壞血酸、尿酸與酪胺酸干擾，可將ABTS再披覆一層帶負電之Nafion® 高分子膜予以排除干擾。浸泡ABTS溶液30分鐘，再披覆60 µL Nafion® 高分子膜，可得最佳效果。
此光學感測器感測肌酸酐具有高度可行性之感測性能，包括靈敏度(0.3216 µmol L-1 mM-1)，偵測下限(67.7 µM)，線性範圍(0- 20.0 mM)，反應時間(150 s)，以及對尿液中常見干擾物具高選擇性。

The reaction order of electrochemically oxidized ABTS and creatinine was found 1 with respect to each reactant. The sensing process at the developed sensing electrode consists of chemical reaction between the electrochemically oxidized ABTS and creatinine. This sensing process suffered serious interferences from tyrosine, uric acid and ascorbic acid, but these interferences can be minimized by coating Nafion® film on the chitosan-SiO2/ABTS layer. The absorbance change at 419 nm was used to correlated to creatinine concentration. This sensor has highly sensible sensing performance, including sensitivity (0.0044 min-1 mM-1), low detection limit (67.7 μM), wide linear range (0-20.0 mM), low response time (150 s), and high selectivity in urine with common interferences.

[1] A. S. Levey, C. Becker, and L. A. Inker, "Glomerular filtration rate and albuminuria for detection and staging of acute and chronic kidney disease in adults: a systematic review," JAMA, vol. 313, no. 8, pp. 837-46, Feb 24 2015.
[2] M. L. Marcovecchio et al., "Renal and Cardiovascular Risk According to Tertiles of Urinary Albumin-to-Creatinine Ratio: The Adolescent Type 1 Diabetes Cardio-Renal Intervention Trial (AdDIT)," Diabetes Care, vol. 41, no. 9, pp. 1963-1969, Sep 2018.
[3] 衛生福利部, https://dep.mohw.gov.tw/DOS/lp-4472-113.html, 2019.
[4] L. C. Clark and C. Lyons, "Electrode systems for continuous monitoring in cardiovascular surgery," Annals of the New York Academy of Sciences, vol. 102, pp. 29-45, 1962.
[5] W. Joseph, "Glucose Biosensors: 40 Years of Advances and Challenges," Electroanalysis, vol. 13, pp. 983-988, 2001.
[6] F. Scheller, F. Schubert, and B. Neumann, "Second generation biosensors," Biosens Bioelectron, vol. 6, pp. 245-253, June 19 1991.
[7] S. P. Mohanty and E. Kougianos, "Biosensors: a tutorial review," IEEE Potentials, vol. 25, no. 2, pp. 35-40, 2006.
[8] C. Karunakaran, R. Rajkumar, and K. Bhargava, "Introduction to Biosensors," in Biosensors and Bioelectronics, 2015, pp. 1-68.
[9] C. H. Nieh, S. Tsujimura, O. Shirai, and K. Kano, "Amperometric biosensor based on reductive H2O2 detection using pentacyanoferrate-bound polymer for creatinine determination," Anal Chim Acta, vol. 767, pp. 128-33, Mar 12 2013.
[10] S. Yadav, R. Devi, P. Bhar, S. Singhla, and C. S. Pundir, "Immobilization of creatininase, creatinase and sarcosine oxidase on iron oxide nanoparticles/chitosan-g-polyaniline modified Pt electrode for detection of creatinine," Enzyme Microb Technol, vol. 50, no. 4-5, pp. 247-54, Apr 5 2012.
[11] P. Luppa, L. Sokoll, and D. Chan, "Immunosensors—principles and applications to clinical chemistry," vol. 314, pp. 1-26, July 20 2001.
[12] A. Hasan et al., "Recent advances in application of biosensors in tissue engineering," Biomed Res Int, vol. 2014, p. 307519, 2014.
[13] M.-H. Lee, D. O'Hare, H.-Z. Guo, C.-H. Yang, and H.-Y. Lin, "Electrochemical sensing of urinary progesterone with molecularly imprinted poly(aniline-co-metanilic acid)s," Journal of Materials Chemistry B, vol. 4, no. 21, pp. 3782-3787, 2016.
[14] A. Diouf, S. Motia, N. El Alami El Hassani, N. El Bari, and B. Bouchikhi, "Development and characterization of an electrochemical biosensor for creatinine detection in human urine based on functional molecularly imprinted polymer," Journal of Electroanalytical Chemistry, vol. 788, pp. 44-53, 2017.
[15] M. Hassanzadeh and M. Ghaemy, "An effective approach for the laboratory measurement and detection of creatinine by magnetic molecularly imprinted polymer nanoparticles," New Journal of Chemistry, vol. 41, no. 6, pp. 2277-2286, 2017.
[16] C. Bayrak and S. Bayari, "Vibrational and DFT studies of creatinine and its metal complexes," vol. 38, pp. 107-118, Mar 4 2010.
[17] P. Nagaraja, K. Avinash, A. Shivakumar, and H. Krishna, "Quantification of creatinine in biological samples based on the pseudoenzyme activity of copper-creatinine complex," Spectrochim Acta A Mol Biomol Spectrosc, vol. 92, pp. 318-24, Jun 15 2012.
[18] J. Raveendran, R. P.E, R. T, B. G. Nair, and T. G. Satheesh Babu, "Fabrication of a disposable non-enzymatic electrochemical creatinine sensor," Sensors and Actuators B: Chemical, vol. 243, pp. 589-595, 2017.
[19] V. Kumar et al., "Creatinine-Iron Complex and Its Use in Electrochemical Measurement of Urine Creatinine," IEEE Sensors Journal, vol. 18, no. 2, pp. 830-836, 2018.
[20] W. Shi, W. Yuting, and M. Qing, "Simultaneous Electrochemical Determination of Dopamine and serotonin in rat ceebrospinal fluid using SPCE modified with MWNT-Sio2-chitosan composite," int.J.Electrochem.Sci., vol. 11, pp. 2360-2376, 2016.
[21] 吳俊億, "2,2, 聯氮-雙(3-乙基苯并噻唑啉-6-磺酸)修飾奈米碳管電極在尿蛋白感測之研究," 成功大學, 2016.
[22] 黃煒婷, "經磷摻雜之還原氧化石墨烯電極於同時偵測多巴胺及尿酸之應用," 成功大學, 2017.
[23] N. K. Guimard, N. Gomez, and C. E. Schmidt, "Conducting polymers in biomedical engineering," Progress in Polymer Science, vol. 32, no. 8-9, pp. 876-921, 2007.
[24] A. Sassolas, L. J. Blum, and B. D. Leca-Bouvier, "Immobilization strategies to develop enzymatic biosensors," Biotechnol Adv, vol. 30, no. 3, pp. 489-511, May-Jun 2012.
[25] M. Pohanka and P. Skladal, "Electrochemical biosensors-principles and applications," vol. 6, pp. 57-64, 2008.
[26] S. V. Marchenko, O. O. Soldatkin, B. O. Kasap, B. A. Kurc, A. P. Soldatkin, and S. V. Dzyadevych, "Creatinine Deiminase Adsorption onto Silicalite-Modified pH-FET for Creation of New Creatinine-Sensitive Biosensor," Nanoscale Res Lett, vol. 11, no. 1, p. 173, Dec 2016.
[27] Y. Zhang and S. Tadigadapa, "Calorimetric biosensors with integrated microfluidic channels," Biosens Bioelectron, vol. 19, no. 12, pp. 1733-43, Jul 15 2004.
[28] S. Narayanan and H. Appleton, "Creatinine: A review," vol. 26, pp. 1119-1126, Feb 20 1980.
[29] A. Killard and M. Smyth, "Creatinine biosensors: principles and designs," Trends in Biotechnology, vol. 18, pp. 433-437, Oct 10 2000.
[30] U. Lad, S. Khokhar, and G. Kale, "Electrochemical creatinine sensors," J. Appl. Biomed., vol. 80, pp. 7910-7917, Nov 21 2008.
[31] E. Mohabbati-Kalejahi, V. Azimirad, M. Bahrami, and A. Ganbari, "A review on creatinine measurement techniques," Talanta, vol. 97, pp. 1-8, Aug 15 2012.
[32] C. S. Pundir, S. Yadav, and A. Kumar, "Creatinine sensors," TrAC Trends in Analytical Chemistry, vol. 50, pp. 42-52, 2013.
[33] E. P. Randviir and C. E. Banks, "Analytical methods for quantifying creatinine within biological media," Sensors and Actuators B: Chemical, vol. 183, pp. 239-252, 2013.
[34] R. Canovas, M. Cuartero, and G. A. Crespo, "Modern creatinine (Bio)sensing: Challenges of point-of-care platforms," Biosens Bioelectron, vol. 130, pp. 110-124, Apr 1 2019.
[35] C. S. Pundir, P. Kumar, and R. Jaiwal, "Biosensing methods for determination of creatinine: A review," Biosens Bioelectron, vol. 126, pp. 707-724, Feb 1 2019.
[36] C. Francoz, D. Glotz, R. Moreau, and F. Durand, "The evaluation of renal function and disease in patients with cirrhosis," Journal of Hepatology, vol. 52, pp. 605-613, Sep 16 2010.
[37] E. G. Marcos et al., "Relation of renal dysfunction with incident atrial fibrillation and cardiovascular morbidity and mortality: The PREVEND study," Europace, vol. 19, no. 12, pp. 1930-1936, Dec 1 2017.
[38] H. Husdan and A. Rapoport, "Estimation of creatinine by the Jaffe reaction," CLIN. CHEM., vol. 14, pp. 222-238, Sep 27 1968.
[39] J. Weber and A. Zanten, "Interferences in current methods for measurements of creatinine," CLIN. CHEM., vol. 37, pp. 695-700, Feb 26 1991.
[40] M. Kroll, N. Roach, and R. EIIN, "Mechanism of interference with the Jaffe reaction for creatinine," CLIN. CHEM., vol. 3, pp. 1129-1131, Apr 7 1987.
[41] P. Fossati, L. Prenclpe, and G. Berti, "Enzymic creatinine assay: A new colorimetric method based on hydrogen peroxide measurement," CLIN. CHEM., vol. 29, pp. 1994-1996, Mar 9 1983.
[42] H. Du, R. Chen, J. Du, J. Fan, and X. Peng, "Gold Nanoparticle-Based Colorimetric Recognition of Creatinine with Good Selectivity and Sensitivity," Industrial & Engineering Chemistry Research, vol. 55, no. 48, pp. 12334-12340, 2016.
[43] A. K. Parmar, N. N. Valand, K. B. Solanki, and S. K. Menon, "Picric acid capped silver nanoparticles as a probe for colorimetric sensing of creatinine in human blood and cerebrospinal fluid samples," Analyst, vol. 141, no. 4, pp. 1488-1498, Feb 21 2016.
[44] M. T. Alula, L. Karamchand, N. R. Hendricks, and J. M. Blackburn, "Citrate-capped silver nanoparticles as a probe for sensitive and selective colorimetric and spectrophotometric sensing of creatinine in human urine," Anal Chim Acta, vol. 1007, pp. 40-49, May 12 2018.
[45] M. Cox and D. Nelson, Lehninger Principles of Biochemistry. 2000.
[46] G. L. Squadrito et al., "Reaction of uric acid with peroxynitrite and implications for the mechanism of neuroprotection by uric acid," Arch Biochem Biophys, vol. 376, no. 2, pp. 333-7, Apr 15 2000.
[47] R. Zuo, S. Zhou, Y. Zuo, and Y. Deng, "Determination of creatinine, uric and ascorbic acid in bovine milk and orange juice by hydrophilic interaction HPLC," Food Chem, vol. 182, pp. 242-5, Sep 1 2015.
[48] M. G. Antoniou and D. D. Dionysiou, "Application of immobilized titanium dioxide photocatalysts for the degradation of creatinine and phenol, model organic contaminants found in NASA's spacecrafts wastewater streams," Catalysis Today, vol. 124, no. 3-4, pp. 215-223, 2007.
[49] C. H. Chen and M. S. Lin, "A novel structural specific creatinine sensing scheme for the determination of the urine creatinine," Biosens Bioelectron, vol. 31, no. 1, pp. 90-4, Jan 15 2012.
[50] A. Bard, "ELECTROCHEMICAL METHODS : FUNDAMENTALS AND APPLICATIONS " Wiley New York, 1980.
[51] O. Thomas and V. Cerdà, "UV-Visible Spectrophotometry of Water and Wastewater," 2007, pp. 21-46.
[52] N. Gohain, Studies on the structure and function of phenazine modifying enzymes PhzM and PhzS involved in the biosynthesis of pyocyanin. 2009.
[53] R. Vidu, M. Rahman, M. Mahmoudi, M. Enachescu, T. Dan Poteca, and I. Opris, Nanostructures: A Platform for Brain Repair and Augmentation. 2014, p. 91.
[54] A. Tiwari and S. R. Dhakate, "Chitosan-SiO2-multiwall carbon nanotubes nanocomposite: a novel matrix for the immobilization of creatine amidinohydrolase," Int J Biol Macromol, vol. 44, no. 5, pp. 408-12, Jun 1 2009.
[55] D. H. Gharib, F. Malherbe, and S. E. Moulton, "Debundling, Dispersion, and Stability of Multiwalled Carbon Nanotubes Driven by Molecularly Designed Electron Acceptors," Langmuir, vol. 34, no. 40, pp. 12137-12144, Oct 9 2018.
[56] A. Garcia-Leis, D. Jancura, M. Antalik, J. V. Garcia-Ramos, S. Sanchez-Cortes, and Z. Jurasekova, "Catalytic effects of silver plasmonic nanoparticles on the redox reaction leading to ABTS (+) formation studied using UV-visible and Raman spectroscopy," Phys Chem Chem Phys, vol. 18, no. 38, pp. 26562-26571, Sep 29 2016.
[57] S. Scott, C. Wen-Jang, A. Bakac, and J. Espenson, "Spectroscopic Parameters, Electrode Potentials, Acid Ionization Constants, and Electron Exchange Rates of the 2,2~-Azinobis(3-ethylbenzothiazolineine-6-sulfonateR)a dicals and Ions.," J. Phys. Chem., vol. 97, pp. 6710-6714, Mar 9 1993.
[58] R. Bourbonnais, D. Leech, and M. Paice, "Electrochemical analysis of the interactions of laccase mediators with lignin model compounds," Biochimica et Biophysica Acta, vol. 1379, pp. 381-390, Mar 3 1998.
[59] J. Gebicki, "Reactions of 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulfonate) Dianion, ABTS2-, with ˙OH,(SCN)2˙-, and Glycine or Valine Peroxyl Radicals," J. Phys. Chem., vol. 111, pp. 2122-2127, Jan 3 2007.
[60] B. Wolfenden and R. Willson, "Radical-cations as Reference Chromogens in Kinetic Studies of One electron Transfer Reactions : Pulse Radiolysis Studies of 2,2'-Azinobisethyl benzthiazoline-6-sulphonate," J. Chem. Soc., Perkin Trans., vol. 2, pp. 805-812, Sep 6 1982.
[61] T. Perkin, "Nitrogen Dioxide and Related Free Radicals: Electron-transfer Reactions with Organic Compounds in Solutions containing Nitrite or Nitrate," J. Chem. Soc., vol. 2, pp. 1-6, 1986.
[62] K. V. Konan, C. Le Tien, and M. A. Mateescu, "Electrolysis-induced fast activation of the ABTS reagent for an antioxidant capacity assay," Analytical Methods, vol. 8, no. 28, pp. 5638-5644, 2016.
[63] Y. Song et al., "ABTS as an Electron Shuttle to Enhance the Oxidation Kinetics of Substituted Phenols by Aqueous Permanganate," Environ Sci Technol, vol. 49, no. 19, pp. 11764-71, Oct 6 2015.
[64] A. Chemie, "Selective Photometric Determination of Peroxycarboxylic Acids in the Presence of Hydrogen Peroxide," vol. 122, pp. 567-571, June 1 1997.
[65] Y. Lee, J. Yoon, and U. von Gunten, "Spectrophotometric determination of ferrate (Fe(VI)) in water by ABTS," Water Res, vol. 39, no. 10, pp. 1946-53, May 2005.
[66] W. Fan, J. Qiao, and X. Guan, "Multi-wavelength spectrophotometric determination of Cr(VI) in water with ABTS," Chemosphere, vol. 171, pp. 460-467, Mar 2017.
[67] U. Pinkernell, B. Nowack, and H. Gallard, "Methds for the photometric determination of reactive bromine and chlorine species with ABTS," Wat. Rws., vol. 34, pp. 4343-4350, Mar 10 2000.
[68] I. Valent, D. Topolska, K. Valachova, J. Bujdak, and L. Soltes, "Kinetics of ABTS derived radical cation scavenging by bucillamine, cysteine, and glutathione. Catalytic effect of Cu(2+) ions," Biophys Chem, vol. 212, pp. 9-16, May 2016.
[69] F. Maggi and E. Daly, "Decomposition pathways and rates of human urine in soils," J Agric Food Chem, vol. 61, no. 26, pp. 6175-86, Jul 3 2013.
[70] D. Sechi, B. Greer, J. Johnson, and N. Hashemi, "Three-dimensional paper-based microfluidic device for assays of protein and glucose in urine," Anal Chem, vol. 85, no. 22, pp. 10733-7, Nov 19 2013.
[71] F. Inam, H. Yan, M. J. Reece, and T. Peijs, "Dimethylformamide: an effective dispersant for making ceramic-carbon nanotube composites," Nanotechnology, vol. 19, no. 19, p. 195710, May 14 2008.
[72] Z. Dlaskova, T. Navratil, M. Heyrovsky, D. Pelclova, and L. Novotny, "Voltammetric determination of thiodiglycolic acid in urine," Anal Bioanal Chem, vol. 375, no. 1, pp. 164-8, Jan 2003.
[73] C. G. Tsiafoulis, M. I. Prodromidis, and M. I. Karayannis, "Development of an amperometric biosensing method for the determination of L-fucose in pretreated urine," Biosens Bioelectron, vol. 20, no. 3, pp. 620-7, Oct 15 2004.
[74] G. Cui, S. Kim, and S. Choi, "A glucose biosensor employing lead oxide as an interference removing agent.pdf>," Anal. Chem., vol. 72, pp. 1925-1929, 2000.
[75] B. Tombach, J. Schneider, F. Matzkies, R. Schaefer, and G. Chemnitius, "Amperometric creatinine biosensor for hemodialysis patients.," Clinica Chimica Acta, vol. 312, pp. 129-134, June 22 2001.
[76] J. Du et al., "Colorimetric Detection of Creatinine Based on Plasmonic Nanoparticles via Synergistic Coordination Chemistry," Small, vol. 11, no. 33, pp. 4104-10, Sep 2 2015.
[77] Y. He, X. Zhang, and H. Yu, "Gold nanoparticles-based colorimetric and visual creatinine assay," Microchimica Acta, vol. 182, no. 11-12, pp. 2037-2043, 2015.
[78] L.-M. Fu, C.-C. Tseng, W.-J. Ju, and R.-J. Yang, "Rapid Paper-Based System for Human Serum Creatinine Detection," Inventions, vol. 3, no. 2, 2018.
[79] C.-C. Tseng, R.-J. Yang, W.-J. Ju, and L.-M. Fu, "Microfluidic paper-based platform for whole blood creatinine detection," Chemical Engineering Journal, vol. 348, pp. 117-124, 2018.