醣化血紅素(Glycated Hemoglobin)的濃度與血糖的控制有非常緊密的關聯性，是監控糖尿病患者血糖值的一個重要指標，醣化血紅素中又以HbA1c (Hemoglobin A1c)含量最高，由於一般紅血球平均壽命約為120天，因此血中HbA1c的濃度可以反映出體內最近2-3個月的血糖控制情況，因此是糖尿病患者的重要血糖指標。本論文的主要目的是要開發出一能簡易快速偵測血中HbA1c的方法，所利用的偵測原理是以醣化胜肽氧化酶 (fructosyl peptide oxidase, FPO)氧化經蛋白水解酵素水解HbA1c所產生的醣化胜肽 (fructosyl valyl histidine, fructosyl dipeptide, FVH) 而產生之H2O2，再利用過氧化酶(horseradish peroxidase, HRP)催化呈色劑呈色，藉由呈色的深淺來推算出檢體中HbA1c的濃度。
本論文先以化學合成之FPO基質醣化胺基酸 (fructosyl valine, FV)加入標準品中，來模擬檢體中不同濃度的HbA1c，探討最終反應產物H2O2之酵素反應呈色劑DA-67的呈色靈敏度，發現其可偵測標準品內FV範圍為0.002 mM~0.01 mM，換算成HbA1c濃度約為4.57%~18.30%，已可滿足一般糖尿病患者之HbA1c濃度範圍。而為改善之前使用蛋白水解酶需要長時間、高溫才能有效水解HbA1c的方式，已找出可以在短時間內以37 oC下有效水解HbA1c的蛋白水解酶與其水解之最佳操作條件。結合呈色劑DA-67、最佳之蛋白酶、與之前實驗室所開發之基因改質FPOX酵素，本論文已成功開發出一套能用酵素呈色達到操作方便且快速檢測標準品中HbA1c含量的方法，可以在8分鐘之內完成檢測，標準品中HbA1c的濃度，偵測線性範圍為5.7%~12.2%之HbA1c濃度。

The concentration of glycated hemoglobin, espcially hemoglobin A1c (HbA1c) in human blood is closely related to the control of blood glucose level and it is an important indicator of monitoring blood glucose level in diabetic patients. Since the average lifetime of red blood cells is about 120 days, the concentration of HbA1c can reflect the blood glucose level of recent 2-3 months. So far there is no oxidase can directly oxidize the sugar residues in HbA1c. Only fructosyl peptide oxidase (FPO) was found to be able to oxidize the fructosyl group attached to the N-terminal of valyl histine dipeptide generated by protease hydrolysis of HbA1c to produce H2O2. By using horseradish peroxidase (HRP) coupled with a chromagen, H2O2 will be detected by the color developed due to the oxidation of chromagen. HbA1c concentration will be determined by knowning the H2O2 concentration generated from the FPO reaction.
The aim of this dissertation is to develop a fast and rapid enzymatic colorimetric based HbA1c detection method which can be easily operated by diabetic patients. First part of this research is to find an ultimate chromagen which has good sensitive and will not be interfered by the background color of blood sample. Fructosyl valine (FV) was syntheized and used as a simulated substrate for FPO to find out DA-67 is the chromagen with good performance that equivalent HbA1c concentration of 4.57% to 18.30% can be measured with good accuracy. In second part of this work, protease and its working condition which can hydrolyze HbA1c at body temperatue within reasonable time to produce FPO recognizable substrate were studied. Neutral protease was found to perform well for HbA1c hydrolysis in 2-(N-morpholino)ethanesulfonic acid (MES) buffer. HbA1c could be effectively hydrolyzed at 37 oC within 5 min for further enzymatic reaction (FPO, HRP and DA-67) to develop color which can be monintored at 665 nm with a linear calibration curve in the range of 5.7% - 12.2%. Finally, recombinant FPO produced in our laboratory was employed in the developed enzymatic colorimetric HbA1c assay kit. The recombinant FPO worked well for HbA1c detection that the whole operation starts from blood sample lysis to final color development could be finished in 8 minutes for the detection of HbA1c concenration 5.7% - 12.2%.

Akazawa, S., Karino, T., Yoshida, N., Katsuragi, T., Tani, Y., 2004. Functional Analysis of Fructosyl Amino Acid Oxidase of Aspergillus oryzae. Environmental Microbiology,70(10):5882-5890.
Amin, A. N. and Morris, R. P., 1983. Glycosylated Hemoglobins: A Review, Can. Fam. Physician, 29:564-568.
Biela, A., 2010. Biomimetic Sensors for HbA1c, Cranfield University Cranfield Health.
Blaedel, W.J., Uhi, J.M., 1975. Nature of Materials in Serum That Interfere in the Glucose Oxidase-Peroxidase--o- Dianisidine Method for Glucose, and Their Mode of Action. Clinical Chemistry, 21(1):119-124.
Bunn, H.F., Haney, D.N., Kamin,S., Gabbay,K.H., Gallop, P.M.,1976. The Biosynthesis of Human Hemoglobin A. Clinical Investigation, 57:1652-1659.
Chien, H.C., Chou, T.C., 2010. Glassy Carbon Paste Electrodes for the Determination of Fructosyl Valine. Electroanalysis, 22(6): 688 – 693.
Emma, E., Garry, J., 2012. HbA1c (glycated haemoglobin) (blood), Association for Clinical Biochemistry.
Ferri, S., Kim, S., M. Eng., Tsugawa, W., Sode, K., 2009. Review of Fructosyl Amino Acid Oxidase Engineering Research: A Glimpse into the Future of Hemoglobin A1c Biosensing. J Diabetes Sci Technol, 3(3):585-592.
Gabriele, H.B., Katzensteiner, S., Schnedl, W.,Pu¨ rstner, P., Pieber, T., and Martie, W.T., 1997. Comparative evaluation of three assay systems for automated determination of hemoglobin A1c. Clinical Chemistry, 43(3):511-517.
Gao, L., Zhuang, J., Nie, L., Zhang, J., Zhang, Y., Gu, N.,Wang, T., Feng, J.,Yang, D.,Perrett, S.,Yan, X., 2007. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nature Nanotechnology, 2:577-583.
Gorestski, J. Hollocher, T. C., 1988, Trapping of Nitric Oxide Produced during Denitrification by Extracellular Hemoglobin, J. Biological Chemistry, 263(5):2316-2323.
Hirokawa, K., Gomi, K., Kajiyama, N., 2003. Molecular cloning and expression of novel fructosyl peptide oxidases and their application for the measurement of glycated protein. Biochemical and Biophysical Research Communications, 311:104–111.
Hirokawa, K., Nakamura, K., Kajiyama, N., 2004. Enzymes used for the determination of HbA1C. FEMS Microbiology Letters, 235:157–162.
Hirokawa, K., Shimoji, K., Kajiyama, N., 2005. An enzymatic method for the determination of hemoglobinA1C. Biotechnology Letters, 27: 963–968.
Hoffbrand, A. V., Pettit, J.E. and Moss, P.A.H., 2001. Haematology. Blackwell Science Ltd., UK.
http://www.toyobousa.com/enzyme-FPO-301.html, [TOYOBO CO., LTD.]
http://www.who.int/mediacentre/factsheets/fs312/en/, [Diabetes, Fact sheet, reviewed November 2016]
International Diabetes Federation. IDF Diabetes Atlas. 6th ed. 2013.
Isarankura-Na-Ayudhya, Chartchalerm1, Tansila1,N., Worachartcheewan, A., Bülow, L., and Prachayasittikul, V.,2010. Biochemical and Cellular Investigation of Vitreoscilla Hemoglobin (VHb) Variants Possessing Efficient Peroxidase Activity. J. Microbiol. Biotechnol, 20(3):532–541.
Jeppsson, J.O., Kobold, U.,Barr, J., Finke, A., Hoelzel, W.,Hoshino, T., Miedema, K., Mosca, A., Mauri, P.,Paroni, R., Thienpont, L., Umemoto., M., Weykamp, C., 2002. Approved IFCC Reference Method for the Measurement of HbA1c in Human Blood. Clin Chem Lab Med, 40(1):78–89.
John, G., 2016. Haemoglobin A1c analysis: Understanding what is measured is fundamental to interpretation, General conference MADRID 2016.
Kim, S., Ferri, S., Tsugawa, W., Mori, K., Sode, K., 2010. Motif-Based Search for a Novel Fructosyl Peptide Oxidase From Genome Databases. Biotechnology and Bioengineering, 106(3):358-366.
Kobold, U., Jeppsson, J.O., Du¨ lffer, T., Finke, A., Hoelzel, W., Miedema, K., 1997. Candidate reference methods for hemoglobin A1c based on peptide mapping. Clinical Chemistry, 43(10):1944-1951.
Liu, L., Hood, S., Wang, Y., Bezverkov, R., Dou, C., Datta, A., Yuan., C., 2008. Direct enzymatic assay for %HbA1c in human whole blood samples. Clinical Biochemistry, 41:576–583.
Miura, S., Ferri, S., Tsugawa,W., Kim ,S., Sode, K., 2008. Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis. Protein Engineering, Design & Selection ,21(4): 233–239.
Miura, S., Ferri, S., Tsugawa,W., Kim ,S., Sode, K., 2008. Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis. Protein Engineering, Design & Selection ,21(4): 233–239.
Nagababu, E., Rifikind, J. M., 2000. Heme Degradtion during Autoxidation of Oxyhemoglobin, Biochemical and Biophysical Research Communication, 273:839-845.
Nanjo, Y., Hayashi, R., Yao, T., 2007. An enzymatic method for the rapid measurement of the hemoglobin A1c by a flow-injection system comprised of an electrochemical detector with a specific enzyme-reactor and a spectrophotometer. Analytica Chimica Acta, 583: 45–54.
Rahbar, S., 1968. An abnormal haemoglobin in red cells of diabetics, Clin Chim Acta, 22:296-298.
Rodríguez-Segade, S., J. Rodríguez, et al. (2009). "Translating the A1C assay into estimated average glucose values." Diabetes Care 32(1).
Ross, J. M., Targeting HbA1c: standardization and clinical laboratory measurement, www.mlo-online.com (MLO: Medical Laboratory Observer), 2008.
Sakurabayashi, I., Watano, T., Yonehara, S., Ishimaru, K., Hirai, K., Komori, T., Yagi, M., 2003. New Enzymatic Assay for Glycohemoglobin. Clinical Chemistry, 49(2):269-274.
Satoshi, Y., Norio, I., Miho, F., and Koji, S., 2015. Use of Fructosyl Peptide Oxidase for HbA1c Assay. Journal of Diabetes and Technology, 9(2):200-205.
Schnedl, W.J., Krause, R., Gabriele, H.B.,Trinker, M., LIPP, R.W., Krejs, G.J., 2000. Evaluation of HbA1c Determination Methods in Patients With Hemoglobinopathies. Diabetes Care,23:339-344.
Sheikholeslam, S., Pritzker, M.D., Chen, P., 2011. Electrochemical Biosensor for Glycated Hemoglobin (HbA1c), Biosensors for Health, Environment and Biosecurity, Pier Andrea Serra (Ed.), ISBN: 978-953-307-443-6, InTech, 13:293-320.
Song, S.Y., Yoon, H.C., 2009. Boronic acid-modified thin film interface for specific binding of glycated hemoglobin (HbA1c) and electrochemical biosensing. Sensors and Actuators B, 140:233–239.
Sylvia, S. M., 1997. Inquiry into Life, 8th edition, The MacGraw-Hill Companies, Ins.
Takagi, K., Nakao, M., Ogura, Y., Nabeshima, T., Kunii, A., 1994. Sensitive colorimetric assay of serum diamine oxidase, Clinica Chimica Acta, 226:67-75.
Weets I., Gorus, F.K., Gerlo, E., 1996. Evaluation of an Immunoturbidimetric Assay for Haemoglobin Alc on a Cobas® Mira S Analyser. Eur J Clin Chem Clin Biochem, 34:449-453.
Weng, C. W., Hsieh, B. C., Hou, Y. T., Cheng, T. J., 2015, Determination of hematocrit by voltage-induced hemolysis on a disposable electrochemical sensing strip, Analyst, 140:6619-6624.
Wolfgang, J.S., Andrea, L., Regina, E. R., Guenter, J. K., 2001. Hemoglobin variants and determination of glycated hemoglobin (HbA1c), Diabetes Metab Res Rev, 17:94-98.
Zang, X., Medzihradszky, K. F., Cunningham, J., Lee, P. D.K., Rognerud, C. L., Ou, C.-N., Harmatz, P., Witkowska, H. E., 2001. Charaterization of glycated hemoglobin in diabetic patients: usefulness of electrospray mass spectrometry in monitoring the extent and distribution of glycation, Journal of Chromatography B, 759:1-15.
王振皓, 2012. FPO重組蛋白於聚多巴胺表面之固定化及在測定糖化血紅素(HbA1c)之應用, 台灣科技大學 碩士學位論文.
何敏夫, 2006 三版五刷. 血液學 第三版, 合記圖書出版社.
巫鎧渝, 2013. 拋棄式糖化血紅素感測試片之製備研究, 台灣科技大學 碩士學位論文.
許富翔, 2012. 高靈敏度糖化血紅素電流式感測器之制備研究, 台灣科技大學 碩士學位論文.
廖貞玟, 2012. 大腸桿菌表現FPO重組蛋白以測定糖化血紅素(HbA1c), 台灣科技大學 碩士學位論文.