本研究利用電性鑑別法結合三明治免疫模型對糖化血色素(Hemoglobin A1c, HbA1c)及血色素(Hemoglobin, Hb)進行濃度的檢測，結合微流道電極晶片與金奈米粒子抗體標定技術，以助於偵側待測檢體免疫反應之阻抗訊號量測並定量分析糖化血色素濃度。實驗中利用電感、電容、電阻量測儀(LCR meter)偵測微流道電極晶片之電性訊號，藉由阻抗變化來判斷免疫反應的情況，並建立三明治免疫分析系統，分別對糖化血色素與血色素(Hemoglobin, Hb)做最適抗體與抗體接金濃度測試，再利用免疫中抗原抗體專一性，對混和糖化血色素與血色素的檢體分別檢測其糖化血色素與血色素之濃度再反推其糖化血色素濃度比例。本實驗利用經氧電漿表面改質後的微流道電極晶片，使用檢體量為10 μL，經三明治免疫反應時間測試，糖化血色素與血色素之三明治免疫反應時間皆可於8分內反應完畢。實驗結果中，糖化血色素的檢測範圍可達到0.1~100 ng/mL，而血色素則是10~500 ng/mL，最後在以整合型檢測晶片實驗中，對混和糖化血色素與血色素之抗原檢體的實驗下，成功對糖化血色素與血色素在濃度比例為2~10%區間之檢體做出判別，為糖尿病檢測提供了一個快速檢測、降低檢體需求以及低成本的構想。

This study presents a method to detect the concentration of glycated hemoglobin (HbA1c) and hemoglobin (Hb) by impedance measurement. Using MEMS and microfluidic theory to design microelectrodes combine with PDMS microchannels into sandwich-immunoassay electrodes chips, and measurement by LCR meter. This experiment is based on the traditional enzyme-linked immunosorbent assay (ELISA), so we first found out the best antibody (first antibody) concentration that is coated on the chips to catch the antigens we want, and then found out the best concentration of antibody labeled by Colloidal gold (second antibody). With these steps, we can make our chips detect the antigens concentration in the range we setup and with the help of microchannels and gold film, we reduce the complicated pre-work of sample preparation and also detect the HbA1c and Hb at the same time in one injection of diluted sample. Finally, our research setup the detecting ranges of our chips are glycated hemoglobin from 0.1~100 ng/mL and hemoglobin from 10~500 ng/mL, then we successful detect the mix sample of glycated hemoglobin and hemoglobin with 2 ~ 10% (ratio of glycated hemoglobin in total hemoglobin).

[1] http://www.bhp.doh.gov.tw/BHPNet/Web/Index/Index.aspx(衛生福利部 國民健康署)
[2] T. Porstmann and S. T. Kiessig, “Enzyme immunoassay techniques and overview,” Journal of Immunological Methods, 150, pp. 5-21, 1992.
[3] T. H. Huisman, E. A. Martis and A. Dozy, “Chromatography of hemoglobin types on carboxymethylcellulose,” Journal of Laboratory and Clinical Medicine, 52, pp. 312–27, 1958.
[4] R. M. Bookchin and P. M. Gallop, “Structure of hemoglobin A1c: nature of the N-terminal beta chain blocking group,” Biochemical and Biophysical Research Communications, 32, pp. 86–93, 1968.
[5] S. Rahbar, O. Blumenfeld and H. M. Ranney, “Studies of an unusual hemoglobin in patients with diabetes mellitus,” Biochemical and Biophysical Research Communications, 36, pp. 838–43, 1969.
[6] H. F. Bunn, D. N. Haney, K. H. Gabbay and P. M. Gallop, “Further identification of the nature and linkage of the carbohydrate in hemoglobin A1c,” Biochemical and Biophysical Research Communications, 67, pp. 103–9, 1975.
[7] R. J. Koenig, C. M. Peterson, R. L. Jones, C. Saudek, M. Lehrman and A. Cerami, “Correlation of glucose regulation and hemoglobin AIc in diabetes mellitus,” The New England Journal of Medicine, 295, pp. 417–420, 1976.
[8] zh.wikipedia.org/wiki/糖化血紅蛋白
[9] 糖化血色素檢測的國際規範及臨床應用 - 義大醫院
[10] A. Chaubey and B. D. Malhotra, “Mediated biosensors,” Biosensors and Bioelectronics, 17, pp. 441-456, 2002.
[11] 何敏夫，臨床生化學，合記圖書出版社， 1992。
[12] R. H. Garrett, and C. M. Grisham, Biochemistry, Saunders College Publishing, 1995.
[13] 廖國棠，金奈米粒子標記物在免疫分析、DNA 序列分析及微管道晶片系統分析上的應用，國立中山大學化學研究所博士論文，民國九十四年。
[14] E. Engvall and P. Perlmann, “Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes,” Journal of Immunology, 109, pp. 129-135, 1972.
[15] T. Watanabe, Y. Ohkuno, H. Matsuoka, H. Kimura, Y. Sakai, Y. Ohkaru, T. Tanaka, and Y. Kitaura, “Development of a simple whole blood panel test for detection of human heart-type fatty acid-binding protein,” Clinical Biochemistry, 34, pp. 257-263, 2001.
[16] M. Hedenfalk, P. Adlercreutz, and B. Mattiasson, “Modulation of the measuring range of a radioimmunoassay using an organic water two phase system,” Analytica Chimica Acta, 341, pp. 269-274, 1997.
[17] F. Hardy, L. Djavadi-Ohaniance and M. E. Goldberg, “Measurement of antibody/antigen association rate constants in solution by a method based on the enzyme-linked immunosorbent assay,” Journal of immunological methods, 200, pp. 155-159, 1997.
[18] P. Onnerfjord, S. Eremin, J. Emneus and G. Marko-Varga, “Fluorescence polarisation for immunoreagent characterization,” Journal of immunological methods, 213, pp. 31-39, 1998.
[19] J. A. Schmid and A. Billich, “Simple method for high sensitivity chemiluminescence ELISA using conventional laboratory equipment,” BioTechniques, 22, pp. 278, 1997.
[20] C. A. Janeway, P. Travers, M. Walport, and M. J. Shlomchik, Immunobiology, 5th edition, Garland Science, 2001.
[21] I. Bronstein, J. C. Voyta, G. H. G. Thorpe, L. J. Kricka and G. Armstrong, “Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin,” Clinical Chemistry, 35, pp. 1441-1446, 1989.
[22] E. Ishikawa, S. Hashida, T. Kohno and K. Hirota, “Ultrasensitive enzyme immunoassay,” Clinica Chimica Acta, 194, pp. 43-55, 1990.
[23] S. A. Berson and R. S. Yalow, “Immunoassay of endogenous plasma insulin in Man,” Annals of the New York Academy of Sciences, 82, pp. 1157-1175, 1959.
[24] G. Sakai, K. Ogata, T. Uda, N. Miura and N. Yamazoe, “A surface plasmon resonance-based immunosensor for highly sensitive detection of morphine,” Sensors And Actuators B-Chemical, 49, pp. 5-12, 1998.
[25] B. K. Oh, Y. K. Kim, W. Lee, Y. M. Bae, W. H. Lee and J. W. Choi, “Immunosensor for detection of legionella pneumophila using surface plasmon resonance,” Biosensors and Bioelectronics, 18, pp. 605-611, 2003.
[26] W. D. Wilson, “Analyzing Biomolecular Interactions,” Science, 295, pp. 2103-2105, 2002.
[27] B. K. Oh, W. Lee, B. S. Chun, Y. M. Bae, W. H. Lee and J. W. Choi, “The fabrication of protein chip based on surface plasmonresonance for detection of pathogens,” Biosensors and Bioelectronics, 20, pp. 1847-1850, 2004.
[28] S. Hearty, P. J. Conroy, B. V. Ayyar, B. Byrne, and R. O’Kennedy, “Surface plasmon resonance for vaccine design and efficacy studies: recent applications and future trends,” Expert Review of Vaccines, 9, pp. 645-664, 2010.
[29] G. U. Lee, D. A. Kidwell and R. J. Colton, “Sensing discrete streptavidin-biotin interactions with atomic force microscopy,” Langmuir, 10, pp. 354-357, 1994.
[30] L. Li, S. Chen, S. Oh and S. Jiang, “In situ single-molecule detection of antibody-antigen binding by tapping-mode atomic force microscopy,” Analytical Chemistry, 74, pp. 6017-6022, 2002.
[31] F. Cecchet, A. S. Duwez, S. Gabriel, C. Jérôme, R. Jérôme, K. Glinel, S. D. Champagne, A. M. Jonas, and B. Nysten, “Atomic Force Microscopy Investigation of the Morphology and the Biological Activity of Protein-Modified Surfaces for Bio- andImmunosensors,” Analytical Chemistry, 79, pp. 6488-6495, 2007.
[32] G. Kada, F. Kienberger, and P. Hinterdorfer, “Atomic force microscopy in bionanotechnology,” Nano Today, 13, pp. 12-19, 2008.
[33] S. H. Lee, D. D. Stubbs, J. Cairney and W. D. Hunt, “Rapid detection of bacterial spores using a quartz crystal microbalance (QCM) immunoassay,” IEEE Sensors Journal, pp. 5, 737-743, 2005.
[34] S. Kurosawa, H. Aizawa, M. Tozuka, M. Nakamura and J.-W. Park, “Immunosensors using a quartz crystal microbalance,” Measurement Science and Technology, 14, pp. 1882-1887, 2003.
[35] S. Schluecker, B. Kuestner, A. Punge, R. Bonfig, A. Marx and P. Stroebel, “Immuno-Raman microspectroscopy: in situ detection of antigens in tissue specimens by surface-enhanced Raman scattering,” Journal of Raman Spectroscopy, 37, pp. 719-721, 2006.
[36] D. A. Stuart, A. J. Haes, C. R. Yonzon, E. M. Hicks and R. P. Van Duyne, “Biological applications of localised surface plasmonic phenomenae,” IEE Proceedings-Nanobiotechnology, 152, pp. 13-32, 2005.
[37] D. S. Grubisha, R. J. Lipert, H.-Y. Park, J. Driskell, and M. D. Porter, “Femtomolar Detection of Prostate-SpecificAntigen: An Immunoassay Based onSurface-Enhanced Raman Scattering andImmunogold Labels,” Analytical Chemistry, 75, pp. 5936-5943, 2003.
[38] J. P. Gosling, “A decade of development in immunoassay methodology,” Clinical Chemistry, 36, pp. 1408-1427, 1990.
[39] J. Kai, A. Puntambekar, N. Santiago, S. Hawan Lee, D. W. Shey, V. Moore, J. Han and C. H. Ahn, “A novel microfluidic microplate as the next generation assay platform for enzyme linked immunoassays (ELISA) ,” Lab on a Chip, 12, pp. 4257-4262, 2012.
[40] W. C. W. Chan and S. M. Nie, “Quantum dot bioconjugates for ultrasensitive nonisotopic detection,” Science, 281, pp. 2016-2018, 1998.
[41] M. Bruchez, M. Moronne, P. Gin, S. Weiss and A. P. Alivisatos, “Semiconductor nanocrystals as fluorescent biological labels,” Science, 281, pp. 2013-2016, 1998.
[42] J. Yakovleva, R. Davidsson, A. Lobanova, M. Bengtsson, S. Eremin, T. Laurell and J. Emneus, “Microfluidic enzyme immunoassay using silicon microchip with immobilized antibodies and chemiluminescence detection,” Analytical Chemistry, 74, pp. 2994-3004, 2002.
[43] C. A. Marquette and L. J. Blum, “Electro-chemiluminescent biosensing,” Analytical and Bioanalytical Chemistry, 390, pp. 155-168, 2003.
[44] C. S. Holgate, P. Jackson, P. N. Cowen and C. C. Bird, “Immunogold-silver staining: New method of immunostaining with enhanced sensitivity,” The Histochemical Society, 31, pp. 938-944, 1983.
[45] S. Kubitschko, J. Spinke, T. B. ckner, S. Pohl, and N. Oranth, “Sensitivity Enhancement of Optical Immunosensorswith Nanoparticles,” Analytical Biochemistry, 253, pp. 112-122, 1997.
[46] R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger and C. A. Mirkin, “Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles,” Science, 277, pp. 1078-1081, 1997.
[47] S. Brakmann, “DNA-based barcodes, nanoparticles, and nanostructures for the ultrasensitive detection and quantification of proteins,” Angewandte Chemie-International Edition, 43, pp. 5730-5734, 2004.
[48] J. M. Nam, C. S. Thaxton and C. A. Mirkin, “Nanoparticle-based bio–bar codes for the ultrasensitive detection of proteins,” Science, 301, pp. 1884-1886, 2003.
[49] X. Chu, X. Fu, K. Chen, G. L. Shen and R. Q. Yu, “An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label,” Biosensors and Bioelectronics, 20, pp. 1805-1812, 2005.
[50] Y. P. Bao, T. F. Wei, P. A. Lefebvre, H. An, L. He, G. T. Kunkel and U. R. Muller, “Detection of protein analytes via nanoparticle-based bio bar code technology,” Analytical Chemistry, 78, pp. 2055-2059, 2006.
[51] C. Grüttner, K. Müller, J. Teller, F. Westphal, A. Foreman and R. Ivkov, “Synthesis and antibody conjugation of magnetic nanoparticles with improved specific power absorption rates for alternating magnetic field cancer therapy,” Journal of Magnetism and Magnetic Materials, 311, pp. 181-186, 2007.
[52] N. T. K. Thanh and Z. Rosenzweig, “Development of an aggregation-based immunoassay for anti-Protein A using gold nanoparticles,” Analytical Chemistry, 74, pp. 1624-1628, 2002.
[53] S. Cobbe, S. Connolly, D. Ryan, L. Nagle, R. Eritja and D. Fitzmaurice, “DNA-controlled assembly of protein-modified gold nanocrystals,” Journal of Physical Chemistry B, 107, pp. 470-477, 2003.
[54] N. L. Binnun, A. B. Lindner, O. Zik, Z. Eshhar and E. Moses, “Quantitative detection of protein arrays,” Analytical Chemistry, 75, pp. 1436-1441, 2003.
[55] Z. F. Ma and S. F. Sui, “Naked-eye sensitive detection of immunoglubulin G by enlargement of Au nanoparticles in vitro,” Angewandte Chemie-International Edition, 41, pp. 2176-2179, 2002.
[56] L. R. Hirsch, J. B. Jackson, A. Lee, N. J. Halas and J. L. West, “A whole blood immunoassay using gold nanoshells,” Analytical Chemistry, 75, pp. 2377-2381, 2003.
[57] M. Lin, Y. C. Lin, K.C. Su, Y. T. Wang, T. C. Chang and H. P. Lin, “A novel real-time immunoassay utilizing an electro-immunosensing microchip and gold nanoparticles for signal enhancement,” Sensors and Actuators B-Chemical, 117, pp. 451-456, 2006.
[58] A. Abera and J.-W. Choi, “Quantitative lateral flow immunosensor using carbon nanotubes as label,” Analytical Methods, 2, pp. 1819-1822, 2010.
[59] H. H. Chen, C. H. Wu, M. L. Tsai, Y. J. Huang and S. H. Chen,“Detection of Total and A1c-Glycosylated Hemoglobin in Human Whole Blood Using Sandwich Immunoassays on Polydimethylsiloxane-Based Antibody Microarrays,” Analytical Chemistry, 84, pp. 8635–8641, 2012
[60] Y. C. Chuang, K. C. Lan, K. M. Hsieh, L. S. Jang and M. K. Chen, “Detection of glycated hemoglobin based on impedance measurement with parallel electrodes integrated into a microfluidic device,”　Sensors and Actuators B-Chemical, 171–172, pp. 1222–1230, 2012
[61] M. A. Hayat (Ed.), Colloidal Gold: Principles, Methods, and Applications, Academic Press, New York, 1989.
[62] O. Niwa, M. Morita, and H. Tabei, “Electrochemical Behavior of Reversible Redox Species at Interdigitated Array Electrodes with Different Geometries: Consideration of Redox Cycling and Collection Efficiency,” Analytical Chemistry, 62, pp. 447-452, 1990.
[63] O. Niwa, M. Morita, and H. Tabei, “Highly sensitive and selective voltammetric detection of dopamine with vertically separated interdigitated array electrodes,” Electroanalysis, 3, pp. 163-168, 1991.
[64] B. H. Jo, L. M. V. Lerberghe, K. M. Motsegood and D. J. Beebe, “Three dimensional nicro-channel gabrication in polydimethylsiloxane (PDMS) elastomer,” Journal of Micro-Electromechanical Systems, 9, pp. 76-81, 2000.
[65] http://sms.kaist.ac.kr/~ischoi/bk/lecture/class%2009.pdf
[66] http://www.mne.umd.edu/LAMP/lamp_msds.htm, Laboratory for advanced materials processing, 2002.