本研究利用螢光粒子製備新一代驗孕檢測試劑，其提高三明治型免疫反應檢測靈敏度，突破傳統金奈米粒子檢測閾值限制，摒除影像擷取環境干擾，使檢測試劑於相同抗原濃度測試時，達到最佳線性度。
研究中各項測試皆使用自製影像擷取裝置與本實驗室開發之T線判讀法，配合Matrix Laboratory (MATLAB)分析軟體進行定量分析，改善定性判讀誤差缺失，最後導入田口方法進行最佳化設計。研究結果顯示，以一步共價法、混和反應儀器強度4及鍵結反應劑反應30分鐘時，鍵結效率可提高42.12%，品質損失降低88.2%；使用PAL視訊標準，亮度提高12.5%，對比提高12.5%，色調降低12.5%時，T線相對灰階值提高41.08%，品質損失降低49%。此外，螢光接hCG二抗濃度為30 μg/mL、樣品墊緩衝液pH = 4與抗原稀釋液為100 μL時，SN比提高了6.308 dB，品質損失降低76.7%；結合最佳化影像擷取裝置，可使hCG檢測試劑檢測極限達6.25 mIU/mL，成功地突破閾值25 mIU/ mL限制，灰階-hCG濃度轉換曲線決定係數(Coefficient of determination)達0.977，代表本研究可利用此轉換曲線進行定量分析。

This thesis presents the key factors influencing the analyte detection limit of the sandwich immunochromatographic assay and reduction of cut-off value using fluorescent particles instead of colloid gold. The combination with image capture device and test line color analysis method. MATLAB was successfully quantitative the concentration of Human Chorionic Gonadotropin antigen transformed from test line color. In the Taguchi method, when we used one-step covalent, Vortex level 4 and EDC reaction 30 minutes, the bonding efficiency can be increased by 42.12%. When we used PAL video standard, brightness increased by 12.5%, contrast increased by 12.5% and hue decreased by 12.5%, and the relative grayscale increased by 41.08%. In addition, we did not freeze-dried test paper when completing the test paper by lateral flow assay process. When we used the hCG 2nd-fluorescence concentration 30 μg/mL, Antigen dilution volume 100 μL and sample pad buffer pH=4, the signal-to-noise ratio (SN ratio) increased by 6.308 dB. With the above conditions and the image capture device, the cut-off value decreased from 25 mIU/mL to 6.25 mIU/mL.

[1] T. Porstmann and S.T. Kiessig, “Enzyme immunoassay techniques and overview,” Journal of Immunological Methods, 150, pp. 5-21, 1992.
[2] 何敏夫，臨床生化學， 合記圖書出版社， 1992。
[3] R. H. Garrett and C. M. Grisham, Biochemistry, Saunders College Publishing, 1995.
[4] 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。
[5] E. Engvall and P. Perlmann, “Enzyme-linked immunosorbent assay, ElISA III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes,” Journal of Immunology, 109, pp. 129-135, 1972.
[6] 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.
[7] 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.
[8] 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.
[9] P. Onnerfjord, S. Eremin, J. Emneus and G. Marko-Varga, “Fluorescence polarisation for immunoreagent characterization,” Journal of immunological methods, 213, pp. 31-39, 1998.
[10] J. A. Schmid and A. Billich, “Simple method for high sensitivity chemiluminescence ELISA using conventional laboratory equipment,” BioTechniques, 22, pp. 278, 1997.
[11] C. A. Janeway, P. Travers, M. Walport, and M. J. Shlomchik, Immunobiology, Garland Science, 2001.
[12] A. J. Lapthorn, D. C. Harris, A. Littlejohn, J. W. Lustbader, R. E. Canfield, K. J. Machin, F. J. Morgan and N. W. Isaacs, “Crystal structure of human chorionic gonadotropin,” Nature, 369, pp.455-461, 1994.
[13] J. G. Pierce and T. F. Parsons, “Glycoprotein Hormones: Structure and Function,” Annual Reviews Biochemistry, 50, pp.465-495, 1981.
[14] S. Birken, Y. Maydelman, M. A. Gawinowicz, A. Pound, Y. Liu and A. S. Hartree, “Isolation and characterization of human pituitary chorionic gonadotropin,” Endocrinology, 137, pp.1402-1411, 1996.
[15] L. A. Cole and A. Kardana, “Discordant Results in Human Chorionic Gonadotropin Assays,” Clinical Chemistry, 38, pp.263-270, 1992.
[16] R. Bellisario, R. B. Carlsen and O. P. Bahl, “Human Chorionic Gonadotropin Linear Amino Acid Sequence of The α Subunit,” Journal of Biological Chemistry, 248, pp.6796-6809, 1973.
[17] R. B. Carlsen, O. P. Bahl and N. Swawinathan, “Human Chorionic Gonadotropin Linear Amino Acid Sequence of The β Subunit,” Journal of Biological Chemistry, 248, pp.6810-6827, 1973.
[18] http://life.nthu.edu.tw/~scwu/ls6342/hcg/hcg.html
[19] goo.gl/eR9hbT
[20] L. A. Cole, “New discoveries on the biology and detection of human chorionic gonadotropin,” Reproductive Biology and Endocrinology, 7, pp. 8, 2009.
[21] R. Hoermann, G. Spoettl, R. Moncayo and K. Mann, “Evidence for the presence of human chorionic gonadotropin (hCG) and free beta-subunit of hCG in the human pituitary,” Journal of Clinical Endocrinology & Metabolism, 71, pp.179-186, 1990.
[22] J. J. Gregory and J. L. Finlay, “Alpha-fetoprotein and beta-human chorionic gonadotropin: their clinical significance as tumour markers,” Drugs, 57, pp.463-467, 1999.
[23] U. A. Kayisli, B. Selam, O. G. Kayisli, R. Demir and A. Arici, “Human chorionic gonadotropin contributes to maternal immunotolerance and endometrial apoptosis by regulating Fas-Fas ligand system,” Journal of Immunology, 171, pp.2305-2313, 2003.
[24] J. Askling, G. Erlandsson, M. Kaijser, O. Akre and A. Ekbom, “Sickness in pregnancy and sex of child,” Lancet, 354, pp.2053, 1999.
[25] X. G. Fan and Z. Q. Zheng, “A Study of Early Pregnancy Factor ctivity in Preimplantation,” American Journal of Reproductive Immunology, 37, pp.359-364, 1997.
[26] http://www.fertilityplus.com/faq/hpt.html
[27] http://www.firstep.com.tw/chinese/images/download0300.pdf
[28] L. A. Cole, Y. X. Wang, M. Elliott, M. Latif, J. T. Chambers, S. K. Chambers and P. E. Schwartz, “Urinary Human Chorionic Gonadotropin Free β-Subunit and β-Core Fragment: A New Marker of Gynecological Cancers,” Cancer Research, 48, pp.1356-1360, 1988.
[29] Y. Kato and G. B. Braunstein, “β-core fragment is a major form of immunoreactive urinary chorionic gonadotropin in uman pregnancy,” Journal of Clinical Endocrinology & Metabolism, 66, pp.1197-1201, 1988.
[30] R. W. McNaught and J. T. France, “Studies of the biochemical basis of steroid sulphatase deficiency: Preliminary evidence suggesting a defect in membrane-enzyme structure,” Journal of Steroid Biochemistry, 13, pp. 363-373, 1980.
[31] 徐敘瑢與陳憲偉，光電材料與顯示技術，五南文化事業出版, 2007.
[32] G. Harrison, P. Haffey and E. E. Golub, “A nanogram-level colloidal gold single reagent quantitative protein assay,” Analytical Biochemistry, 380, pp.1-4, 2008.
[33] W. Leung, C. P. Chan, T. H. Rainer, M. Ip, G. W.H. Cautherley and R. Renneberga, “InfectCheck CRP barcode-style lateral flow assay for semi-quantitative detection of C-reactive protein in distinguishing between bacterial and viral infections,” Journal of Immunological Methods, 336, pp.30-36, 2008.
[34] C. Abe, K. Hirano and T. Tomiyama, “Simple and Rapid Identification of the Mycobacterium tuberculosis Complex by Immunochromatographic Assay Using Anti-MPB64 Monoclonal Antibodies,” Journal of Clinical Microbiology, 37, pp.3693-3697, 1999.
[35] W. N. Zhang and Z. Y. Zhao, “Three-color Data Reproduction Algorithm and Implementation for the Color Image Based on CCD and DSPs,” Computer Engineering, 28, pp.111-113, 2002.
[36] R. Z. Zhou, J. He and Z. L. Hong, “Adaptive Algorithm of Auto White Balance for Digital Camera,” Journal of Computer Aided Design & Computer Graphics, 17, pp.529-533, 2005.
[37] D. Z. Zheng, Z. L. Wu and S. Wang, “Detecting Method of Quantitative Colloidal Gold Test Strip Concentration Based on the DSP Image Processing,” Proc. IEEE 4th International iCBBE Conference (IEEE iCBBE 2010), pp.1-4, 2010.
[38] H. Y. Jiang and M. Du, “Research on the Detection of Gold Immunochromatographic Assay by the Image Histogram Feature Vectors and Fuzzy C-means,” Proc. IEEE 8th International FSKD Conference (IEEE FSKD 2011), pp.467-471, 2011.
[39] http://alumni-voice.nctu.edu.tw/Alumni404/404-27-31.pdf
[40] J. L. Rosa, A. Robin, M. B. Silva, C. A. Baldan and M. P. Peres, “Electrodeposition of copper on titanium wires: Taguchi experimental design approach,” Journal of Materials Processing Technology, 209, pp. 1181-1188, 2009.
[41] R. S. Rao, R. S. Parkasham, K. K. Prasad, S. Rajesham, P. N. Sama and L. V. Rao, “Xylitol production by Candida sp.: parameter optimization using Taguchi approach,” Process Biochemistry, 39, pp. 951-956, 2004.
[42] 陳東煌，功能性粉末：複合奈米粒子–有趣的人造原子，科學發展，408期，40 ~ 45頁，2006年12月
[43] Fluoro-Max Fluorescent Beads with Europium Chelate, Thermo Fisher, Inc
[44] Particle Reagent Optimization - Recommended Adsorption and Covalent Coupling Procedures, Thermo Fisher,Inc. , 2010
[45] Pierce BCA Protein Assay Kit instruction, Thermo Fisher, Inc.