本研究提出新型阻抗免疫分析方法，利用微阻抗晶片偵測免疫反應，使用金奈米粒子作為抗體之標定物，並扮演催化劑的角色，催化銀析出反應，配合銀析出反應放大電性偵測訊號。有別於傳統酵素聯結免疫吸附分析法(enzyme-linked immunosorbent assay, ELISA)，採用現行傳統的抗原抗體固定技術，將抗原抗體固定於晶片表面，並加入銀訊號增強技術，以增強金奈米粒子產生的反應偵測訊號。

利用電感、電容、電阻量測儀(LCR meter)偵測阻抗晶片之電性訊號，並應用在免疫分析是一個全新的偵測方法，在本研究中使用兩種不同的分析模型：(a)直接型免疫分析法(兩層式)是將抗原直接固定在晶片表面，作為定性實驗，尋求較佳的實驗參數；(b)三明治型免疫分析法(三層式)使用抗體檢測待測抗原，並對抗原作定量分析。在阻抗免疫分析法中，結合濕式與乾式兩種不同的阻抗偵測方法。

放大、偵測並分析電性訊號，利用其阻抗改變的情況，判斷免疫反應的發生，建構出全新的免疫分析方法，所需整體反應偵測時間約為30分鐘，濕式阻抗免疫分析方法之靈敏度為10-8 g/mL，而乾式阻抗免疫分析方法之靈敏度則可達10-9 g/mL。本研究設計不同的電極間距，用以討論電極間距與偵測靈敏度之間的關係。除此之外，在阻抗免疫分析方法中，將討論專一性、靈敏度等問題；利用阻抗分析晶片、奈米粒子技術與銀訊號增強方法，提供了免疫分析一個全新的方向與思維。

This study reports a novel immunoassay using an electro-microchip to detect the immuno-reaction signal, a gold nanoparticles (ANPs) as a label of antigen or antibody and as a catalyst for silver precipitation, and the silver enhancement reaction to magnify the detection signal. Compared with traditional enzyme-linked immunosorbent assay (ELISA), we adopt the immobilization of antigen or antibody on the chip surface, and introduce silver enhancement method to magnify the detection signal generated by the gold nanoparticles.

The LCR meter instrument, a new detection method for immunoassay, is used to detect the electric single. There are two major formats developed in this study: (a) direct immunoassay (two-layer format), the antigen is immobilized on the chip directly to optimize the assay conditions; (b) sandwich immunoassay (sandwich format), first and secondary antibodies are used to quantify and qualify antigens. Two different electro-detection methods are used in electro-immunoassay: wet type electro-detection and dry type electro-detection.
The change of the impedance to corroborate immuno-reaction is detected, amplified and analyzed to construct this new immunoassay. The total detection time of the immunoassay is about 30 minutes. The sensitivity of the wet type immunoassay is about 10-8 g/mL for detected antigen and in dry type immunoassay the sensitivity is reached 10-9 g/mL. Different sizes of the electrodes are designed to discuss the relationship between gap size and sensitivity. In addition, the specificity and sensitivity of the electro-immunoassay are discussed. Using electro-microchip, nanoparticles and silver enhancement provided a new immunoassay method.

[1] M. Hedenfalk, P. Adlercreutz, B. Mattiasson, “Modulation of the measuring range of a radioimmunoassay using an organic water two phase system,” Analytica Chimica Acta, 341, 269, 1997.
[2] 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, 155, 1997.
[3] P. Onnerfjord, S. Eremin, J. Emneus and G. Marko-Varga, “Fluorescence polarisation for immunoreagent characterization,” Journal of immunological methods, 213, 3, 1998.
[4] J. A. Schmid and A. Billich, “Simple method for high sensitivity chemiluminescence ELISA using conventional laboratory equipment,” BioTechniques, 22, 278, 1997.
[5] J. P. Gosling, “A decade of development in immunoassay methodology,” Clinical Chemistry, 36, 1408, 1990.
[6] K. Nielsen, M. Lin, D. Gall and M. Jolley, “Fluorescence polarization immunoassay: Detection of antibody to brucella abortus,” Methods, 22, 71, 2000.
[7] J. Yakovleva, R. Davidsson, A. Lobanova, M. Bengtsson, S. Eremin, T. Laurell and J. Emne´us, “Microfluidic enzyme immunoassay using silicon microchip with immobilized antibodies and chemiluminescence detection,” Analytical Chemistry, 74, 2994, 2002.
[8] M. Dequaire, C. Degrand and B. Limoges, “An electrochemical metalloimmunoassay based on a colloidal gold label,” Analytical Chemistry, 72, 5521, 2000.
[9] N. Kangatharalingam and M. Essenberg, “Modified pyrogallol-initiated immunogold–silver enhancement technique applicable to prokaryotes,” Journal of Microbiological Methods, 41, 211, 2000.
[10] J. S. Ko, H. C. Yoon, H. Yang, H. B. Pyo, K. H. Chung, S. J. Kim and Y. T. Kim, “Polymer-based microfluidic device for immunosensing biochips,” Lab on a Chip, 3, 106, 2003.
[11] B. H. Schneider, E. L. Dickinson, M. D. Vach, J. V. Hoijer and L. V. Howard, “Optical chip immunoassay for hCG in human whole blood,” Biosensors & Bioelectronics, 15, 597, 2000.
[12] N. Lochner, C. Lobmaier, M. Wirth, A. Leitner, F. Pittner and F. Gabor, “Silver nanoparticle enhanced immunoassays: One step real time kinetic assay for insulin in serum,” European Journal of Pharmaceutics and Biopharmaceutics, 56, 469, 2003.
[13] E. Matveeva, Z. Gryczynski, J. Malicka, I. Gryczynski and J. R. Lakowicz, “Metal-enhanced fluorescence immunoassays using total internal reflection and silver island-coated surfaces,” Analytical Biochemistry, 34, 303, 2004.
[14] W. Laureyn, D. Nelis, P. V. Gerwen, K. Baert, L. Hermans, R. Magnee, J. J. Pireaux and G. Maes, “Nanoscaled interdigitated titanium electrodes for impedimetric biosensing,” Sensors and Actuators B, 68, 360, 2000.
[15] W. Laureyn, F. Frederix, P. V. Gerwen and G. Maes, “Nanoscaled interdigitated gold electrodes for impedimetric immunosensing,” Transducers ’99, Digest of Technical Papers, Sendai, Japan, 1884, 1999.
[16] P. V. Gerwen, W. Laureyn, W. Laureys, G. Huyberechts, M. O. D. Beeck, K. Baert, J. Suls, W. Sansen, P. Jacobs, L. Hermans, and R. Mertens, “Nanoscaled interdigitated electrode arrays for biochemical sensors,” Sensors and Actuators B, 49, 73, 1999.
[17] Y. Huang, K. L. Ewalt, M. Tirado, R. Haigis, A. Forster, D. Ackley, M. J. Heller, J. P. O’Connell and M. Krihak, “Electric manipulation of bioparticles and macromolecules on microfabricated electrodes,” Analytical Chemistry, 73, 1549, 2001.
[18] G. Liu, J. Wang, J. Kim, and M. R. Jan, “ Electrochemical coding for multiplexed immunoassays of proteins,” Analytical Chemistry, 76, 7126, 2004.
[19] 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, 1805. 2005.
[20] M. A. Hayat (Ed.), Colloidal Gold: Principles, Methods, and Applications, 1, 52, Academic Press, New York, 1989.
[21] 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, 938,1983.
[22] M. Moeremans, G Daneels, D. V. Dijck, G. Langanger and J. D. Mey, “Sensitive visualization of antigen– antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silver staining,” Journal of immunological methods, 74, 353, 1984.
[23] H. Yi, Jan L. M. Leunissen, G. M. Shi, C. A. Gutekunst and S. M. Hersch, “A novel procedure for pre-embedding double immunogold–silver labeling at the ultrastructural level,” The Journal of Histochemistry & Cytochemistry, 49, 279, 2001.
[24] P. M. Lackie, “Immunogold silver staining for light microscopy,” Histochemistry and Cell Biology, 106, 9, 1996.
[25] R. Q. Liang, C. Y. Tan and K. C. Ruan, “Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement,” Journal of Immunological Methods, 285, 157, 2004.
[26] 洪青元，應用奈米粒子標記與銀析出偵測法於免疫分析之研究，國立成功大學碩士論文，民國九十四年。
[27] I. Alexandre, S. Hamels, S. Dufour, J. Collet, N. Zammatteo, F. D. Longueville, J. L. Gala, and J. Remacle, “Colorimetric silver detection of DNA microarrays,” Analytical Biochemistry, 295, 1, 2001.
[28] X. Ji, S. Xu, L. Wang, M. Liu, K. Pan, H. Yuan, L. Ma, W. Xu, J. Li, Y. Bai and T. Li, “Immunoassay using the probe-labeled Au/Ag core-shell nanoparticles based on surface-enhanced Raman scattering,” Colloids and Surfaces A, 257-258, 171, 2005.
[29] S. J. Park, T. A. Taton and C. A. Mirkin, “Array-based electrical detection of DNA with nanoparticle probes,” Science, 295, 1503, 2002.
[30] T. A. Taton, C. A. Mirkin and R. L. Letsinger, “Scanometric DNA array detection with nanoparticle probes,” Science, 289, 1757, 2000.
[31] T. C. Charles, “The active site in nanoparticle gold catalysis,” Science, 306, 234, 2004.
[32] http://www.devicelink.com/ivdt/archive/01/03/002.html, Handling false signals in gold-based rapid tests, 2001.
[33] http://www.devicelink.com/ivdt/archive/00/03/004.html, The place of gold in rapid tests, 2000.
[34] K. C. Ho, P. J. Tsai, Y. S. Lin and Y. C. Chen, “Using biofunctionalized nanoparticles to probe,” Analytical Chemistry, 76, 7162, 2004.
[35] http://www.deltaunited.com.tw/, deltaunited, 1998.
[36] T. Kotnik and D. Miklavcic, “Analytical description of transmembrane voltage induced by electric fields on spheroidal cells,” Journal of Biophysical, 79, 670, 2000.
[37] P. V. Gerwen, W. Laureys, G. Huyberechts, M. O. Beeck, K. Baert, J. Suls, A. Varian, W. Sansen, L. Hermans and R. Mertens, “Nanoscaled interdigitated electrode arrays for biochemical sensor,” Transducers `97, International Conference on Solid-State Sensors and Actuators, Chicago, 907, 1997.
[38] M. J. Madou, Fundamentals of Microfabrication, 97, CRC Press, Florida, 1997.
[39] S. M. Sze, VLSI Technology, 386, McGraw-Hill, New York, 1998.
[40] 莊達人，VLSI 製造技術，高立圖書，235，民國九十六年。
[41] 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, 76, 2000.
[42] http://www.agac.umn.edu/microarray/protocols/protocols.htm, Microarray protocols, 2000.
[43] http://sms.kaist.ac.kr/~ischoi/bk/lecture/class%2009.pdf
[44] http://www.mne.umd.edu/LAMP/lamp_msds.htm, Laboratory for advanced materials processing, 2002.
[45] M. A. Hayat (Ed.), Colloidal Gold: Principles, Methods, and Applications, 1, 15, Academic Press, New York, 1989.
[46] Y. C. Lin, M. Li, C. Y. Wu, W. C. Hsiao and Y. C. Chung, “Microchips for cell-type identification,” The seventh International Symposium on Micro Total Analysis System, μTAS 2003, California, USA, 729, 2003.
[47] http://members.aol.com/logan20/red_tabl.html, Standard reduction potentials in aqueous solution at 25C, 1997.