近來胜肽晶片(peptide array)運用在蛋白質體學的研究上愈來愈受重視，像是應用在大量掃描分析蛋白質激酶、蛋白酶活性分析及蛋白質的指紋辨識，胜肽晶片提供一個方便實用的分析平台。傳統上，胜肽晶片大部分皆以玻璃或紙做為基材，但它們分別存在著不具撓曲性及低透光度、高螢光背景值的缺點。因此在本研究中，我們利用低價、高光穿透性且生物相容性高的聚雙甲基矽氧烷(PDMS)聚合物做為基材，並結合原位(in situ)合成法製備胜肽晶片的分析平台上。進行完原位合成金奈米粒子於PDMS表面後，我們利用分子自組裝的方法在金奈米粒子上進行化學修飾，讓金奈米粒子表面同時帶有具活性端能與胺基酸進行共價鍵及能抗非專一性吸附的官能基聚乙二醇。在原位合成胜肽晶片的部分，我們先最佳化能與PDMS相容之有機溶劑，並使用在特定位置植入光起始試劑(photoinitiator, PI)以避免點與點間的汙染，再結合PGA-P (photogenerated acid precursor)進行光化學反應的技術，確保合成在晶片上的胜肽序列的正確性。最後，我們利用自家合成N端具有保護基且側鏈帶有螢光分子Cy5的離胺酸(Lys)組件(Boc-Lys-Cy5)驗證此原位合成胜肽於PDMS基材上的策略，我們成功地藉由五個光化學合成的步驟於基材上接上五個離胺酸(Lys)組件形成聚離胺酸(poly-Lys)。此外，原位合成在PDMS上的奈米金也藉由其高表面積的特性增強整體偵測的靈敏度。

Peptide arrays are becoming popular for conducting proteomics studies such as the large scale screening for kinase or protease activity and protein finger printing. Although cellulose membranes and glass slides are popular substrates for peptide arrays, they suffer from one or two drawbacks including low transparency, high background fluorescence, and low flexibility. Poly(dimethylsiloxane) (PDMS) is a good alternative due to its low cost, high optical transparency, and biocompatibility. In this study, we aim to develop a PDMS-based peptide arrays via in-situ synthesis. We first utilized an in-situ synthesis method to form a AuNP layer on PDMS surface and then modified the AuNP surface with active groups and the shorter polyethylene glycol which could minimize nonspecific bindings. The active groups were shown to form covalent binding with aminoacids via EDC/NHS chemistry. For in-situ peptide synthesis, PDMS-compatible solvent was judiciously chosen for photopolymerization reaction and the photoinitiator (PI) was implanted into the substrate before the first photo-reaction to minimize cross contaminations between adjacent aminoacid layers. Using Boc-Lys-Cy5 as the building block, we showed the method could successfully complete a 5-step synthesis for a 5-mer of poly-Lys (Lys-Lys-Lys-Lys-Lys) molecule. Furthermore, the AuNP layer was shown to increase the detection sensitivity by increasing the surface area.

1.Merrifield, R. B., Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J Am Chem Soc 1963, 85 (14), 2149-2154.
2.Frank, R., Spot-Synthesis - an Easy Technique for the Positionally Addressable, Parallel Chemical Synthesis on a Membrane Support. Tetrahedron 1992, 48 (42), 9217-9232.
3.Frank, R., The SPOT synthesis technique - Synthetic peptide arrays on membrane supports - principles and applications. J Immunol Methods 2002, 267 (1), 13-26.
4.Hilpert, K.; Winkler, D. F. H.; Hancock, R. E. W., Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion. Nat Protoc 2007, 2 (6), 1333-1349.
5.Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D., Light-Directed, Spatially Addressable Parallel Chemical Synthesis. Science 1991, 251 (4995), 767-773.
6.Pellois, J. P.; Zhou, X. C.; Srivannavit, O.; Zhou, T. C.; Gulari, E.; Gao, X. L., Individually addressable parallel peptide synthesis on microchips. Nat Biotechnol 2002, 20 (9), 922-926.
7.Gao, X. L.; Zhou, X. C.; Gulari, E., Light directed massively parallel on-chip synthesis of peptide arrays with t-Boc chemistry. Proteomics 2003, 3 (11), 2135-2141.
8.Cretich, M.; Damin, F.; Pirri, G.; Chiari, M., Protein and peptide arrays: Recent trends and new directions. Biomol Eng 2006, 23 (2-3), 77-88.
9.Xu, Q.; Lam, K. S., Protein and Chemical Microarrays-Powerful Tools for Proteomics. J Biomed Biotechnol 2003, 2003 (5), 257-266.
10.Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew Chem Int Ed 2001, 40 (11), 2004-2021.
11.Thiele, A.; Zerweck, J.; Schutkowski, M., Peptide arrays for enzyme profiling. Methods Mol Biol 2009, 570, 19-65.
12.Yeatman, T. J., A renaissance for SRC. Nat Rev Cancer 2004, 4 (6), 470-80.
13.Toker, A.; Newton, A. C., Cellular signaling: Pivoting around PDK-1. Cell 2000, 103 (2), 185-188.
14.Lu, P. J.; Zhou, X. Z.; Shen, M. H.; Lu, K. P., Function of WW domains as phosphoserine- or phosphothreonine-binding modules. Science 1999, 283 (5406), 1325-1328.
15.Li, T.; Liu, D. J.; Wang, Z. X., Screening Kinase Inhibitors with a Microarray-Based Fluorescent and Resonance Light Scattering Assay. Anal Chem 2010, 82 (7), 3067-3072.
16.Han, X.; Shigaki, S.; Yamaji, T.; Yarnanouchi, G.; Mori, T.; Niidome, T.; Katayama, Y., A quantitative peptide array for evaluation of protein kinase activity. Anal Biochem 2008, 372 (1), 106-115.
17.Uttamchandani, M.; Yao, S. Q., Peptide microarrays: Next generation biochips for detection, diagnostics and high-throughput screening. Curr Pharm Des 2008, 14 (24), 2428-2438.
18.Collet, B. Y. M.; Nagashima, T.; Yu, M. S.; Pohl, N. L. B., Fluorous-based peptide microarrays for protease screening. J Fluorine Chem 2009, 130 (11), 1042-1048.
19.Gershoni, J. M.; Roitburd-Berman, A.; Siman-Tov, D. D.; Tarnovitski Freund, N.; Weiss, Y., Epitope Mapping: The First Step in Developing Epitope-Based Vaccines. BioDrugs 2007, 21 (3), 145-156.
20.Andresen, H.; Zarse, K.; Grotzinger, C.; Hollidt, J. M.; Ehrentreich-Forster, E.; Bier, F. F.; Kreuzer, O. J., Development of peptide microarrays for epitope mapping of antibodies against the human TSH receptor. J Immunol Methods 2006, 315 (1-2), 11-18.
21.Min, D. H.; Mrksich, M., Peptide arrays: towards routine implementation. Curr Opin Chem Biol 2004, 8 (5), 554-558.
22.Ahmed, S.; Mathews, A. S.; Byeon, N.; Lavasanifar, A.; Kaur, K., Peptide Arrays for Screening Cancer Specific Peptides. Anal Chem 2010, 82 (18), 7533-7541.
23.Su, J.; Mrksich, M., Using mass spectrometry to characterize self-assembled monolayers presenting peptides, proteins, and carbohydrates. Angew Chem Int Ed 2002, 41 (24), 4715-4718.
24.Min, D. H.; Tang, W. J.; Mrksich, M., Chemical screening by mass spectrometry to identify inhibitors of anthrax lethal factor. Nat Biotechnol 2004, 22 (6), 717-723.
25.Chen, H. H.; Sung, W. C.; Liang, S. S.; Chen, S. H., Functional fluorinated modifications on a polyelectrolyte coated polydimethylsiloxane substrate for fabricating antibody microarrays. Anal Chem 2010, 82 (18), 7804-13.
26.Sung, W. C.; Chang, C. C.; Makamba, H.; Chen, S. H., Long-term affinity modification on poly(dimethylsiloxane) substrate and its application for ELISA analysis. Anal Chem 2008, 80 (5), 1529-1535.
27.McDonald, J. C.; Whitesides, G. M., Poly(dimethylsiloxane) as a material for fabricating microfluidic devices. Acc Chem Res 2002, 35 (7), 491-9.
28.Liu, Y.; Fanguy, J. C.; Bledsoe, J. M.; Henry, C. S., Dynamic coating using polyelectrolyte multilayers for chemical control of electroosmotic flow in capillary electrophoresis microchips. Anal Chem 2000, 72 (24), 5939-44.
29.Lahann, J.; Balcells, M.; Lu, H.; Rodon, T.; Jensen, K. F.; Langer, R., Reactive polymer coatings: a first step toward surface engineering of microfluidic devices. Anal Chem 2003, 75 (9), 2117-22.
30.Yang, T.; Baryshnikova, O. K.; Mao, H.; Holden, M. A.; Cremer, P. S., Investigations of bivalent antibody binding on fluid-supported phospholipid membranes: the effect of hapten density. J Am Chem Soc 2003, 125 (16), 4779-84.
31.Sung, W. C.; Chang, C. C.; Makamba, H.; Chen, S. H., Long-term affinity modification on poly(dimethylsiloxane) substrate and its application for ELISA analysis. Anal Chem 2008, 80 (5), 1529-35.
32.Sung, W. C.; Chen, H. H.; Makamba, H.; Chen, S. H., Functionalized 3D-hydrogel plugs covalently patterned inside hydrophilic poly(dimethylsiloxane) microchannels for flow-through immunoassays. Anal Chem 2009, 81 (19), 7967-73.
33.Chen, S. H.; Chen, H. H.; Sung, W. C.; Hang, S. S., Functional Fluorinated Modifications on a Polyelectrolyte Coated Polydimethylsiloxane Substrate for Fabricating Antibody Microarrays. Anal Chem 2010, 82 (18), 7804-7813.
34.Lee, J. N.; Park, C.; Whitesides, G. M., Solvent compatibility of poly(dimethylsiloxane)-based microfluidic devices. Anal Chem 2003, 75 (23), 6544-6554.
35.Schneider, M. H.; Tran, Y.; Tabeling, P., Benzophenone Absorption and Diffusion in Poly(dimethylsiloxane) and Its Role in Graft Photo-polymerization for Surface Modification. Langmuir 2011, 27 (3), 1232-1240.
36.Wang, Y. L.; Lai, H. H.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. L., Covalent micropatterning of poly(dimethylsiloxane) by photografting through a mask. Anal Chem 2005, 77 (23), 7539-7546.
37.Nie, Z.; Kumacheva, E., Patterning surfaces with functional polymers. Nat Mater 2008, 7 (4), 277-290.
38.Liu, S.; Zhu, T.; Hu, R.; Liu, Z., Evaporation-induced self-assembly of gold nanoparticles into a highly organized two-dimensional array. Phys Chem Chem Phys 2002, 4 (24), 6059-6062.
39.Kim, Y. P.; Oh, Y. H.; Kim, H. S., Protein kinase assay on peptide-conjugated gold nanoparticles. Biosens Bioelectron 2008, 23 (7), 980-986.
40.Huang, H.; Liu, Z.; Yang, X., Application of electrochemical impedance spectroscopy for monitoring allergen-antibody reactions using gold nanoparticle-based biomolecular immobilization method. Anal Biochem 2006, 356 (2), 208-14.
41.Jena, B. K.; Raj, C. R., Enzyme-free amperometric sensing of glucose by using gold nanoparticles. Chemistry 2006, 12 (10), 2702-8.
42.Li, Y. T.; Li, C. W.; Sung, W. C.; Chen, S. H., Heme Protein Assisted Dispersion of Gold Nanoparticle Multilayers on Chips: From Stabilization to High-Density Double-Stranded DNAs Fabricated in Situ for Protein/DNA Binding. Anal Chem 2009, 81 (10), 4076-4081.
43.Qi, Z.; Honma, I.; Ichihara, M.; Zhou, H., Layer-by-Layer Fabrication and Characterization of Gold-Nanoparticle/Myoglobin Nanocomposite Films. Adv Funct Mater 2006, 16 (3), 377-386.
44.Zhang, Q.; Xu, J. J.; Liu, Y.; Chen, H. Y., In-situ synthesis of poly(dimethylsiloxane)-gold nanoparticles composite films and its application in microfluidic systems. Lab Chip 2008, 8 (2), 352-357.
45.Wu, W. Y.; Bian, Z. P.; Wang, W.; Wang, W.; Zhu, J. J., PDMS gold nanoparticle composite film-based silver enhanced colorimetric detection of cardiac troponin I. Sens Actuators, B 2010, 147 (1), 298-303.
46.Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J., Preparation and Characterization of Au Colloid Monolayers. Anal Chem 1995, 67 (4), 735-743.
47.Wang, Y. L.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. L., Simple photografting method to chemically modify and micropattern the surface of SU-8 photoresist. Langmuir 2006, 22 (6), 2719-2725.