免疫生物感測晶片(Immunosensor chip)需致力改善的部分有二：
一為微晶片儲存期的增長，二為靈敏度的增加。為了進行短鏈胜肽親
合配位子的設計，我們首先在抗體Fc區域的底部尋找較疏水的區域，
然後分析疏水區域的電荷分佈。同時利用疏水作用力以及靜電吸引力
去設計胜肽配位子，預期與Fc 區域底部產生較強的親和力。本研究
使用的抗原為人類前列腺特異抗原(Prostate specific antigen，PSA)並
用兔子免疫球蛋白IgG( Rabbit IgG ) 為目標抗體。利用上述方式設計
出可能的的胜肽配體，再利用分子動態模擬(Molecular dynamic
simulation，MD)，尋找出一條與Rabbit IgG具有高親和力的胜肽配體。
接著將胜肽配體接枝於金片表面上，使用表面電漿共振儀測(Surface
plasmon resonance，SPR)量測兔子免疫球蛋白IgG、PSA 以及兔子免
疫球蛋IgG 的二抗(Secondary antibody，2nd antibody)在金片表面的吸
附量，藉此得到兔子IgG 的抗原辨識效率與位向因子。我們也將以
Mouse IgG2a 拿來做測試，看所設計胜肽配位子是否對於Rabbit IgG
具有專一性。結果發現一短鏈胜肽，對於兔子IgG 具有高親和性與專
一性，對PSA 的抗原辨識效果有不錯的成效。因此依據我們提出之
策略所設計出的胜肽配體，確實有效地使抗體位向性固定化於表面。

There are two drawbacks of Immunosensor Chip required to be
improved, one is the shelf life of microchip and the other is sensitivity
enhancement. For the peptide ligand design, we searched for the
hydrophobic patch around the bottom Fc region of antibody and analyzed
the charge distribution among the hydrophobic patch. On the other hand,
we designed another peptide ligands based on the electrostatic interaction
and hydrophobicity which bind strongly to the bottom Fc region of
antibody, as well. In this study, we focused on the Rabbit IgG as the
antibody and the prostate specific antigen (PSA) as the antigen. To find a
peptide ligand with high affinity to Rabbit IgG, we designed all the
possible peptide sequences according to the previous methods and
assisted with molecular dynamics simulation (MD). After immobilizing
peptide ligands onto gold chip surface, we measured the binding
capacities of Rabbit IgG, PSA, and the secondary antibody (2nd antibody)
on gold chip by surface plasmon simulation (SPR). Also, we could obtain
the recognition efficiency of Rabbit IgG to antigen and the orientation
factor by SPR measurement. Besides, on specificity test, we performed
the designed peptide ligands on Mouse IgG2a.The result revealed that a
peptide ligand exhibits high affinity and specificity to Rabbit IgG and has
well effect on the recognition efficiency to PSA antigen. Therefore,
according to the strategy we proposing on peptide ligand design, we can
definitely immobilize the antibody orientedly onto the sensor chip and
further enhance the sensitivity of antibody to antigen detection.

1. Sullivan F. http://www.frost.com/prod/servlet/frost-home.pag.
2. J. M. Nelson EGG. ADSORPTION OF INVERTASE. J Am Chem Soc. 1916;38(5):1109-15.
3. Dan H. Campbell EL, and Leonard S. Lerman. Immunologic Adsorbents. Proc Natl Acad Sci USA. 1951;37(9):575-8.
4. N. Grubhofer LS. Modifizierte Ionenaustauscher als spezifische Adsorbentien. ERSTES OKTOBER. 1953;40(19):508.
5. Taylor. RF. Protein immobilization fundamental and application. Marcel Dekker, INC Hong Kong. 1991:3-4,15-29,105-33.
6. Huaying Zhao IIG, Gregory L. Fu, Peter Schuck. A comparison of binding surfaces for SPR biosensing using an antibody–antigen system and affinity distribution analysis. Biophysical Methods for the Study of Protein Interactions. 2013;559(3):328-35.
7. Winzor DJ. An analytical solution to the characterization of antigen–antibody interactions by kinetic exclusion assay. Analytical Biochemistry. 2013;438(1):42-6.
8. Lu B, M.R. Smyth, and R. Okennedy. Oriented immobilization of antibodies and its applications in immunoassays and immunosensors. Analyst. 1996;121(3):R29-R32.
9. Smith CL, J. S.Milea, Nguyen, G. H. Immobilization of nucleic acids using biotin-strept(avidin) systems. Immobilization of DNA on Chips Ii. 2005;261:63-90.
10. Yongwon Jung JYJaBHC. Recent advances in immobilization methods of antibodies on solid supports. Analyst. 2008;133:697-701.
11. Bilitewski U. Protein-sensing assay formats and devices. Analytica Chimica Acta. 2006;568:232-47.
12. F. Rusmini ZYZ, J. Feijen. Protein immobilization strategies for protein biochips. Biomacromolecules. 2007;8:1775-89.
13. Huihui Wanga HO, Hideaki Endob, Mitsuru Izumia. Effects of self-assembled monolayers on amperometric glucose biosensors based on an organic–inorganic hybrid system. Sensors and Actuators B: Chemical. 2012;168(20):249-55.
14. S.P. Silvia Ferretti DAR, Kim E. Sapsford. Self-assembled monolayers: a versatile tool for the formulation of bio-surfaces. trends in analytical chemistry 2000;19:530-40.
15. Busch RTK. Single molecule research on surfaces: from analytics to construction and back. Reviews in Molecular Biotechnology 2001;82:3-24.
16. Hang T. Ta KP, Christoph E. Hagemeyer. Enzymatic Antibody Tagging: Toward a Universal Biocompatible Targeting Tool. Trends in Cardiovascular Medicine. 2012;22(4):105-11.
17. N.K. Jo Tominagaa SD, Hirofumi Ichinoseb, Masahiro Gotoa. An enzymatic strategy for site-specific immobilization of functional proteins using microbial transglutaminase. Enzyme and Microbial Technology. 2004;35:613-8.
18. A. Kausaite-Minkstimiene AR, J. Kirlyte and A. Ramanavicius. Comparative Study of Random and Oriented Antibody Immobilization Techniques on the Binding Capacity of Immunosensor. Anal Chem. 2010;82(15):6401-8.
19. A.A. Karyakin GVP, M.Y. Rubtsova, A.M. Egorov. Oriented immobilization of antibodies onto the gold surfaces via their native thiol groups. Analytical Chemistry. 2000;72:3805-11.
20. Kronvall LBaG. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. The Journal of Immunology. 1984;133(2):969-74.
21. AM Gronenborn DF, NZ Essig, A Achari, M Whitlow, PT Wingfield, GM Clore. A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science. 1991;253(5020):657-61.
22. B.G. Mathias Uhlen BN, Gatenbeck, LennarPt hilipson, and, M. Lindberg. Complete Sequence of the Staphylococcal Gene EncodinPg rotein A. THE JOURNAOFL BIOLOGICAL CHEMISTRY 1984;259:1695-702.
23. Gagnon P. Technology trends in antibody purification. Journal of Chromatography A. 2012;1221:57-70.
24. C.D. Kang SWL, T.H. Park, S.J. Sim. Performance enhancement of real-time detection of protozoan parasite, Cryptosporidium oocyst by a modified surface plasmon resonance (SPR) biosensor. Enzyme and Microbial Technology. 2006;39:387-90.
25. N.-P. Huang JV, S.M. De Paul, M. Textor, N.D. Spencer. Biotin-Derivatized Poly(L-lysine)-g-poly(ethylene glycol): A Novel Polymeric Interface for Bioaffinity Sensing. Langmuir. 2002;18(1):220-30.
26. Kimihiro Susumu ILM, James B. Delehanty , Kelly Boeneman and Hedi Mattoussi. Modification of Poly(ethylene glycol)-Capped Quantum Dots with Nickel Nitrilotriacetic Acid and Self-Assembly with Histidine-Tagged Proteins. J Phys Chem C. 2010;114(32):13526-31.
27. M.C. Cheeksa NK, A. Sorrella, D. Darlingb, F. Farzanehb, N.K.H. Slatera. Immobilized metal affinity chromatography of histidine-tagged lentiviral vectors using monolithic adsorbents. Journal of Chromatography A. 2009;1216(13):2705-11.
28. Wen-Pin Hua L-YH, Tai-Chih Kuoc, Wei-Wen Hub, Yung Changd, Chien-Sheng Chene, Hong-Cheng Chenb, Wen-Yih Chenb. Optimization of DNA-directed immobilization on mixed oligo(ethylene glycol) monolayers for immunodetection. Analytical Biochemistry. 2012;423(1):26-35.
29. H. Yang PVG, R.G. Carbonell. Hexamer peptide affinity resins that bind the Fc region of human immunoglobulin G. J Pept Res. 2005;66:120-37.
30. G. Fassina AV, M.R. Odierna, M. Ruvo, G. Cassini. Protein a mimetic peptide ligand for affinity purification of antibodies. Journal of Molecular Recognition. 1996;9:564-9.
31. Y. Jung HJK, J.M. Lee, S.O. Jung, W.S. Yun, S.J. Chung, B.H. Chung. Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide. Anal Biochem. 2008;374:99-105.
32. B.M. Inbal Halperin H, and Ruth Nussinov. Principles of Docking: An Overviewof Search Algorithms and a Guide to Scoring Functions. PROTEINS: Structure, Function, and Genetics. 2002;47:409-43.
33. 林榮信. 計算統計物理與藥物設計. 物理雙月刊. 2007;廿九卷:920-8.
34. Goldblum BGaA. High quality binding modes in docking ligands to proteins. Proteins. 2008;71:1373-86.
35. Alder BJaTEW. Studies in Molecular Dynamics. I. General Method. The Journal of Chemical Physics. 1959;31(2):459-66.
36. Stillinger FHaAR. Improved simulation of liquid water by molecular dynamics. The Journal of Chemical Physics. 1974;60(4):1545-57.
37. McCammon JA, B.R. Gelin, and M. Karplus. Dynamics of folded proteins. Nature. 1977;267(5612):585-90.
38. Brooks BR, et al. CHARMM: The Biomolecular Simulation Program. Journal of Computational Chemistry. 2009;30(10):1545-614.
39. England P, F. Bregegere, and H. Bedouelle. Energetic and kinetic contributions of contact residues of antibody D1.3 in the interaction with lysozyme. Biochemistry. 1997;36(1).
40. Wu ZN, et al. LIGAND-BINDING ANALYSIS OF SOLUBLE INTERLEUKIN-2 RECEPTOR COMPLEXES BY SURFACE-PLASMON RESONANCE. Journal of Biological Chemistry. 1995;270(27):16045-51.
41. Boozer C, et al. DNA-directed protein immobilization for simultaneous detection of multiple analytes by surface plasmon resonance biosensor. Analytical Chemistry. 2006;78(5):1515-9.
42. Ladd J, et al. DNA-directed protein immobilization on mixed self-assembled monolayers via a Streptavidin bridge. Langmuir. 2004;20(19):8090-5.
43. Ansorena P, et al. Comparative analysis of QCM and SPR techniques for the optimization of immobilization sequences. Sensors and Actuators B-Chemical. 2011;155(2):667-72.
44. Otto A. Excitation of nonradiative surface plasma waves in silver by the method of frustrated total reflection. Zeitschrift für Physik. 1968;216(4):398-410.
45. Kretschmann EaHR. Radiative decay of non radiative surface plasmons excited by light. Radiative Decay of Nonradiative Surface Plasmons Excited by Light. 1968:2135-6.
46. Haes AJ, et al. Detection of a biomarker for Alzheimer's disease from synthetic and clinical samples using a nanoscale optical biosensor. Journal of the American Chemical Society. 2005;127(7):2264-71.
47. Inamori K, et al. Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule. Analytical Chemistry. 2005;77(13):3979-85.
48. Gobi KV, H. Iwasaka, and N. Miura. Self-assembled PEG monolayer based SPR immunosensor for label-free detection of insulin. Biosens Bioelectron. 2007;22(7):1382-9.
49. Cimitan S, et al. Early absorption and distribution analysis of antitumor and anti-AIDS drugs: Lipid membrane and plasma protein interactions. Journal of Medicinal Chemistry. 2005;48(10):3536-46.
50. Reddy TRK, et al. Library design, synthesis, and screening: Pyridine dicarbonitriles as potential prion disease therapeutics. Journal of Medicinal Chemistry. 2006;49(2):607-15.
51. Dillon PP, et al. Development and use of antibodies in surface plasmon resonance-based immunosensors for environmental monitoring. International Journal of Environmental Analytical Chemistry. 2003;83(7-8):525-43.
52. Gobi KV, et al. Highly sensitive regenerable immunosensor for label-free detection of 2,4-dichlorophenoxyacetic acid at ppb levels by using surface plasmon resonance imaging. Sensors and Actuators B-Chemical. 2005;111:562-71.
53. Haasnoot W, et al. Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder. Journal of Agricultural and Food Chemistry. 2001;49(11):5201-6.
54. Haasnoot WaRV. A direct (Non-Competitive) immunoassay for gentamicin residues with an optical biosensor. Food and Agricultural Immunology. 2001;13(2):131-4.
55. Mader C, et al. Interaction of the crystalline bacterial cell surface layer protein SbsB and the secondary cell wall polymer of Geobacillus stearothermophilus PV72 assessed by real-time surface plasmon resonance biosensor technology. Journal of Bacteriology. 2004;186(6):1758-68.
56. Oh BK, et al. Surface plasmon resonance immunosensor for the detection of Yersinia enterocolitica. Colloids and Surfaces a-Physicochemical and Engineering Aspects. 2005;257(58):369-74.
57. Dhesingh Ravi SaMN. Trends in interfacial design for surface plasmon resonance based immunoassays. Journal of Physics D: Applied Physics. 2007;40(23):7187.