本研究利用高生物相容性 (biocompatibility) 之幾丁聚醣 (chitosan) 還原具有表面電漿共振效應 (surface plasmon resonance effect) 的奈米銀做為肉眼辨識的生物感測器。且在此研究中利用不同濃度的人類血清白蛋白 (human serum albumin, HSA) 與chitosan進行接合來驗證此生物感測器檢測蛋白質的可行性，並使用穿透式電子顯微鏡 (transmission electron microscopy, TEM) 與紫外/可見光吸收光譜儀 (UV-Vis) 來探討其檢測機制。此外，本實驗亦將UV-Vis之光吸收圖譜轉化為色座標 (chromatic diagram) 來定量HSA的濃度。
本研究中首先探討chitosan還原奈米銀的反應機制，接著探討接合HSA之chitosan對還原奈米銀反應的影響。隨chitosan接合不同濃度的HSA，其所還原出奈米銀之粒徑分布及數量亦不相同，而造成奈米銀在顏色上會根據HSA的接合濃度而有明顯差異。且本研究也透過TEM和UV-Vis分析，進一步了解HSA影響chitosan還原奈米銀的機制。
本研究利用chitosan還原奈米銀的反應應用於比色法生物感測器 (colorimetric biosensor)。其機制為當HSA接合於chitosan上時會阻斷chitosan上能還原奈米銀的位置，且接合越多HSA，奈米銀就無法被還原。另外，亦可以透過改變chitosan濃度來調控HSA檢測區間。
本研究中，以HSA為待測物並利用chitsan接合HSA之後會生成不同顏色之奈米銀之特性，期望可以發展出肉眼辨識、方便使用、快速檢測且低成本讓每個人都能擁有之生物感測器，得以隨時了解自己身體的健康狀況。

In this study, silver nanoparticles (Ag NPs), which were synthesized by biocompatible chitosan, showed strong surface plasmon resonance (SPR) effect and were used to develop a naked-eye biosensor. Human serum albumin (HSA) with different concentrations were conjugated with chitosan to demonstrate the feasibility of protein sensing. The mechanism of Ag NP synthesis by HSA-conjugated chitosan was studied via UV-Vis spectra and TEM analyses. Furthermore, the UV-Vis spectra were transformed to chromatic diagram to characterize the color of synthesized Ag NPs to correlate the HSA concentration.
In this work, the mechanism of Ag NP synthesis by chitosan was discussed in the first instance, followed by the study on the effects of HSA-conjugated chitosan. The result showed that HSA with different concentrations restrained the reaction of Ag NP formation to different extent and therefore led to the color changes. Furthermore, UV-Vis and transmission electron microscopy (TEM) were used to analyze the Ag NPs to demonstrate the restraining effect caused by HSA.
Moreover, the effect of HSA on restraining the chemical reduction to form Ag NPs was applied to develop colorimetric biosensor. Through synthesis of Ag NPs in chitosan, which was conjugated with HSA of different concentrations, the lighter colors of Ag NPs were observed when the conjugating concentration of HSA is higher. The severe color change occurred when the formation of Ag NPs was completely retarded. Furthermore, the detecting range is adjustable through tuning the concentration of chitosan.
In summary, this study addresses a new mechanism that the synthesis of Ag NPs by chitosan would be restrained by conjugating proteins on chitosan to provide a naked-eye, convenient, instant-detecting and cost-effective biosensor for everyone.

[1] L. C. Clark, Jr., and C. Lyons, “Electrode Systems for Continuous Monitoring in Cardiovascular Surgery.”, Ann. NY. Acad. Sci., 1965, 102(1), 29-45

[2] J. H. T. Luong, K.B. Male, and J. D. Glennon, “Biosensor Technology: Technology Push versus Market Pull”, Biotechnol. Adv.,2008, 26, 492-500

[3] “Wikipedia, http://zh.wikipedia.org/wiki/%E8%85%8E”

[4] Khetani, R. Salman, Sangeeta N. Bhatia. "Microscale culture of human liver cells for drug development." Nature biotechnology, 2007, 26 (1), 120-126.

[5] Rockey, C. Don, D. Montgomery Bissell. "Noninvasive measures of liver fibrosis." Hepatology , 2006, 43 (1), 113-120.

[6] “Wikipedia, http://zh.wikipedia.org/wiki/%E8%82%9D”

[7] I. Moser, G. Jobst, P. Svasek, M. Varahram, G. Urban, “Rapid Liver Enzyme Assay with Miniaturized Liquid Handling System Comprising Thin Film Biosensor Array”, Sensor Actuat. B, 1997, 44, 377-380.

[8] T. M. Tolaymat,A. M. El Badawy, A. Genaidy, K. G. Scheckel, T. P. Luxton, M. Suidan, “An evidence-based environmental perspective of manufactured silver nanoparticle in syntheses and applications: A systematic review and critical appraisal of peer-reviewed scientific papers”, Science of the Total Environment, 2010, 408, 999–1006.

[9] R. de Lima, A. B. Seabrac, N. Durán, “Silver nanoparticles: a brief review of cytotoxicity and genotoxicity of chemically and biogenically synthesized nanoparticles”, J. Appl. Toxicol., 2012, 32, 867–879.
[10]Cytodiagnostics,http://www.cytodiagnostics.com/silver_nanoparticle_properties.php
[11]Nanocomposix,http://nanocomposix.com/collections/silver-spheres/products/100-nm-silver-nanospheres
[12] M. Bauch, K. Toma, M. Toma, Q. Zhang, J. Dostalek, “Plasmon-Enhanced Fluorescence Biosensors: a Review”, Plasmonics, 2013, 1-19.

[13] L. Hui, Danke Xu, “Silver nanoparticles as labels for applications in bioassays”, TrAC Trends in Anal. Chem., 2014, accepted manuscript.

[14] C. Marambio-Jones, M. V. Hoek, “A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment”, J. Nanopart Res, 2010, 12, 1531-1551.

[15] Sondi, Ivan, and B. Salopek-Sondi. "Silver nanoparticles as antimicrobial agent: a case study on E. coli. as a model for Gram-negative bacteria.", Journal of colloid and interface science, 2004, 275 (1), 177-182.

[16] J-S. Kim, “Antibacterial Activity of Ag+ Ion-Containing Silver Nanoparticles Prepared Using the Alcohol Reduction Method”, J. Ind. Eng. Chem., 2007, 4, 718-722.

[17] S. Pal, Y. K. Tak, J. M. Song, “Does the Antibacterial Activity of Silver Nanoparticles Depend on the Shape of the Nanoparticle? A Study of the Gram-Negative Bacterium Escherichia coli.”, APPLIED AND ENVIRONMENTAL MICROBIOLOGY , 2007, 73, 1712-1720.

[18] C. Burda, X. Chen, R. Narayanan, M. A. El-Sayed, “Chemistry and Properties of Nanocrystals of Different Shapes”, Chem. Rev. 2005, 105, 1025-1102.

[19] P. M. Tessier, O. D. Velev, A. T. Kalambur, J. F. Rabolt, A. M. Lenhoff, E. W. Kaler, “Assembly of Gold Nanostructured Films Templatedby Colloidal Crystals and Use in Surface-Enhanced Raman Spectroscopy”, J. Am. Chem. Soc. 2000, 122, 9554-9555.

[20] N. Misraa, V. Kumara, L. Bordeb, L. Varshneya, “Localized surface plasmon resonance-optical sensors based on radiolytically synthesized silver nanoparticles for estimation of uric acid”, Sensors and Actuators B, 2013, 178, 371– 378.

[21] J. Zhang, Yi Fu, D. Liang, R. Y. Zhao, J. R. Lakowicz, “Fluorescent Avidin-Bound Silver Particle: A Strategy for Single Target Molecule Detection on a Cell Membrane”, Anal. Chem., 2009, 81, 883–889.

[22] K. Ogawa, T. Yui, M. Miya, “Dependence on the Preparation Procedure of the Polymorphism and Crystallinity of Chitosan Membranes”, Biosci.Biotech .Biochem, 1992, 56 (6), 858-862.

[23] M. Rinaudo, “Chitin and chitosan: Properties and applications”, Prog. Polym. Sci., 2006, 31, 603–632

[24] Venkatesham, Maragoni, et al. “A novel green one-step synthesis of silver nanoparticles using chitosan: catalytic activity and antimicrobial studies.”, Applied Nanoscience , 2014, 4 (1), 113-119.
[25] Z. Y.Huang, G. Mills, B. Hajek., “Spontaneous formation of silver particles in basic 2-propanol.”,The Journal of Physical Chemistry, 1993, 97 (44), 11542-11550.

[26] Z-J. Jiang, C-Y. Liu, and Y. Liu., “Formation of silver nanoparticles in an acid-catalyzed silica colloidal solution.”, Applied surface science, 2004, 233 (1), 135-140.

[27] Wei, Dongwei, et al. “Chitosan as an active support for assembly of metal nanoparticles and application of the resultant bioconjugates in catalysis.”, Carbohydrate research, 2010, 345 (1), 74-81.

[28] K. L. Kelly, E. Coronado, L. L. Zhao, G. C. Schatz, “The Optical Properties of Metal Nanoparticles:  The Influence of Size, Shape, and Dielectric Environment”, J. Phys. Chem. B, 2003, 107, 668-677.

[29] Lim, K. Rae, et al. "Gold glyconanoparticle-based colorimetric bioassay for the determination of glucose in human serum." Microchemical Journal, 2013, 106, 154-159.

[30] P. Angenendt, “Progress in Protein and Antibody Microarray Technology”, Drug Discov. Today, 2005, 10, 503-511.

[31] W. P. Blackstock, M. P. Weir, “Proteomics: Quantitative and Physical Mapping of Cellular Proteins”, Trends Biotechnol., 1999, 17, 121-127.

[32] S. Liu, Z. Zhang, M. Han., "Gram-scale synthesis and bio-functionalization of silica-coated silver nanoparticles for fast colorimetric DNA detection." Analytical chemistry, 2005, 77 (8), 2595-2600.
[33] T. M. Chinowsky, J. G. Quinn, D.U. Bartholomew, R. Kaiser, J. L. Elkind, “Performance of the Spectra 2000 integrated surface plasmon resonance affinity sensor.”, Sens. Actuators, B, 2003, 91, 266-274.

[34] B. N. Feltis, B. A. Sexton, F. L. Glem, M. J. Best, M. Wilkins, T. J. Davis, “A hand-hold surface plasmon resonance biosensor for the detection of ricin and other biological agents.”, Biosens. Bioelectron., 2008, 23, 1131-1136.

[35] S. Heng, et al. "A symmetrical optical waveguide based surface plasmon resonance biosensing system.", Sensors and Actuators B: Chemical, 2013, 185, 91-96.

[36] “Wikipedia, http://en.wikipedia.org/wiki/Human_serum_albumin”

[37] S. Curry, H. Mandelkow, P. Brick, N. Franks, “Crystal Structure of Human Serum Albumin Complexed with Fatty Acid Reveals an Asymmetric Distribution of Binding Sites”, Nat. Struct. Biol., 1998, 5, 827-835.

[38] C. E. Ha, J. S. Ha, A. G. Theriault, N. V. Bhagavan, “Effects of Statins on the Secretion of Human Serum Albumin in Cultured HepG2 Cells”, J. Biomed. Sci., 2009, 16, 1-10.