在亞洲地區流行的腸病毒71型(Enterovirus 71, EV71)，是一種生長於腸道的RNA病毒，是一種手足口症(hand-foot-mouth disease, HFMD)，這類感染病出現於神經系統的主要病徵有：呼吸困難、抽蓄及腦膜炎等症狀。近幾年的研究也證實P-選擇素糖蛋白配體-1(p-selectin glycoprotein ligand-1, PSGL-1)是其載體，藉由接附於其上使人體發病。表面增顯拉曼散射光譜偵測生物分子已被證實其可行性，檢測所帶有胜肽(peptide)官能基之文獻指出於檢測基板材料之選擇，利用金奈米多孔洞結構可快速辨識出表面活性之蛋白質結構，因此選擇利用製備金奈米孔洞結構作為表面增顯拉曼散射基板，應用拉曼光譜於快速辨識PSGL-1以利往後進行更深入的研究致病源與其載體之間所進行的反應。
本研究使用物理氣象沉積儀(PVD)沉積金銀合金於預先以電子束蒸鍍機沉積金膜於其上的矽基板，以硝酸進行不同時間的蝕刻，而得不同孔徑大小的奈米孔洞結構，討論電磁效應傳遞之影響。以羅丹明6G(Rhodamine 6g, R6G)作為待測分子評估表面增顯拉曼散射基板。結果顯示，當蝕刻時間為120秒，金奈米多孔洞尺寸分布約50-60 nm，雷射波長為633 nm時有最佳增顯效果，其偵測極限可達10-12 M而增顯因子為4.67*108。
應用表面增顯拉曼散射光譜於金奈米多孔洞結構做為檢測PSGL-1與硫酸化EV-71抗體(Anti-sulfotyrosine EV-71 antibody)。結果顯示，於拉曼位移1000-1600 cm-1區間，可辨識其特定分子震動結構，如羰基(Carbonyl, C=O, 1584 cm-1 for PSGL-1)、磺胺基(Sulfonamide, -SO3, 1072-1268 cm-1)。因此以金奈米多孔洞結構作為表面增顯拉曼散射基板應用於生物分子的偵測，能精準地確認其表面活性蛋白質結構，達到快速篩選的目的。

Surface-enhanced Raman scattering (SERS) substrates with hot spots generating from large-scale massive nanogaps between gold nanoporous and the gold film. In bio-sensing detection such as protein, virus, and bacteria, Raman signals can’t get a very strong characteristic peak for recognition. Therefore, the nanoporous structure is prepared as SERS substrate. The three-dimensional structure can generate high density plasmonic hotspots excited very strong electromagnetic field enhancement. The nanoporous gold SERS-active substrates are used to detect the Raman signals of P-selectin glycoprotein ligand-1(PSGL-1) and anti-sulfotyrosine EV-71 antibody. Raman spectral of rhodamine 6g shows as average pore size 50-60 nm high enhancement factor (4.67x108 ) and detect limit(10-12 M) can be achieved. By self-assembled process, the nanoporous gold substrate identify characteristic peak of biomolecules separately. This research provides a simple method to fabricate nanoporous gold structure and possibility to in vitro detect of virus.

[1]Lyon, L. A. et al. Raman Spectroscopy. Analytical Chemistry 70, 341-362, doi:10.1021/a1980021p (1998).
[2]Schuster, K. C., Urlaub, E. & Gapes, J. R. Single-cell analysis of bacteria by Raman microscopy: spectral information on the chemical composition of cells and on the heterogeneity in a culture. Journal of Microbiological Methods 42, 29-38, doi:10.1016/s0167-7012(00)00169-x (2000).
[3]Campion, A. & Kambhampati, P. Surface-enhanced Raman scattering. Chemical Society Reviews 27, 241, doi:10.1039/a827241z (1998).
[4]Nie, S. Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering. Science 275, 1102-1106, doi:10.1126/science.275.5303.1102 (1997).
[5]Ferreira, J. et al. Attomolar protein detection using in-hole surface plasmon resonance. J Am Chem Soc 131, 436-437, doi:10.1021/ja807704v (2009).
[6]Kneipp, K. et al. Surface-Enhanced Raman Spectroscopy in Single Living Cells Using Gold Nanoparticles. Applied Spectroscopy 56, 150-154, doi:10.1366/0003702021954557 (2002).
[7]Zeiri, L., Bronk, B. V., Shabtai, Y., Czégé, J. & Efrima, S. Silver metal induced surface enhanced Raman of bacteria. Colloids and Surfaces A: Physicochemical and Engineering Aspects 208, 357-362, doi:10.1016/s0927-7757(02)00162-0 (2002).
[8]Lin, M. et al. Detection of melamine in gluten, chicken feed, and processed foods using surface enhanced Raman spectroscopy and HPLC. J Food Sci 73, T129-134, doi:10.1111/j.1750-3841.2008.00901.x (2008).
[9]Shanmukh, S. et al. Rapid and sensitive detection of respiratory virus molecular signatures using a silver nanorod array SERS substrate. Nano Lett 6, 2630-2636, doi:10.1021/nl061666f (2006).
[10]Fang, C. et al. DNA detection using nanostructured SERS substrates with Rhodamine B as Raman label. Biosens Bioelectron 24, 216-221, doi:10.1016/j.bios.2008.03.032 (2008).
[11]Tripp, R. A., Dluhy, R. A. & Zhao, Y. Novel nanostructures for SERS biosensing. Nano Today 3, 31-37, doi:10.1016/s1748-0132(08)70042-2 (2008).
[12]Sepúlveda, B., Angelomé, P. C., Lechuga, L. M. & Liz-Marzán, L. M. LSPR-based nanobiosensors. Nano Today 4, 244-251, doi:10.1016/j.nantod.2009.04.001 (2009).
[13]Brown, R. J. C. & Milton, M. J. T. Nanostructures and nanostructured substrates for surface-enhanced Raman scattering (SERS). Journal of Raman Spectroscopy 39, 1313-1326, doi:10.1002/jrs.2030 (2008).
[14]Hering, K. et al. SERS: a versatile tool in chemical and biochemical diagnostics. Anal Bioanal Chem 390, 113-124, doi:10.1007/s00216-007-1667-3 (2008).
[15]Stewart, M. E. et al. Nanostructured plasmonic sensors. Chem Rev 108, 494-521, doi:10.1021/cr068126n (2008).
[16]Albrecht, M. G. & Creighton, J. A. ChemInform Abstract: ANOMALOUSLY INTENSE RAMAN SPECTRA OF PYRIDINE AT A SILVER ELECTRODE. Chemischer Informationsdienst 8, no-no, doi:10.1002/chin.197744044 (1977).
[17]Zhang, J., Zhang, L. & Xu, W. Surface plasmon polaritons: physics and applications. Journal of Physics D: Applied Physics 45, 113001, doi:10.1088/0022-3727/45/11/113001 (2012).
[18]Liu, Y. C. & McCreery, R. L. Raman spectroscopic determination of the structure and orientation of organic monolayers chemisorbed on carbon electrode surfaces. Anal Chem 69, 2091-2097, doi:10.1021/ac961305s (1997).
[19]Xia, L. et al. Visualized method of chemical enhancement mechanism on SERS and TERS. Journal of Raman Spectroscopy 45, 533-540, doi:10.1002/jrs.4504 (2014).
[20]Campion, A., Ivanecky, J. E., Child, C. M. & Foster, M. On the Mechanism of Chemical Enhancement in Surface-Enhanced Raman Scattering. Journal of the American Chemical Society 117, 11807-11808, doi:10.1021/ja00152a024 (1995).
[21]Feng, J. J. et al. Novel Au-Ag hybrid device for electrochemical SE(R)R spectroscopy in a wide potential and spectral range. Nano Lett 9, 298-303, doi:10.1021/nl802934u (2009).
[22]Szunerits, S., Spadavecchia, J. & Boukherroub, R. Surface plasmon resonance: signal amplification using colloidal gold nanoparticles for enhanced sensitivity. Reviews in Analytical Chemistry 33, doi:10.1515/revac-2014-0011 (2014).
[23]Ye, Q., Fang, J. & Sun, L. Surface-Enhanced Raman Scattering from Functionalized Self-Assembled Monolayers. 2. Distance Dependence of Enhanced Raman Scattering from an Azobenzene Terminal Group. The Journal of Physical Chemistry B 101, 8221-8224, doi:10.1021/jp970869c (1997).
[24]Chen, S. et al. Electromagnetic Enhancement in Shell-Isolated Nanoparticle-Enhanced Raman Scattering from Gold Flat Surfaces. The Journal of Physical Chemistry C 119, 5246-5251, doi:10.1021/acs.jpcc.5b01254 (2015).
[25]Connor, E. E., Mwamuka, J., Gole, A., Murphy, C. J. & Wyatt, M. D. Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity. Small 1, 325-327, doi:10.1002/smll.200400093 (2005).
[26]Liu, H. et al. Single molecule detection from a large-scale SERS-active Au(7)(9)Ag(2)(1) substrate. Sci Rep 1, 112, doi:10.1038/srep00112 (2011).
[27]Reinhard, B. r. M., Champion, P. M. & Ziegler, L. D. Engineered SERS Substrates with Multiscale Signal Enhancement: Nanoparticle Cluster Arrays. 952-953, doi:10.1063/1.3482909 (2010).
[28]Oli, M. Aptamer conjugated gold nanorods for targeted nanothermal radiation of Glioblastoma cancer cells (A novel selective targeted approach to cancer treatment). Young Scientists Journal 2, 18, doi:10.4103/0974-6102.68740 (2009).
[29]Daggumati, P., Matharu, Z. & Seker, E. Effect of nanoporous gold thin film morphology on electrochemical DNA sensing. Anal Chem 87, 8149-8156, doi:10.1021/acs.analchem.5b00846 (2015).
[30]Murphy, C. J. et al. ChemInform Abstract: Gold Nanoparticles in Biology: Beyond Toxicity to Cellular Imaging. ChemInform 40, doi:10.1002/chin.200916266 (2009).
[31]Levard, C., Hotze, E. M., Lowry, G. V. & Brown, G. E., Jr. Environmental transformations of silver nanoparticles: impact on stability and toxicity. Environ Sci Technol 46, 6900-6914, doi:10.1021/es2037405 (2012).
[32]Dondapati, S. K. et al. Label-free biosensing based on single gold nanostars as plasmonic transducers. ACS Nano 4, 6318-6322, doi:10.1021/nn100760f (2010).
[33]Yang, L. et al. Engineering Nanoparticle Cluster Arrays for Bacterial Biosensing: The Role of the Building Block in Multiscale SERS Substrates. Advanced Functional Materials 20, 2619-2628, doi:10.1002/adfm.201000630 (2010).
[34]Lin, Y. Y., Liao, J. D., Ju, Y. H., Chang, C. W. & Shiau, A. L. Focused ion beam-fabricated Au micro/nanostructures used as a surface enhanced Raman scattering-active substrate for trace detection of molecules and influenza virus. Nanotechnology 22, 185308, doi:10.1088/0957-4484/22/18/185308 (2011).
[35]Dorofeeva, T. S. & Seker, E. In situ electrical modulation and monitoring of nanoporous gold morphology. Nanoscale 8, 19551-19556, doi:10.1039/c6nr07237b (2016).
[36]Zhao, F. et al. Monolithic NPG nanoparticles with large surface area, tunable plasmonics, and high-density internal hot-spots. Nanoscale 6, 8199-8207, doi:10.1039/c4nr01645a (2014).
[37]Arnob, M. M. et al. Laser rapid thermal annealing enables tunable plasmonics in nanoporous gold nanoparticles. Nanoscale 6, 12470-12475, doi:10.1039/c4nr03672g (2014).
[38]Zeng, J. et al. Morphological control and plasmonic tuning of nanoporous gold disks by surface modifications. J. Mater. Chem. C 3, 247-252, doi:10.1039/c4tc02328e (2015).
[39]Qi, J. et al. Label-free, in situ SERS monitoring of individual DNA hybridization in microfluidics. Nanoscale 6, 8521-8526, doi:10.1039/c4nr01951b (2014).
[40]Shih, W.-C., Bechtel, K. L. & Feld, M. S. Noninvasive Glucose Sensing with Raman Spectroscopy. 391-419, doi:10.1002/9780470567319.ch14 (2009).
[41]Sudheendran, N. et al. Line-scan Raman microscopy complements optical coherence tomography for tumor boundary detection. Laser Physics Letters 11, 105602, doi:10.1088/1612-2011/11/10/105602 (2014).
[42]Santos, G. M., Zhao, F., Zeng, J., Li, M. & Shih, W. C. Label-free, zeptomole cancer biomarker detection by surface-enhanced fluorescence on nanoporous gold disk plasmonic nanoparticles. J Biophotonics 8, 855-863, doi:10.1002/jbio.201400134 (2015).
[43]Li, M. et al. Microfluidic surface-enhanced Raman scattering sensor with monolithically integrated nanoporous gold disk arrays for rapid and label-free biomolecular detection. J Biomed Opt 19, 111611, doi:10.1117/1.JBO.19.11.111611 (2014).
[44]Jia, F., Yu, C., Deng, K. & Zhang, L. Nanoporous Metal (Cu, Ag, Au) Films with High Surface Area:  General Fabrication and Preliminary Electrochemical Performance. The Journal of Physical Chemistry C 111, 8424-8431, doi:10.1021/jp071815y (2007).
[45]Deng, Y., Huang, W., Chen, X. & Li, Z. Facile fabrication of nanoporous gold film electrodes. Electrochemistry Communications 10, 810-813, doi:10.1016/j.elecom.2008.03.003 (2008).
[46]Lu, Y., Wang, Q., Sun, J. & Shen, J. Selective dissolution of the silver component in colloidal Au and Ag multilayers: a facile way to prepare nanoporous gold film materials. Langmuir 21, 5179-5184, doi:10.1021/la0500878 (2005).
[47]Zhang, R., Hummelgard, M. & Olin, H. Large area porous gold films deposited by evaporation-induced colloidal crystal growth. J Colloid Interface Sci 340, 58-61, doi:10.1016/j.jcis.2009.08.006 (2009).
[48]Wi, J. S., Tominaka, S., Uosaki, K. & Nagao, T. Porous gold nanodisks with multiple internal hot spots. Phys Chem Chem Phys 14, 9131-9136, doi:10.1039/c2cp40578d (2012).
[49]Schneider-Hohendorf, T. et al. VLA-4 blockade promotes differential routes into human CNS involving PSGL-1 rolling of T cells and MCAM-adhesion of TH17 cells. J Exp Med 211, 1833-1846, doi:10.1084/jem.20140540 (2014).
[50]Tinoco, R. et al. PSGL-1 Is an Immune Checkpoint Regulator that Promotes T Cell Exhaustion. Immunity 44, 1190-1203, doi:10.1016/j.immuni.2016.04.015 (2016).
[51]Nishimura, Y. et al. Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71. Nat Med 15, 794-797, doi:10.1038/nm.1961 (2009).
[52]Larkin, P. Basic Principles. 7-25, doi:10.1016/b978-0-12-386984-5.10002-3 (2011).
[53]Fleischmann, M., Hendra, P. J. & McQuillan, A. J. Raman spectra of pyridine adsorbed at a silver electrode. Chemical Physics Letters 26, 163-166, doi:10.1016/0009-2614(74)85388-1 (1974).
[54]Jeanmaire, D. Surface Raman spectroelectrochemistry Part I. Heterocyclic, aromatic, and aliphatic amines adsorbed on the anodized silver electrode. Journal of Electroanalytical Chemistry 84, 1-20, doi:10.1016/s0022-0728(77)80224-6 (1977).
[55]Wang, D. S. & Kerker, M. Enhanced Raman scattering by molecules adsorbed at the surface of colloidal spheroids. Physical Review B 24, 1777-1790, doi:10.1103/PhysRevB.24.1777 (1981).
[56]Kerker, M., Wang, D. S. & Chew, H. Surface enhanced Raman scattering (SERS) by molecules adsorbed at spherical particles. Appl Opt 19, 3373-3388, doi:10.1364/AO.19.003373 (1980).
[57]Xu, H., Aizpurua, J., Käll, M. & Apell, P. Electromagnetic contributions to single-molecule sensitivity in surface-enhanced Raman scattering. Physical Review E 62, 4318-4324, doi:10.1103/PhysRevE.62.4318 (2000).
[58]Gao, P. et al. Defects evolution in nanoporous Au(Pt) during dealloying. Scripta Materialia 113, 68-70, doi:10.1016/j.scriptamat.2015.10.006 (2016).
[59]Lin, C.-H. et al. Surface composition and interactions of mobile charges with immobilized molecules on polycrystalline silicon nanowires. Sensors and Actuators B: Chemical 211, 7-16, doi:10.1016/j.snb.2015.01.052 (2015).
[60]Spanier, E. J. & MacDiarmid, A. G. The Conversion of Silane to Higher Silanes in a Silent Electric Discharge. Inorganic Chemistry 1, 432-433, doi:10.1021/ic50002a051 (1962).