Characterization of trypsin immobilized on the functionable alkylthiolate self-assembled monolayers: A preliminary application for trypsin digestion chip on protein identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

被引:10
作者
Tyan, YC
Liao, JD
Jong, SB
Liao, PC
Yang, MH
Chang, YW
Klauser, R
Himmelhaus, M
Grunze, M
机构
[1] Natl Cheng Kung Univ, Dept Environm & Occupat Hlth, Tainan 701, Taiwan
[2] Natl Cheng Kung Univ, Dept Mat Sci & Engn, Tainan 701, Taiwan
[3] Kaohsiung Med Univ, Dept Nucl Med, Kaohsiung 807, Taiwan
[4] Texas Christian Univ, Dept Chem, Ft Worth, TX 76129 USA
[5] Natl Synchrotron Radiat Res Ctr, Hsinchu 300, Taiwan
[6] Univ Heidelberg, Lehrstuhl Angew Phys Chem, D-69120 Heidelberg, Germany
关键词
D O I
10.1007/s10856-005-5987-6
中图分类号
R318 [生物医学工程];
学科分类号
0831 ;
摘要
Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition and other interfacial phenomena. Recently the bonding of enzyme to SAMs of alkanethiols onto Au electrode surfaces was exploited to produce a bio-sensing system. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble N-ethyl-N'-(3-dimetliyiaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agent. The thickness of SAMs was determined by optical ellipsometer; contact angles of the modified Au surfaces were measured in air using a goniometer. The Second Harmony Generation data displays the last few percents of the alkylthiol molecules adsorbed and produced the complete monolayer by inducing the transition from a high number of gauche defects to an all-trans conformation. Using X-ray Photoelectron Spectroscopy (XPS) and Fourier-Transformed Infrared Reflection-Absorption and Attenuated Total Reflection Spectroscopes (FTIR-RAS and ATR), we examined the chemical structures of samples with different treatments. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we demonstrated the digestion of bovine serum albumin (BSA) on the trypsin-immobilized SAMs surface. Experimental results have revealed that the XPS Cls core levels at 286.3 and 286.5 eV (Amine bond), 288.1 eV (Amide bond) and 289.3 eV (Carboxylic acid) illustrate the immobilization of trypsin. These data were also in good agreement with FTIR-ATR spectra for the peaks valued at 1659.4 cm(-1) (Amide 1) and 1546.6 cm(-1) (Amide II). Using MALDI-TOF MS observations, analytical results have demonstrated the BSA digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, BSA was digested on the trypsin-immobilized SAMs surface, which shows the enzyme digestion ability of the immobilized trypsin. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials. (C) 2005 Springer Science + Business Media, Inc.
引用
收藏
页码:135 / 142
页数:8
相关论文
共 38 条
[1]   FORMATION OF MONOLAYER FILMS BY THE SPONTANEOUS ASSEMBLY OF ORGANIC THIOLS FROM SOLUTION ONTO GOLD [J].
BAIN, CD ;
TROUGHTON, EB ;
TAO, YT ;
EVALL, J ;
WHITESIDES, GM ;
NUZZO, RG .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1989, 111 (01) :321-335
[2]  
Beamson G., 1992, ADV MATER, DOI DOI 10.1002/ADMA.19930051035
[3]   GROWTH OF SELF-ASSEMBLED N-ALKYLTRICHLOROSILANE FILMS ON SI(100) INVESTIGATED BY ATOMIC-FORCE MICROSCOPY [J].
BIERBAUM, K ;
GRUNZE, M ;
BASKI, AA ;
CHI, LF ;
SCHREPP, W ;
FUCHS, H .
LANGMUIR, 1995, 11 (06) :2143-2150
[4]  
BUERK DG, 1993, BIOSENSOR THEORY APP
[5]   CHEMICAL FUNCTIONALITY IN SELF-ASSEMBLED MONOLAYERS - STRUCTURAL AND ELECTROCHEMICAL PROPERTIES [J].
CHIDSEY, CED ;
LOIACONO, DN .
LANGMUIR, 1990, 6 (03) :682-691
[6]   Self-assembly of n-alkanethiols:: A kinetic study by second harmonic generation [J].
Dannenberger, O ;
Buck, M ;
Grunze, M .
JOURNAL OF PHYSICAL CHEMISTRY B, 1999, 103 (12) :2202-2213
[7]  
DELDEN CJ, 1997, BIOMATERIALS, V18, P845
[8]   SYNTHESIS, STRUCTURE, AND PROPERTIES OF MODEL ORGANIC-SURFACES [J].
DUBOIS, LH ;
NUZZO, RG .
ANNUAL REVIEW OF PHYSICAL CHEMISTRY, 1992, 43 :437-463
[9]   SELF-ASSEMBLED DISULFIDE-FUNCTIONALIZED AMPHIPHILIC COPOLYMERS ON GOLD [J].
ERDELEN, C ;
HAUSSLING, L ;
NAUMANN, R ;
RINGSDORF, H ;
WOLF, H ;
YANG, JL ;
LILEY, M ;
SPINKE, J ;
KNOLL, W .
LANGMUIR, 1994, 10 (04) :1246-1250
[10]   Covalent attachment and derivatization of poly(L-lysine) monolayers on gold surfaces as characterized by polarization-modulation FT-IR spectroscopy [J].
Frey, BL ;
Corn, RM .
ANALYTICAL CHEMISTRY, 1996, 68 (18) :3187-3193