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.
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-dimethylaminopropyl)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 C1s 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 I) 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.
Tyan
Yu-Chang
YC
Department of Enviornmental & Occupational Health, National Cheng Kung University, No.1, Ta-Hsueh Road, Tainan, 701, Taiwan, Republic of China. yctyan@mail.ncku.edu.tw
Liao
Jiunn-Der
JD
Jong
Shiang-Bin
SB
Liao
Pao-Chi
PC
Yang
Ming-Hui
MH
Chang
Yin-Wei
YW
Klauser
Ruth
R
Himmelhaus
Michael
M
Grunze
Michael
M
D015374:Biosensing Techniques / Q000379:methods; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D003460:Crystallization / Q000379:methods; D005227:Fatty Acids / Q000737:chemistry; D005240:Feasibility Studies; D008422:Materials Testing; D010865:Pilot Projects; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000032:analysis / Q000737:chemistry*
D020099:Coated Materials, Biocompatible; D005227:Fatty Acids; C016173:undecanoic acid; D012710:Serum Albumin, Bovine; D014357:Trypsin
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