Modified fabrication process of protein chips using a short-chain self-assembled monolayer

In previous work a short chain SAM, 4,4-Dithiodibutyric Acid (DTBA) was found to be a thin monolayer in protein chips. However, obtaining uniform fluorescent intensity remains difficult because water-soluble carbodiimides (EDC) in an aqueous system cause the hydrolysis of N-hydroxysuccinimide ester (NHS esters). The hydrolysis of NHS esters reduces coupling yields and therefore reduces the fluorescent intensity of protein chips. The NHS can increase the stability of active intermediate resulting from the reaction of EDC and NHS, but the ratio of the concentration of EDC to that of NHS strongly affects this stability. The effects of the solvents used in the washing step are studied to solve this problem. The results reveal that PBST (PBS + 5% Tween20) is more effective in reducing the hydrolysis of NHS esters than deionized water. Additionally, the effects of 3:1 and 5:2 EDC/NHS ratios on the chips are examined. The 3:1 EDC/NHS ratio yields a higher fluorescent intensity than the 5:2 ratio. The effects on the chips of dissolving EDC in DI water, DI water + 0.1 M MES and alcohol are also investigated. The results show that alcohol provides higher fluorescent intensity than other solvents and the reaction time of 4 h yields a high fluorescent intensity with 3:1 EDC/NHS ratio. A modified fabrication process of protein chips using 4,4-DTBA is developed. In this work, 160 mM 4,4-DTBA is used as a self-assembled monolayer in the fabrication of protein chips. Experiments to characterize 4,4-DTBA are performed by contact angle goniometry and Fourier transform infrared spectroscopy (FTIR). Furthermore, the immobilized protein A-FITC (fluorescein isothiocyanate) is adopted in fluorescent assays.

[1]  R. W. Wright,et al.  Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. , 1986, Analytical biochemistry.

[2]  B. Ratner,et al.  Protein adsorption on 18-alkyl chains immobilized on hydroxyl-terminated self-assembled monolayers. , 2005, Biomaterials.

[3]  J. V. Staros,et al.  N-hydroxysulfosuccinimide active esters: bis(N-hydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linkers. , 1982, Biochemistry.

[4]  Gwo-Bin Lee,et al.  Micromachined polymerase chain reaction system for multiple DNA amplification of upper respiratory tract infectious diseases. , 2005, Biosensors & bioelectronics.

[5]  M. Jennings,et al.  Localization of a site of intermolecular cross-linking in human red blood cell band 3 protein. , 1985, The Journal of biological chemistry.

[6]  O'sullivan,et al.  Human cytomegalovirus detection by a quartz crystal microbalance immunosensor. , 2000, Enzyme and microbial technology.

[7]  E. Fukusaki,et al.  Real-time quantification of methanol in plants using a hybrid alcohol oxidase-peroxidase biosensor. , 2004, Analytical chemistry.

[8]  W. Andreoni,et al.  Thiols and Disulfides on the Au(111) Surface: The Headgroup−Gold Interaction , 2000 .

[9]  Hendrik Neubert,et al.  Enhanced affinity capture MALDI-TOF MS: orientation of an immunoglobulin G using recombinant protein G. , 2002, Analytical chemistry.

[10]  A. Asanov,et al.  A total internal reflection fluorescence biosensor for aluminum (III) , 2001 .

[11]  Jeong-Woo Choi,et al.  Deposition behavior and photoelectrochemical characteristics of chlorophylla Langmuir-Blodgett films , 2001 .

[12]  Jing-Min Hwang,et al.  Development of an immunosensor for human ferritin, a nonspecific tumor marker, based on surface plasmon resonance. , 2004, Biosensors & bioelectronics.

[13]  M Aizawa,et al.  Assembling of engineered IgG-binding protein on gold surface for highly oriented antibody immobilization. , 2000, Journal of biotechnology.

[14]  Michael R. Malone,et al.  Novel surface coatings for antibody attachment to surface plasmon resonance sensors , 2005, SPIE Optics East.

[15]  G. Whitesides,et al.  Self-Assembly of Mesoscale Objects into Ordered Two-Dimensional Arrays , 1997, Science.

[16]  C. Nicolini,et al.  Immunological activity of IgG Langmuir films oriented by protein A sublayer , 1995 .

[17]  Jeong-Woo Choi,et al.  Nanoscale fabrication of protein A on self-assembled monolayer and its application to surface plasmon resonance immunosensor , 2004 .

[18]  Tien,et al.  Design and self-assembly of open, regular, 3D mesostructures , 1999, Science.

[19]  M. Grunze,et al.  Modification of aliphatic monomolecular films by free radical dominant plasma: The effect of the alkyl chain length and the substrate , 2003 .

[20]  T. Matsunaga,et al.  Fully automated chemiluminescence immunoassay of insulin using antibody-protein A-bacterial magnetic particle complexes. , 2000, Analytical chemistry.

[21]  J. V. Staros.  Membrane-impermeant crosslinking reagents: probes of the structure and dynamics of membrane proteins , 1988 .

[22]  J. Gergely,et al.  Zero-length crosslinking procedure with the use of active esters. , 1990, Analytical biochemistry.

[23]  George M. Whitesides,et al.  The interaction of proteins and cells with self-assembled monolayers of alkanethiolates on gold and silver , 1999 .

[24]  Jang Ling-Sheng,et al.  Development and Characterization of 4,4-Dithiodibutyric Acid as a Monolayer for Protein Chips , 2006 .

[25]  D. Wong,et al.  Direct application strategy to immobilise a thioctic acid self-assembled monolayer on a gold electrode , 2004 .