Abstract
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.
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Acknowledgements
The authors would like to thank the National Science Council of Taiwan, the Republic of China, for financially supporting this research under Contract number NSC 95-2622-E-006-039-CC3 and NSC 94-2218-E-006-043. The authors also would like to thank the Center for Micro/Nano Science and Technology, National Cheng Kung University, Tainan, Taiwan, for access to equipment and technical support. Furthermore, this work made use of Shared Facilities supported by the Program of Top 100 Universities Advancement, Ministry of Education, Taiwan.
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Jang, LS., Keng, HK. Modified fabrication process of protein chips using a short-chain self-assembled monolayer. Biomed Microdevices 10, 203–211 (2008). https://doi.org/10.1007/s10544-007-9126-7
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DOI: https://doi.org/10.1007/s10544-007-9126-7