Poly-L-Lysine玻片在寡核苷酸芯片制备中的改进

摘要/Abstract

摘要： 目的为了制得适合固定未修饰寡核苷酸的芯片,提高检测灵敏性,对Patrick Brown 实验室的多聚左旋赖氨酸包被玻片的方法进行改进。方法玻片经清洗后用缩水甘油-丙氧基三甲氧基硅烷进行硅烷化,然后应用Poly-L-Lysine在玻片表面形成聚合物涂层,经次亚苯基二异硫氰酸盐表面活化后可使寡核苷酸共价连接在芯片表面。设计了各种实验考察方法改进前后芯片表面的性能,并将改进后的玻片初步应用于SARS冠状病毒寡核苷酸芯片检测中。结果方法改进后芯片表面性能优良:固定效率高、点的同一性好、杂交效率和热稳定性好、寡核苷酸结合牢固、芯片可以重复利用。结论利用共价连接,方法改进后的芯片表面适合固定未修饰的寡核苷酸,解决了寡核苷酸与玻片之间物理结合不稳定、易剥离的缺陷,提高了芯片检测的灵敏性。

Abstract: Objective To modify conventional poly-L-lysine coating for oligonucleotide microarray preparation so as to enhance the sensitivity of the microarray.Methods The proposed chemical approaches included silanizing the slides with 3-glycid-oxypropyltrimethoxysilane (GOPS) after cleanling, followed by slide coating with polymers (poly-L-lysine) that was covalently bound to the modified glass. Subsequent attachment of the oligonucleotide to the modified slide surface was achieved after 1,4-phenylene diisothiocyanate (PDITC) activation of the surface. Various experiments were carried out, such as the immobilization efficiency and hybridization assays to test the modified slides, which were then used tentatively in the preparation of microarrays for SARS coronavirus detection.Results The improved surface had high immobilization efficiency, good uniformity and satisfactory hybridization efficiency, better than those slides with conventional poly-L-lysinecoating. In addition, such modified slides rendered the microarrays more resistant to consecutive probing/stripping cycles.Conclusion The modified slide surface is satisfactory to immobilize unmodified oligonucleotide by covalent binding, which enhances not only the sensitivity of the prepared oligonucleotide microarray but also the binding of the oligonucleotide to the slide surface.

中图分类号:

吴清华¹, 马文丽¹, 石嵘¹, 郭秋野¹, 张宝¹, 李凌¹, 张海燕¹, 郑文岭². Poly-L-Lysine玻片在寡核苷酸芯片制备中的改进[J]. 南方医科大学学报, 2004, 24(11): 1236-1241.

WU Qing-hua¹, MA Wen-li¹, SHI Rong¹, GUO Qiu-ye¹, ZHANG Bao¹, LI Ling¹, ZHANG Hai-yan¹, ZHENG Wen-ling². Oligonucleotide microarray preparation using enhanced poly-L-lysine glass slides[J]. Journal of Southern Medical University, 2004, 24(11): 1236-1241.

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