Wong April K Y, Krull Ulrich J
Chemical Sensors Group, University of Toronto Mississauga, 3359 Mississauga Rd. N., Mississauga, ON, Canada L5L 1C6.
Anal Chim Acta. 2009 Apr 20;639(1-2):1-12. doi: 10.1016/j.aca.2009.03.005. Epub 2009 Mar 16.
Covalently immobilized mixed films of oligonucleotide and oligomer components on glass and silicon surfaces are reported. This work has investigated how such films can improve selectivity for the detection of multiple base-pair mismatches. The intention was to introduce a "matrix isolation" effect on oligonucleotide probe molecules by surrounding the probes with oligomers, thereby reducing oligonucleotide-to-oligonucleotide and/or oligonucleotide-to-surface interactions. Thiol-functionalized oligonucleotide was coupled onto (3-aminopropyl)trimethoxysilane (APTMS) via a heterobifunctional linker, succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Using a variety of monomers such as 2-hydroxyethyl methacrylate (HEMA), oligomers were grown by surface-initiated atom transfer radical polymerization (ATRP) from a bromoisobutyryl NHS ester initiator which was immobilized onto APTMS sites that coated glass and oxidized silicon substrates. Various surface modification steps on silicon substrates were characterized by ellipsometry, wettability, atomic force microscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry. Polymerized HEMA (PHEMA) in mixture with oligonucleotide probes was evaluated for fluorescence transduction of hybridization. The presence of PHEMA was found to provide a sharper melt curve for hybrids containing both fully complementary and three base-pair mismatched targets, and this surface derivatization also minimized non-selective adsorption. The maximum increase in slope was improvement by a factor of 3-fold. An increase of up to 30% in difference of melting temperatures between fully complementary and 3 base-pair mismatched targets was achieved using PHEMA. The results suggest that the presence of oligomers dispersed among DNA hybrids can improve selectivity through what is believed to be a reduction of dispersity of interactions of probes with targets, and probes within their local environment at a surface.
报道了寡核苷酸和低聚物组分在玻璃和硅表面的共价固定混合膜。这项工作研究了此类膜如何提高对多个碱基对错配检测的选择性。目的是通过用低聚物包围探针，对寡核苷酸探针分子引入一种“基质隔离”效应，从而减少寡核苷酸与寡核苷酸和/或寡核苷酸与表面的相互作用。硫醇功能化的寡核苷酸通过异双功能连接剂琥珀酰亚胺基 4 - [N - 马来酰亚胺甲基]环己烷 - 1 - 羧酸酯（磺基 - SMCC）与（3 - 氨丙基）三甲氧基硅烷（APTMS）偶联。使用各种单体，如甲基丙烯酸 2 - 羟乙酯（HEMA），通过表面引发的原子转移自由基聚合（ATRP）从固定在涂覆玻璃和氧化硅基板的 APTMS 位点上的溴异丁酰基 NHS 酯引发剂生长低聚物。通过椭偏仪、润湿性、原子力显微镜、X 射线光电子能谱和飞行时间二次离子质谱对硅基板上的各种表面改性步骤进行了表征。评估了聚合 HEMA（PHEMA）与寡核苷酸探针混合物用于杂交的荧光转导。发现 PHEMA 的存在为包含完全互补和三个碱基对错配靶标的杂交体提供了更尖锐的熔解曲线，并且这种表面衍生化还使非选择性吸附最小化。斜率的最大增加提高了 3 倍。使用 PHEMA 实现了完全互补和 3 个碱基对错配靶标之间的熔解温度差异增加高达 30%。结果表明，分散在 DNA 杂交体中的低聚物的存在可以通过减少探针与靶标以及表面局部环境中探针之间相互作用的分散性来提高选择性。
