Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays
Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays
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DOI：
10.1021/ac0010431
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发表时间：
2001-01-01
影响因子：
7.4
通讯作者：
Corn, RM
中科院分区：
文献类型：
--
作者：
Nelson, BP;Grimsrud, TE;Corn, RM
表面等离子体共振（SPR）成像是一种表面敏感的光谱技术，用于测量未标记的生物分子与表面结合物质阵列之间的相互作用。在本文中，SPR成像用于定量检测杂交吸附短（18碱基）未标记的DNA寡核苷酸在低浓度下，以及，首次，杂交吸附未标记的RNA寡核苷酸和较大的16 S核糖体RNA（rRNA）分离的微生物大肠杆菌到DNA阵列。对于DNA和RNA寡核苷酸的杂交吸附，报告的检测限为10 nM;对于大的（1500个碱基）16 S rRNA分子，检测到的浓度低至2 nM。巯基-DNA探针与金表面的共价连接导致高表面探针密度（10（12）分子/cm（2））和优异的探针稳定性，其能够进行超过25个循环的杂交和变性而不损失信号或特异性。菲涅耳计算显示，通过SPR成像测量的反射率百分比的变化相对于吸附的DNA寡核苷酸的表面覆盖率是线性的。从SPR成像的数据被用来构建一个定量的吸附等温线上的表面上的杂交吸附。发现DNA和RNA 18-mer寡核苷酸杂交吸附遵循Langmuir等温线，吸附系数为1.8 × 10（7）M-1。
Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique for measuring interactions between unlabeled biological molecules with arrays of surface-bound species. In this paper, SPR imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array. For the hybridization adsorption of both DNA and RNA oligonucleotides, a detection limit of 10 nM is reported; for large (1500-base) 16S rRNA molecules, concentrations as low as 2 nM are detected. The covalent attachment of thiol-DNA probes to the gold surface leads to high surface probe density (10(12) molecules/cm(2)) and excellent probe stability that enables more than 25 cycles of hybridization and denaturing without loss in signal or specificity. Fresnel calculations are used to show that changes in percent reflectivity as measured by SPR imaging are linear with respect to surface coverage of adsorbed DNA oligonucleotides. Data from SPR imaging is used to construct a quantitative adsorption isotherm of the hybridization adsorption on a surface. DNA and RNA 18-mer oligonucleotide hybridization adsorption is found to follow a Langmuir isotherm with an adsorption coefficient of 1.8 x 10(7) M-1.