Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada V5A 1S6.
Anal Biochem. 2010 May 15;400(2):282-8. doi: 10.1016/j.ab.2010.01.017. Epub 2010 Jan 18.
A microfluidic microarray device, which has been developed for parallel DNA detection, is now further optimized for more rapid and sensitive DNA detection and for the single-base-pair discrimination of two fungal pathogenic PCR products. Two poly(dimethylsiloxane) (PDMS)-based microfluidic chips consist of radial and spiral microchannels in which flexible probe creation and convenient sample delivery have been achieved by centrifugal pumping. The microarray hybridizations occurred at the cross sections within the spiral channels intersecting the preprinted radial probe lines. The centrifugal pumping method showed advantages over the vacuum suction method in terms of parallel solution delivery and less signal variations between replicate samples. The effect of microchannel depth was studied, and hybridization time is predictable at a certain rotation speed. Cy5 dye labels were proved to show much higher hybridization efficiency as well as less photobleaching effect as compared with the fluorescein dye labels used in our previous work. With these optimized conditions, the method was applied to the detection of three fungal pathogenic polymerase chain reaction (PCR) products with a sample load of 0.2 ng (in 1 microl). Furthermore, the single-base-pair discrimination between the PCR products of two relevant Botrytis species (B. cinerea and B. squamosa) was achieved in a duration as short as 3 min.
一种用于并行 DNA 检测的微流控微阵列器件，现进一步优化，以实现更快速、更灵敏的 DNA 检测，以及对两种真菌病原 PCR 产物的单碱基对鉴别。两个基于聚二甲基硅氧烷 (PDMS) 的微流控芯片包含放射状和螺旋状微通道，通过离心泵送实现了灵活的探针创建和方便的样品输送。微阵列杂交发生在螺旋通道的交叉点处，与预印的放射状探针线相交。与真空抽吸法相比，离心泵送法在并行溶液输送和重复样品之间的信号变化方面具有优势。研究了微通道深度的影响，并且在一定的转速下可以预测杂交时间。与我们之前工作中使用的荧光染料标记相比，Cy5 染料标记显示出更高的杂交效率和更少的光漂白效应。在这些优化条件下，该方法用于检测三种真菌病原聚合酶链反应 (PCR) 产物，样品负荷为 0.2ng（1 微升）。此外，在短短 3 分钟内即可实现两种相关葡萄孢属（B. cinerea 和 B. squamosa）PCR 产物的单碱基对鉴别。
