Department of Nanoscience and Engineering, Myongji University, Yongin, Gyeonggi, Korea.
Lab Chip. 2012 Nov 7;12(21):4455-64. doi: 10.1039/c2lc40727b.
A fully integrated microchip for performing cell lysis, polymerase chain reaction (PCR) and quantitative analysis of DNA amplicons in a single step is described herein. The chip was built on glass substrate using an indium-tin-oxide (ITO) microheater and PDMS engraved microchannels, which integrated an electrochemical cell lysis zone, a continuous flow PCR module and capillary electrophoresis amperometric detection (CE-AD) system. The total length of the microchannel was 4625 mm for performing 25 cycles of flow-through PCR and was laid on a handheld form factor of 96 × 96 mm(2) area. The key to the fabrication of such a device lies in the use of a single medium to carry out different kinds of biochemical reactions and hence, a reagentless electrochemical cell lysis protocol was integrated on the microchip which was capable of lysing most cell types, including difficult to lyse gram positive bacteria. The lysate contained genomic DNA from a sample which was proven to be suitable for PCR reactions. Genetic analysis was successfully performed on the microchip with purified lambda phage genomic DNA and various cell types, including non-tumorigenic MCF-10A and tumorigenic MCF-7 human cell lines, gram negative bacteria Escherichia coli O157:H7, and gram positive bacteria Bacillus subtilis, at an optimized flow rate of 5 μl min(-1). For the detection of amplicon DNA, a CE-AD system was used, with semisolid alkaline agarose within the capillary microchannel to minimize interference from cell debris and for efficient resolution of DNA fragments. High signal to noise ratio during amperometric detection and the use of online FFT filtering protocol enhanced the limit of detection of DNA amplicons. Therefore, with a combination of portability, cost-effectiveness and performance, the proposed integrated PCR microchip can be used for one step genetic analysis of most of the cell types and will enable more accessible healthcare.
本文介绍了一种用于在单个步骤中进行细胞裂解、聚合酶链反应 (PCR) 和 DNA 扩增子定量分析的完全集成微芯片。该芯片是在玻璃基板上使用氧化铟锡 (ITO) 微加热器和 PDMS 刻蚀微通道构建的，集成了电化学细胞裂解区、连续流动 PCR 模块和毛细管电泳安培检测 (CE-AD) 系统。微通道的总长度为 4625mm，用于进行 25 个循环的流动 PCR，并铺设在 96×96mm(2) 的手持形式因子上。制造这种装置的关键在于使用单一介质进行不同类型的生化反应，因此，在微芯片上集成了无试剂的电化学细胞裂解方案，该方案能够裂解大多数细胞类型，包括难以裂解的革兰氏阳性菌。裂解液包含来自样品的基因组 DNA，经证明适合 PCR 反应。使用微芯片成功地对纯化的噬菌体基因组 DNA 和各种细胞类型进行了基因分析，包括非致瘤 MCF-10A 和致瘤 MCF-7 人细胞系、革兰氏阴性菌大肠杆菌 O157:H7 和革兰氏阳性菌枯草芽孢杆菌，在优化的流速为 5μl min(-1) 时。为了检测扩增子 DNA，使用了 CE-AD 系统，在毛细管微通道内使用半固态碱性琼脂糖，以最大限度地减少细胞碎片的干扰，并实现 DNA 片段的有效分离。在安培检测过程中具有高信噪比和使用在线 FFT 滤波协议增强了 DNA 扩增子的检测限。因此，该集成的 PCR 微芯片具有便携性、成本效益和性能的优势，可用于大多数细胞类型的一步基因分析，并将实现更便捷的医疗保健。
