Department of Biological and Environmental Engineering, Cornell University , Ithaca, NY 14853, United States.
Anal Chem. 2014 Jan 7;86(1):849-56. doi: 10.1021/ac403417z. Epub 2013 Dec 13.
The major modules for realizing molecular biological assays in a micro-total analysis system (μTAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic mRNA within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)25, and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid nonspecific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 10(15) fold and still result in successful on-chip reamplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to benchtop devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes.
为了实现微全分析系统（μTAS）中的分子生物学检测，开发了主要模块来检测病原体。具体重点是在简单的单通道设备中分离和扩增真核 mRNA，该设备可用于非常低的 RNA 浓度，然后与检测模块集成。微小隐孢子虫（Cryptosporidium parvum）的 hsp70 mRNA 被用作模型分析物。研究的重点是聚甲基丙烯酸甲酯（PMMA）微流控通道内的表面化学，这些表面化学能够实现非常低的 mRNA 浓度下的特异性和灵敏性 mRNA 分离和扩增反应。当通道表面通过 UV/臭氧处理羧化，然后在表面固定聚酰胺-胺（PAMAM）树状大分子时，可实现最佳条件，从而提高胸腺嘧啶寡核苷酸 oligo（dT）25 的固定效率，并为扩增反应提供可靠的表面，重要的是，无需使用封闭剂。还研究了剩余活性表面基团的其他化学修饰，以避免非特异性捕获核酸并在低 RNA 浓度下阻碍 mRNA 扩增。使用核酸序列为基础的扩增（NASBA）来完成 mRNA 的扩增，这是一种等温、引物依赖性技术。由先前生成的 NASBA 扩增子组成的阳性对照可以稀释 10(15)倍，仍然可以在芯片上成功再扩增。最后，直接在芯片上证明了从低至 30 个微小隐孢子虫卵囊中分离和扩增 mRNA 的成功，并与台式设备进行了比较。这是首次在相关分析体积的简单微流控设备中成功实现 mRNA 分离和基于 NASBA 的 mRNA 扩增的证明。
