精准医疗急需更加精确、灵敏、方便快捷的核酸定量方法.设计开发了一种结合双荧光等温扩增的高密度皮升级核酸定量微流控芯片.芯片采用多层软光刻技术制成,有3个反应通道,每通道有 40 000 个反应小室,共 120 576 个皮升级反应小室,反应小室的密度达 7 000/cm2.芯片利用负压驱动样品精确分配,热凝固油相封闭小室,无需阀门及其他仪器辅助.芯片中嵌入的氟硅烷纳米涂层能有效地阻止反应过程中水蒸气的散失.以乙肝病毒(hepatitis B virus, HBV)质粒作为模板,进行等温多底物自配引发扩增(isothermal multiple self-matching-initiated amplification, IMSA),测试芯片定量性能.芯片检测模板的动态范围达6个数量级,可检测的最大模板量为36 μL中1.13×106拷贝数核酸.该装置具有定量精确、灵敏、快捷、操作简单等优势,可用于精准检测.%More accurate, sensitive, and convenient methods are eagerly expected for quantifying nucleic acid in precision medicine.Therefore, in this study, a high-density picoliter-level microfluidic chip is developed for the precise detection of nucleic acid, which is combined with a dual fluorescence system based on isothermal multiple self-matching-initiated amplification (IMSA).Multilayer soft lithography technique is applied to the fabrication of the mold of chip.Then, the chip is produced by molding with the material of polydimethylsiloxane (PDMS).In addition, a simple nano-waterproof layer lying up on the microcell array is able to efficiently prevent the water from evaporation during the IMSA.On the chip, a total of 120 000 reactors with picoliter volume for each are integrated and the density reaches up to 7 000 reactors per cm2.This device allows three samples counted through partitioning each into 40 000 reactors at the same time.The sample can be sucked into each microcell of the chip by negative pressure equally without external power.Then, thermo-coagulation oil fills the channels to separate microcells automatically, avoiding the need of any valve.The microcells which contain the target are distinguished by a dual fluorescence system based on IMSA.Then, the accurate concentration of target can be calculated by the formula of Poisson distribution.As a proof-of-concept assay, the IMSA-based quantitative detection of hepatitis B virus (HBV) artificial plasmid DNAs by using this chip device was conducted.As indicated by the results, this device achieves a dynamic range of 106 and the upper limit of quantitation reaches 1.13×106 copies nucleic acid in 36 μL sample.Accordingly, this portable device with accurate quantitation ability and fast sample processing is very beneficial for precision detection.
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