Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells.
Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells.
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DOI：
10.1038/s41598-020-78996-7
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发表时间：
2020-12-14
影响因子：
4.6
通讯作者：
Castell OK
中科院分区：
文献类型：
--
作者：
Hardinge P;Baxani DK;McCloy T;Murray JAH;Castell OK
微流体液滴生成为分子诊断提供了精确、低体积、高通量的机会。等温DNA扩增与生物发光检测是一种快速，低成本，高特异性的分子诊断技术，是由温度可重复。将环介导的等温核酸扩增（LAMP）和生物发光测定真实的时间（BART）与液滴微流体相结合，应该能够通过简单的光发射进行高通量、低拷贝、序列特异性DNA检测。用低成本设备产生稳定、均匀的LAMP-BART液滴。这些液滴的组成和规模是可控的，并且可以对DNA扩增期间的生物发光输出进行成像和定量。此外，这些液滴容易地结合到包封的液滴界面双层（eDIB）或人工细胞中，并且生物发光被真实的跟踪以用于芯片外的精确定量。具有高稳定性和尺度均匀性以及高通量和低成本产生的微流体LAMP-BART液滴适合于在低模板浓度和体积下的数字DNA定量，其中需要多个测量分区。eDIB核心中的可重复反应可用于研究液滴与环境的相互关系，也可用于通过自包含的液滴网络进行更复杂的化学处理，为智能软物质诊断铺平道路。
Microfluidic droplet generation affords precise, low volume, high throughput opportunities for molecular diagnostics. Isothermal DNA amplification with bioluminescent detection is a fast, low-cost, highly specific molecular diagnostic technique that is triggerable by temperature. Combining loop-mediated isothermal nucleic acid amplification (LAMP) and bioluminescent assay in real time (BART), with droplet microfluidics, should enable high-throughput, low copy, sequence-specific DNA detection by simple light emission. Stable, uniform LAMP–BART droplets are generated with low cost equipment. The composition and scale of these droplets are controllable and the bioluminescent output during DNA amplification can be imaged and quantified. Furthermore these droplets are readily incorporated into encapsulated droplet interface bilayers (eDIBs), or artificial cells, and the bioluminescence tracked in real time for accurate quantification off chip. Microfluidic LAMP–BART droplets with high stability and uniformity of scale coupled with high throughput and low cost generation are suited to digital DNA quantification at low template concentrations and volumes, where multiple measurement partitions are required. The triggerable reaction in the core of eDIBs can be used to study the interrelationship of the droplets with the environment and also used for more complex chemical processing via a self-contained network of droplets, paving the way for smart soft-matter diagnostics.
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