Liu Zhenhua, Fornell Anna, Tenje Maria
Department of Materials Science and Engineering, Science for Life Laboratory, Uppsala University, SE-752 37 Uppsala, Sweden.
Biomicrofluidics. 2021 May 17;15(3):034103. doi: 10.1063/5.0050440. eCollection 2021 May.
Droplet microfluidics is a powerful method used to characterize chemical reactions at high throughput. Often detection is performed via in-line optical readout, which puts high demands on the detection system or makes detection of low concentration substrates challenging. Here, we have developed a droplet acoustofluidic chip for time-controlled reactions that can be combined with off-line optical readout. The principle of the platform is demonstrated by the enzymatic conversion of fluorescein diphosphate to fluorescein by alkaline phosphatase. The novelty of this work is that the time of the enzymatic reaction is controlled by physically removing the enzymes from the droplets instead of using chemical inhibitors. This is advantageous as inhibitors could potentially interact with the readout. Droplets containing substrate were generated on the chip, and enzyme-coupled microbeads were added into the droplets via pico-injection. The reaction starts as soon as the enzyme/bead complexes are added, and the reaction is stopped when the microbeads are removed from the droplets at a channel bifurcation. The encapsulated microbeads were focused in the droplets by acoustophoresis during the split, leaving the product in the side daughter droplet to be collected for the analysis (without beads). The time of the reaction was controlled by using different outlets, positioned at different lengths from the pico-injector. The enzymatic conversion could be measured with fluorescence readout in a separate PDMS based assay chip. We show the ability to perform time-controlled enzymatic assays in droplet microfluidics coupled to an off-line optical readout, without the need of enzyme inhibitors.
液滴微流控技术是一种用于高通量表征化学反应的强大方法。通常通过在线光学读出进行检测，这对检测系统提出了很高的要求，或者使得低浓度底物的检测具有挑战性。在此，我们开发了一种用于时间控制反应的液滴声流控芯片，该芯片可与离线光学读出相结合。通过碱性磷酸酶将二磷酸荧光素酶促转化为荧光素，证明了该平台的原理。这项工作的新颖之处在于，酶促反应的时间是通过从液滴中物理去除酶来控制的，而不是使用化学抑制剂。这具有优势，因为抑制剂可能会与读出过程相互作用。在芯片上生成含有底物的液滴，并通过皮升注射将酶偶联的微珠添加到液滴中。一旦添加酶/珠复合物，反应就开始，当微珠在通道分叉处从液滴中去除时，反应停止。在分离过程中，通过声泳将封装的微珠聚焦在液滴中，将产物留在侧子液滴中以便收集进行分析（不含珠子）。通过使用距离皮升注射器不同长度的不同出口来控制反应时间。酶促转化可以在基于聚二甲基硅氧烷（PDMS）的单独检测芯片中通过荧光读出进行测量。我们展示了在与离线光学读出相结合的液滴微流控中进行时间控制酶促测定的能力，而无需酶抑制剂。
