在液滴微流体高通量筛选中,毒性产物的合成会抑制细胞生长,导致筛选准确度下降。该研究建立了一种基于跨油相扩散诱导的动态调控系统,实现了液滴微流体中细胞生长与产物合成的解偶联。首先将单个细胞封装于液滴中进行培养,实现细胞生长;然后将诱导剂IPTG封装于液滴中得到诱导液滴;再利用IPTG的油水双相亲和性实现IPTG跨越油相扩散至细胞液滴中完成诱导。以大肠杆菌(Escherichia coli)异源表达毒性蛋白Phi29 DNA聚合酶(Phi29 DNA polymerase,Phi29 Pol)为例,构建Phi29 pol-超折叠绿色荧光蛋白(super-fold green fluorescent protein,sfGFP)融合蛋白,利用sfGFP的荧光信号强度做表征;通过单因素优化确定了当诱导液滴与细胞液滴的比例为1∶100、IPTG终浓度为1.2 mmol/L时,液滴中Phi29 pol-sfGFP融合蛋白的表达水平最高,达到了单细胞的2.73倍。总的来说,该系统利用油水界面扩散实现液滴中单细胞的动态调控,有效缓解了毒性产物合成对细胞生长的抑制,为液滴微流体高通量筛选提供了方法支撑。
In droplet microfluidics high-throughput screening process, toxic product synthesis often inhibits cell growth, reducing screening accuracy.This study established a dynamic regulation method using trans-oil-phase diffusion in droplets to decouple cell growth and product synthesis.Single cells were first encapsulated and cultured within droplets to promote proliferation.Then, the inducer IPTG was encapsulated.The oil-water amphiphilicity of the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) enabled its diffusion across the oil phase into cell-containing droplets for induction.Using Escherichia coli expressing the toxic protein Phi29 DNA polymerase (Phi29 pol) as a model, a Phi29 pol-super-fold green fluorescent protein (sfGFP) fusion reporter was constructed to monitor expression via sfGFP fluorescence intensity.Single-factor optimization identified that a 1∶100 ratio of induction-to-cell droplets with a final IPTG concentration of 1.2 mmol/L achieved the highest Phi29 pol-sfGFP expression level within droplets.This condition achieved a 2.73-fold that of single-cell controls.In general, this system utilized oil-water interfacial diffusion dynamics to minimize growth inhibition caused by toxic product accumulation.It provides a methodological support for high-throughput screening in droplet microfluidics.
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