Droplet microfluidics with picoinjection provides significant advantages to multistep reactions and screenings. The T-junction design for picoinjection is convenient in adding picoliter reagents into passing droplets to initiate reactions. However, conventional picoinjectors face difficulties in eliminating cross-contamination between droplets, preventing them from widespread use in sensitive biological and molecular assays. Here, we introduce stepinjection, which uses a T-junction with a stepped channel design to elevate the diffusional buffer zone into the main channel and consequently increases the pressure difference between droplets and the inlet of the injection channel. To demonstrate the stepinjector’s ability to perform contamination-sensitive enzymatic assays, we inject casein fluorescein isothiocyanate (FITC-casein) into a mixture of savinase and savinase-free (labeled with a red fluorescent dye) droplets. We observe no cross-contamination using stepinjection but find a severe cross-talk using an optimal picoinjection design. We envision that the simple, tunable, and reliable stepinjector can be easily integrated in various droplet processing devices, and facilitate various biomedical and biochemical applications including multiplex digital PCR, single-cell sequencing, and enzymatic screening.

中文翻译：

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通过 Stepinjection 使用微流体进行可调谐且无污染的注射

具有 picoinjection 的液滴微流体为多步反应和筛选提供了显着优势。用于 picoinjection 的 T 型接头设计便于将皮升试剂添加到通过的液滴中以引发反应。然而，传统的 picoinjectors 在消除液滴之间的交叉污染方面面临困难，从而阻碍了它们在敏感的生物和分子检测中的广泛使用。在这里，我们介绍了阶梯喷射，它使用带有阶梯通道设计的 T 形接头将扩散缓冲区提升到主通道中，从而增加液滴和喷射通道入口之间的压力差。为了证明 stepinjector 执行污染敏感酶分析的能力，我们将酪蛋白异硫氰酸荧光素 (FITC-酪蛋白) 注入到 savinase 和无 savinase（用红色荧光染料标记）液滴的混合物中。我们使用 stepinjection 没有观察到交叉污染，但使用最佳 picoinjection 设计发现了严重的串扰。我们设想，简单、可调谐、可靠的步进注射器可以轻松集成到各种液滴处理设备中，并促进各种生物医学和生化应用，包括多重数字 PCR、单细胞测序和酶筛选。