Droplet-based digital PCR has emerged as a powerful platform for nucleic acid–based detection. However, the formation of droplet compartments and the subsequent amplification process in oil present significant drawbacks: instability under harsh thermal conditions, high background fluorescent noise inside droplets, and major difficulty in supporting multistep assays. Alternatively, droplets made of a hydrogel, or other advanced materials, have been adopted and demonstrate promising improvement over conventional droplet-based platforms. In this context, we present permeability-engineered compartmentation system-enabled digital PCR (PECS-dPCR), a novel digital platform that facilitates multistep biomolecular assays with thermal stability, minimized background noise, and long-term preservation capability. We achieve compartmentalization by forming a core–shell structure using the aqueous two-phase system (ATPS). The hydrogel shell provides exceptional mechanical strength and thermal stability to these compartments. The permeability of the shell can be fine-tuned to retain larger DNA targets while sieving out smaller ancillary molecules. Therefore, we can significantly improve the signal-to-noise ratio inside the compartments by washing out fluorescent background. Furthermore, these core–shell compartments remain intact in aqueous solution and are able to exchange materials with the ambient environment. This critical feature offers the capability to execute multistep assays in simple operational settings, enabling the demonstration of multitarget single-bacteria quantification in our platform. We further show that the assay can be paused with samples preserved for >2 weeks between different detection steps thanks to the excellent biochemical stability offered by the core–shell compartments. We envision PECS-dPCR becoming a versatile platform supporting multiple-reaction-step digital assays, offering high-quality detection signals and long-term stability.

中文翻译：

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渗透性工程区室系统支持的数字 PCR （PECS-dPCR）：多步骤生物分子检测的数字平台


基于液滴的数字 PCR 已成为基于核酸的检测的强大平台。然而，液滴室的形成和随后在油中的扩增过程存在明显的缺点：在恶劣的热条件下不稳定，液滴内部的背景荧光噪声高，以及支持多步骤分析的主要困难。或者，已经采用了由水凝胶或其他先进材料制成的液滴，并且与传统的基于液滴的平台相比，显示出有希望的改进。在此背景下，我们提出了渗透性工程区室系统启用的数字 PCR （PECS-dPCR），这是一种新颖的数字平台，有助于实现具有热稳定性、最小背景噪声和长期保存能力的多步骤生物分子测定。我们通过使用水性两相系统 （ATPS） 形成核壳结构来实现区室化。水凝胶外壳为这些隔室提供了卓越的机械强度和热稳定性。可以微调壳的通透性，以保留较大的 DNA 靶标，同时筛出较小的辅助分子。因此，我们可以通过洗掉荧光背景来显著提高隔室内的信噪比。此外，这些核壳隔室在水溶液中保持完整，并且能够与周围环境交换材料。这一关键功能提供了在简单的操作设置中执行多步骤分析的能力，从而能够在我们的平台中演示多靶标单细菌定量。 我们进一步表明，由于核壳隔室提供出色的生化稳定性，可以在不同检测步骤之间将样品保存 >2 周的情况下暂停测定。我们设想 PECS-dPCR 成为一种支持多反应步骤数字检测的多功能平台，提供高质量的检测信号和长期稳定性。