Due to its high efficiency and selectivity, cell-free biosynthesis has found broad utility in the fields of bioproduction, environment monitoring, and disease diagnostics. However, the practical application is limited by its low productivity. Here, we introduce the entropy-driven assembly of transcription templates as dynamic amplifying modules to accelerate the cell-free transcription process. The catalytic DNA circuit with high sensitivity and enzyme-free format contributes to the production of large amounts of transcription templates, drastically accelerating the as-designed cell-free transcription system without interference from multiple enzymes. The proposed approach was successfully applied to the ultrasensitive detection of SARS-CoV-2, improving the sensitivity by 3 orders of magnitude. Thanks to the high programmability and diverse light-up RNA pairs, the method can be adapted to multiplexing detection, successfully demonstrated by the analysis of two different sites of the SARS-CoV-2 gene in parallel. Further, the flexibility of the entropy-driven circuit enables a dynamic responding range by tuning the circuit layers, which is beneficial for responding to targets with different concentration ranges. The strategy was also applied to the analysis of clinical samples, providing an alternative for sensitively detecting the current SARS-CoV-2 RNA that quickly mutates.

中文翻译：

---

通过转录模板的熵驱动装配加速无细胞生物传感器

由于其高效率和选择性，无细胞生物合成已在生物生产、环境监测和疾病诊断等领域得到广泛应用。然而，实际应用受到其低生产率的限制。在这里，我们引入了熵驱动的转录模板组装作为动态放大模块来加速无细胞转录过程。具有高灵敏度和无酶格式的催化 DNA 电路有助于产生大量转录模板，极大地加速了设计的无细胞转录系统，而不受多种酶的干扰。所提出的方法已成功应用于 SARS-CoV-2 的超灵敏检测，将灵敏度提高了 3 个数量级。由于高度的可编程性和多样化的发光 RNA 对，该方法可以适应多重检测，通过并行分析 SARS-CoV-2 基因的两个不同位点成功证明了这一点。此外，熵驱动电路的灵活性可以通过调整电路层来实现动态响应范围，这有利于响应不同浓度范围的目标。该策略还应用于临床样本的分析，为灵敏检测当前快速变异的 SARS-CoV-2 RNA 提供了一种替代方法。熵驱动电路的灵活性可以通过调整电路层来实现动态响应范围，这有利于响应不同浓度范围的目标。该策略还应用于临床样本的分析，为灵敏检测当前快速变异的 SARS-CoV-2 RNA 提供了一种替代方法。熵驱动电路的灵活性可以通过调整电路层来实现动态响应范围，这有利于响应不同浓度范围的目标。该策略还应用于临床样本的分析，为灵敏检测当前快速变异的 SARS-CoV-2 RNA 提供了一种替代方法。