The analysis of proteins at the single-molecule level reveals heterogeneous behaviours that are masked in ensemble-averaged techniques. The digital quantification of enzymes traditionally involves the observation and counting of single molecules partitioned into microcompartments via the conversion of a profluorescent substrate. This strategy, based on linear signal amplification, is limited to a few enzymes with sufficiently high turnover rate. Here we show that combining the sensitivity of an exponential molecular amplifier with the modularity of DNA–enzyme circuits and droplet readout makes it possible to specifically detect, at the single-molecule level, virtually any D(R)NA-related enzymatic activity. This strategy, denoted digital PUMA (Programmable Ultrasensitive Molecular Amplifier), is validated for more than a dozen different enzymes, including many with slow catalytic rate, and down to the extreme limit of apparent single turnover for Streptococcus pyogenes Cas9. Digital counting uniquely yields absolute molar quantification and reveals a large fraction of inactive catalysts in all tested commercial preparations. By monitoring the amplification reaction from single enzyme molecules in real time, we also extract the distribution of activity among the catalyst population, revealing alternative inactivation pathways under various stresses. Our approach dramatically expands the number of enzymes that can benefit from quantification and functional analysis at single-molecule resolution. We anticipate digital PUMA will serve as a versatile framework for accurate enzyme quantification in diagnosis or biotechnological applications. These digital assays may also be utilized to study the origin of protein functional heterogeneity.

单分子水平的蛋白质分析揭示了被整体平均技术掩盖的异质行为。传统上，酶的数字定量涉及通过荧光底物的转换来观察和计数分配到微区室中的单分子。这种基于线性信号放大的策略仅限于具有足够高周转率的少数酶。在这里，我们表明，将指数分子放大器的灵敏度与 DNA 酶电路和液滴读数的模块化相结合，使得在单分子水平上特异性检测几乎任何 D(R)NA 相关酶活性成为可能。这种策略被称为数字 PUMA（可编程超灵敏分子放大器），已针对十多种不同的酶进行了验证，其中包括许多催化速率较慢的酶，并且对于化脓性链球菌 Cas9的表观单周转率低至极限。数字计数独特地产生绝对摩尔定量，并揭示所有测试的商业制剂中大部分非活性催化剂。通过实时监测单个酶分子的扩增反应，我们还提取了催化剂群体中的活性分布，揭示了各种压力下的替代失活途径。我们的方法极大地增加了可以受益于单分子分辨率的定量和功能分析的酶的数量。我们预计数字 PUMA 将成为诊断或生物技术应用中精确酶定量的多功能框架。这些数字化验还可用于研究蛋白质功能异质性的起源。