Single cell analysis preserves the heterogeneity information of target cell population in search of rare biomarkers for disease diagnosis. Microfluidic technology facilitates single cell analysis through its high integrability with multi-functionalities, high sensitivity, precision and dynamic range for digital assays. However, demonstrated microfluidic devices for single cell analysis suffer from low throughput, the need of external instruments and complicated control system. Herein, we present a self-driven high-density microwell array for quantitative analysis of single-cell metabolic activity. 38,400-microwell array (density: 25,000/cm²) is achieved through two features: (1) Two-layered vertical design of microchannels to provide more space for microwell integration; and (2) Doping of carbon powder in microwell wall to block stray light transmission and improve signal-to-noise ratio, decreasing the interval between microwells down to 30 µm. Moreover, the chip is powered by pre-stored negative pressure without the need of external pump. Our microwell array significantly reduces the assay time from over 24–3 h in Escherichia coli quantitative analysis (6-order dynamic range). We also demonstrated the viability assay and metabolic heterogeneity of single bacteria, envisioning that the microwell array could be applied for other target cells and extended to different molecular techniques such as digital PCR.

单细胞分析保留目标细胞群的异质性信息，以寻找用于疾病诊断的稀有生物标志物。微流控技术通过其与多功能的高度可集成性、高灵敏度、精确度和数字化分析的动态范围来促进单细胞分析。然而，已证明的用于单细胞分析的微流控装置存在通量低、需要外部仪器和复杂的控制系统等问题。在此，我们提出了一种用于定量分析单细胞代谢活性的自驱动高密度微孔阵列。38,400 微孔阵列（密度：25,000/cm ²)是通过两个特点实现的：（1）微通道的两层垂直设计，为微孔集成提供更多空间；(2)在微孔壁中掺杂碳粉，阻断杂散光传输，提高信噪比，将微孔间距减小到30μm。此外，芯片由预存负压供电，无需外接泵。我们的微孔阵列显着减少了大肠杆菌定量分析（6 级动态范围）中超过 24-3 小时的测定时间。我们还展示了单个细菌的活力测定和代谢异质性，设想微孔阵列可应用于其他靶细胞并扩展到不同的分子技术，如数字 PCR。