Microbial detection at the single-cell level offers a novel approach for understanding intricacies of biological systems at the most fundamental level, influencing the advances in therapeutics, drug discoveries, and bioenergy. However, achieving high throughput, high accuracy, specificity, and low damage in sorting and detecting microbial cells have been the most significant hurdles. In this study, we introduced a laser-induced forward transfer (LIFT) with functionalized microwell arrays, called functionalized microwell laser sorting (FMLS). The microwell ejection chip (M-chip) cut the liquid surface tension to form femtoliter droplet arrays by hundreds of thousands of microwell arrays, which enabled efficient capture of single microbial cells and enhanced throughput of microbial detection. The FMLS has achieved over 80 % capture efficiency for individual microorganisms such as Escherichia coli, Saccharomyces cerevisiae, Cyanobacteria spp., and Chlamydomonas spp. Additionally, it integrated bright-field, fluorescence, and Raman identification methods to enhance specificity for microbial detection. FMLS system exhibited nearly 100 % single-cell sorting efficiency without affecting adjacent cells. The sorted single cells were validated through PCR, confirming the accuracy of single-cell capture and sorting. Through simulations, we optimized the microwell thickness to minimize the required sorting energy, enabling over 95 % cell viability and over 88 % genome coverage of single cells. These highlight the flexibility and technical capabilities of the FMLS system, which will become attractive and invaluable sorting and detection tools for single-cell research, driving forward advancements in diagnostics, environmental science, and biotechnology.
bioengineering
,biotechnology
