Wang Xixian, Wang Sen, Diao Zhidian, Hou Xibao, Gong Yanhai, Sun Qing, Zhang Jiaping, Ren Lihui, Li Yuandong, Ji Yuetong, Shen Wei, Yin Yifeng, Huang Shi, Song Xiaojin, Cui Qiu, Feng Yingang, Xu Jian, Ma Bo
State Key Laboratory of Photoelectric Conversion and Utilization of Solar Energy, Key Laboratory of Shandong Energy Biological Genetic Resources, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China.
Shandong Energy Institute, Qingdao 266101, China.
Proc Natl Acad Sci U S A. 2025 Jun 3;122(22):e2503641122. doi: 10.1073/pnas.2503641122. Epub 2025 May 30.
A full spontaneous single-cell Raman spectrum captures the metabolic phenome in a label-free and noninvasive manner. However, Raman-activated cell sorting (RACS) of rare target cells from highly heterogeneous systems has remained largely conceptual. Here, we present a positive dielectrophoresis-induced deterministic lateral displacement (pDEP-DLD)-based RACS (pDEP-DLD-RACS), in which a modulated pDEP-DLD force is applied to focus, trap, and functionally sort fast-moving single cells in a wide channel. For pigment- and oil-producing yeasts, pDEP-DLD-RACS shows high sorting accuracy (>90%), high throughput (600 events min), high yield (>85%), and long stable running time (10 h), and can sort rare cells while preserving full cellular vitality. Moreover, label-free sorting directly from a genome-wide random mutagenesis library with >10 sp. Mutants, based on intracellular docosahexaenoic acid (DHA) content, produces mutant cells with 58% higher DHA productivity in just two RACS runs over two days, representing two-orders-of-magnitude higher time- and cost-efficiency than conventional approaches. This superior trait arises from global remolding of transcriptomes, including enhanced carbon metabolism, reduced intracellular NADPH synthesis rates, and increased triacylglycerol (TAG) synthesis. By enabling direct screening of metabolic traits from genome-wide mutagenesis libraries, pDEP-DLD-RACS is a powerful platform for synthetic biology.
完整的自发单细胞拉曼光谱以无标记和非侵入性方式捕获代谢表型。然而，从高度异质系统中对稀有靶细胞进行拉曼激活细胞分选（RACS）在很大程度上仍停留在概念层面。在此，我们提出一种基于正介电泳诱导确定性横向位移（pDEP-DLD）的RACS（pDEP-DLD-RACS），其中施加调制的pDEP-DLD力以在宽通道中聚焦、捕获并对快速移动的单细胞进行功能分选。对于产色素和产油酵母，pDEP-DLD-RACS显示出高分选精度（>90%）、高通量（约600个事件/分钟）、高产量（>85%）和长稳定运行时间（约10小时），并且能够在保持细胞全部活力的同时分选稀有细胞。此外，基于细胞内二十二碳六烯酸（DHA）含量，直接从包含>10^6个菌株突变体的全基因组随机诱变文库中进行无标记分选，在两天内仅通过两次RACS运行就产生了DHA生产力高58%的突变细胞，这比传统方法的时间和成本效率高出两个数量级。这种卓越特性源于转录组的全局重塑，包括增强的碳代谢、降低的细胞内NADPH合成速率以及增加的三酰甘油（TAG）合成。通过能够直接从全基因组诱变文库中筛选代谢特性，pDEP-DLD-RACS是合成生物学的一个强大平台。
