Department of Chemistry , Tsinghua University , Beijing 100084 , China.
Department of Precision Instrument , Tsinghua University , Beijing 100084 , China.
Anal Chem. 2019 May 7;91(9):5613-5620. doi: 10.1021/acs.analchem.8b05226. Epub 2019 Apr 22.
Changes of metabolite concentrations in single cells are significant for exploring the dynamic regulation of important biological processes, such as cell development and differentiation. Accurate quantitation of metabolites is essential for single cell analysis. In this work, we proposed a quantitative method for single-cell metabolites by combining microwell array with droplet microextraction-mass spectrometry. The microwell can confine both single cells and extraction solvent in defined space, avoiding the irregular spread of trace internal standard solution during microextraction, which was the key to improve the precision and accuracy of quantification in extremely small-volume single-cell samples. Glucose-phosphate as a crucial metabolite in glycolysis was detected and quantified in single cells at this work. The calibration curve of glucose-phosphate was obtained with a linear range from amol (10 mol) to fmol (10 mol), providing the foundation of metabolite quantitation of single cells. We applied this method to investigate the changes of metabolites including glucose-phosphate, 2-deoxy-d-glucose-phosphate, and ribose-phosphate in single K562 cells stimulated by 2-deoxy-d-glucose. With the robust quantitative capabilities, the developed method holds great potential for studying a drugs' mechanism of action and resistance at single cell level.
单细胞内代谢物浓度的变化对于探索细胞发育和分化等重要生物过程的动态调控具有重要意义。准确地定量代谢物对于单细胞分析至关重要。在这项工作中，我们提出了一种通过微流控芯片与液滴微萃取-质谱联用定量单细胞代谢物的方法。微流控芯片可以将单细胞和萃取溶剂限定在一个确定的空间内，避免了痕量内标溶液在微萃取过程中的不规则扩散，这是提高在极小体积单细胞样品中进行定量的精密度和准确性的关键。本工作以糖酵解过程中的关键代谢物葡萄糖-6-磷酸作为检测对象，在单细胞水平实现了对葡萄糖-6-磷酸的检测和定量。葡萄糖-6-磷酸的校准曲线在 amol（10 mol）到 fmol（10 mol）范围内具有良好的线性关系，为单细胞代谢物定量提供了基础。我们应用该方法研究了 2-脱氧-d-葡萄糖刺激的单个 K562 细胞中包括葡萄糖-6-磷酸、2-脱氧-d-葡萄糖-6-磷酸和核糖-5-磷酸在内的代谢物的变化。该方法具有强大的定量能力，在研究药物作用机制和耐药性方面具有很大的潜力。
