Single-cell metabolomics could be used to discover the chemical strategies of cells for coping with chemical or environmental stress because metabolomics provides a more immediate and dynamic picture of cell functionality. However, these small-molecule metabolites are the most difficult to measure because they characterize rapid metabolic dynamics, structural diversity, and incapacity to achieve signal amplification or labeling. In order to solve above problems, we presented a surface-enhanced Raman scattering (SERS)-microfluidic droplet platform to realize the label-free simultaneous analysis of multiplexed metabolites at the single-cell level via a versatile magnetic SERS substrate composed by silver nanoparticles (AgNPs, 30 nm)-decorated 400 nm Fe 3 O 4 magnetic microspheres. This metal-magnetic composite substrate is beneficial to efficient adsorption of single cell metabolites, fast separation from complex matrixes, and high SERS sensitivity. Also, the endocytosis effect can be fully prohibited due to its relatively large size. This work achieves labelfree, nondestructive, simultaneous determination of three single-cell metabolites, pyruvate, adenosine triphosphate (ATP), and lactate, owing to their intrinsic SERS fingerprints. The "hotspots" effect induced by the magnetic aggregation of Fe 3 O 4 @AgNPs allows highly sensitive SERS detection. Encapsulating metabolites in such a limited and isolated droplet accelerates the process of diffusion and adsorption equilibriums. Activation with saponin for metabolites was assessed on different cell lines. The SERS-microdroplet platform is a powerful tool for exploring single-cell heterogeneity at the metabolic level.

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A surface-enhanced Raman scattering (SERS)-microfluidic droplet platform for the rapid, ultrasensitive and simultaneous detection of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) secreted by a single cell is presented. The high-throughput water-in-oil droplets containing an individual cell along with four kinds of immune-particles (antibody-conjugated silver nanoparticles or magnetic beads, AgNPs@Ab1 and MNs@Ab2) in each were achieved by a cross-typed microfluidic chip, and then they were captured by a collection channel array for SERS measurements. In the appearance of cytokines secreted by one cell, AgNPs@Ab1 can be linked onto the surface of MNs@Ab2 through the immune-recognition to form an immune-sandwich, which makes the “turn on” SERS signal of the Raman reporters previously laid on the surface of MNs due to the adjacent AgNPs. Furthermore, the second SERS signal amplification is from the magnetic field-induced spontaneous collection effect, which brings 75 times enhancement for SERS signal. Additionally, the encapsulation of cytokines in an isolated droplet permits an accumulation effect of targets with time. Owing to the dual signal enhancement and the accumulation effect, such few cytokines secreted by a single cell become detectable and a limit of detection is achieved as 1.0 fg/mL in one droplet. By using this ultrasensitive SERS-microdroplet method, the VEGF and IL-8 secretions from several cells in one droplet were explored and the data show that the cell–cell interaction may promote angiogenesis of cancer cells through the up-regulation of VEGF and IL-8.

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