| Droplets as an important branch of microfluidic chip research have developed rapidly due to their great application potential in single-cell analysis,high-throughput,high-efficiency biochemical reactions and screening research.Among them,the significant application of droplets in the field of biomedicine is single-cell analysis.It has the following advantages:(1)the droplet can provide an independent closed reaction chamber to avoid interference from the surrounding environment.(2)Its small size makes it is suitable for encapsulating a single cell and can quickly accumulate cell secretions to a detectable level,while also greatly reducing the consumption of reagents.(3)The method of generating droplets is high-throughput.(4)The special fluidic environment in the droplet can enhance the mixing efficiency of reagent and speed up the reaction.(5)Droplets have good monodispersity,which can ensure that the volume of each droplet is identical,contributing to precise quantitative analysis,cell or drug screening within the droplets.The droplet needs to combine with some detection technology to realize the qualitative analysis and quantitative detection of single-cell nucleic acid,protein and other metabolites.The detection methods currently used to analyze droplets mainly include non-optical methods(electrochemistry,mass spectrometry,nuclear magnetic resonance spectroscopy,etc.)and optical methods(fluorescence and Raman spectroscopy).Each method has its own advantages and applications.Among them,surface-enhanced Raman scattering(SERS)spectroscopy is an in-situ,noninvasive,label-free,fingerprint technology for biochemical analysis of cells.The combination of SERS and droplet microfluidic technology not only provides a good platform for studying single cells,but also solves some of the core problems in SERS detection.For example,the integrated platform of both can precisely control the aggregation time of metal nanoparticles and adjust the mixing efficiency of metal colloids and analytes via the regulation of flow rate and the design of channel structure,which can solve the low reproducibility of SERS in quantitative analysis.In addition,the droplet microfluidic system can generate a uniform SERS active substrate in a high-throughput manner and complete a symmetrical sample preparation process to achieve more stable and reliable analysis.Therefore,with the combination of SERS and droplet microfluidic technology,it is expected to develop new and efficient biological analysis methods.Based on the above advantages,we developed the SERS-microdroplet platform(combined with three SERS signal amplification strategies)to study proteins,cytokines and metabolites in single cells.The three strategies are:the development of resonance Raman enhanced probes to evaluate the activity of single-cell alkaline phosphatase;the magnetic field-induced SERS signal amplification strategy is used to detect the two cytokines secreted by single cells at the same time;the construction of multiple SERS hot spots to achieve ultrasensitive and simultaneous trace multiple metabolites secreted by a single cell.In order to further improve the problem of poor reproducibility of SERS detection,we used droplets as a template to prepare functional hydrogel microparticles that serve as SERS substrates for the highly sensitive direct detection of small molecules in complex biological samples.Since the microfluidic device can generate uniform droplets,the droplets can be used as the operating unit to prepare SERS active substrates with uniform size and high repeatability in a high-throughput manner.The main content of the paper including the following four aspects:(1)Surface enhanced resonance Raman scattering(SERRS)can significantly enhance the Raman signal of the analyte.In this work,we introduced the SERRS effect into the droplet microfluidic platform to achieve ultrasensitive analysis of very small amounts of alkaline phosphatase(ALP)in a single cell.On the one hand,droplets can encapsulate individual cells and nanoprobes in a highly controlled manner.On the other hand,5-bromo-4-chloro-3-indolyl phosphate(BCIP)can be specifically hydrolysed by ALP and then oxidized by oxygen to form a blue indigo dye derivative(BCI).It is found to exhibit high SERS-activity and a Raman resonance effect when excited under a 632.8 nm laser.The ALP activity of single cells can be evaluated by monitoring the SERRS spectra of the catalytic product BCI,and the heterogeneity of cells is further revealed.(2)In order to detect the small amount of cytokines secreted by single cells in the droplets,we constructed a composite metal nanostructure with high-efficiency plasmonic coupling effect(magnetic-silver sandwich nanostructure).The dynamic aggregation of the composite nanostructures induced by the magnetic field reduces the gap of the nanoparticles and improves the detection sensitivity.The droplets in the system can not only encapsulate a single cell but also quickly accumulate the cytokines secreted by a single cell to a detectable level.The double SERS signal amplification caused by the magnetic field induces the aggregation of nanoparticles and the droplet accumulation effect enables this method to achieve rapid,ultrasensitive and simultaneous detection of two cytokines secreted by single cells.(3)The plasmonic "hot spot" structure constructed by noble metal nanoparticles has been widely used in the highly sensitive SERS detection of biomolecules.In this work,the dual hotspot structure formed by the multifunctional Fe3O4@AgNPs composite nanomaterial realizes the simultaneous and label-free analysis of multiple metabolites secreted by single cells.A variety of metabolites secreted by cells(pyruvate,lactate and adenosine triphosphate)can be directly adsorbed on Fe3O4@AgNPs.These particles can spontaneously aggregate in the droplets due to the strong magnetic field of Fe3O4,resulting in the formation of a large number of hot spots between adjacent silver nanoparticles on the same magnetic particle and silver nanoparticles on different magnetic particles,which greatly enhances the Raman signal of metabolites.Based on the different and distinguishable SERS spectra of the three metabolites,we successfully tracked the three metabolites produced by a single cell in the droplet at the same time.(4)The detections of significant small molecules in biological fluids is always challenging due to the complicated sample pretreatment.Here,a universal SERS-hydrogel micropellet was developed for pretreatment-free,reliable detections of small molecules(glucose and melamine)in complex sample(whole blood and milk).The SERS-hydrogel micropellet has an adjustable pore size,which is acquired by the ultraviolet light solidification of the water-in-oil microdroplets in which the hydrogel monomers and the SERS-active metal nanoparticles(MNPs)were encapsulated.These micropellets have a pore size selectivity to allow small molecules to access in and exclude larger molecules,which is helpful for the high selective,high sensitivity SERS determinations of small molecules.This SERS substrate ensures high reproducibility of SERS detections since MNPs are uniformly dispersed in each micropellet.The hydrogel matrix can well protect MNPs from the surrounding environments to guarantee long-term stability.This method avoids the complicated preprocessing steps,requires a small volume of samples,has a fast response time and low-cost,which provides the possibility for multiplex SERS detections in liquid biopsy. |
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