Droplet Microfluidic Based On Pickering Emulsion For High-Throughput Single Molecule/Cell Analysis

With the development of single molecule and single cell technology,a new situation of biology reaction and life action research is springing up.Comparing with the conventional bulk analysis,single molecule and cell technology understand the heterogeneity of them thoroughly,which provided abundant and precision information.Droplet microfluidics,owning to its advantage of small volume,high throughput,speed analysis,convenient integration etc.,provided a good platform for single molecule and cell analysis.However,the existed molecular surfactants limited its application due to the shortcomings of tedious synthesis,poor compatibility and easy cross-talk.Therefore,we developed a new nanoparticle surfactant(F-SiO2 NPs)to generate colloidosomes by self-assembly of nanoparticle surfactant based on Pickering emulsion,solved above shortcomings of molecular surfactant.In addition,we combined F-SiO2 NPs with microfluidic technology and applied it into the single molecule and single cell research.(1)colloidosomes for single molecule detectionNucleic acid is the genetic material of all life and its detection is meaningful to understand the essence of life.Digital PCR as the third generation of nucleic acid amplification technology enabling to perform digital detection and absolute quantification,accelerating the research of nucleic acid.However,the deficiency of surfactant limited its wider application.Hence,we developed a facile method to synthesize a new nanoparticle surfactant to overcome the shortcomings of difficult synthesis,insufficient stabilization,and poor compatibility of molecular surfactant.In addition,we exploited a colloidosome digital PCR(cdPCR)method using F-SiO2 NPs,and realized nucleic acid digital detection.Our studies show that the cdPCR have better compatible with the commercial PCR Master Mix,comparing with the molecular surfactant.In addition,the results of digital detection present a good linear relation when a series of concentration of DNA templates encapsulated in the droplets,and are consistent with the commercial digital platform(Bio-Rad),which verified the precision and accuracy of this methodNucleic is the genetic information carrier,while the protein is the function executor in life.Single protein analysis facilitates to comprehend the phenotype of life.Therefore,we developed a colloidosomes based protein digital detection method,which solved the cross-talk problem between the droplets.According to generating massively monodispersed colloidosomes by microfluidics that encapsulated the single molecule level(3-galactosidase and probes,and utilizing the catalytic of enzyme to produced intense fluorescence signals,we realized the high fidelity of protein digital detection.In addition,we find that the fluorescence intensity of colloidosomes that encapsulated the β-galactosidase increased difference,indicating the heterogeneity of the catalytic activity of β-galactosidase,which verified the colloidosome based system could using for the dynamitic analysis of enzyme.(2)colloidosome for single cell analysisSingle molecular technology makes scientist understand the biology reaction better,however,plenty of life actions are conducted based on the single cell.Therefore,it is important to study the life action in single cell level.In this paper,we firstly introduced F-SiO2 NPs into the heterogenetic analysis of the secretory protein MMP-9 at the single cell level.Firstly,the tumor cells and probes were encapsulated in the colloidosomes,and making use of the MMP-9 capability of hydrolysis probe to enhance the fluorescence signals.The heterogeneity of MMP-9 secreted by every single cell encapsulated in the droplets can be detection according to analyzing the fluorescence intensity of the droplets.The droplet-based method solved that the secretory protein analysis based on the flow cytometry are difficult to addressable,which provided a good platform to study the cell heterogeneity of secretory protein.