Amplifying rare cell surface markers

Abstract

This invention relates generally to a microfluidic device for encapsulation, incubation, and analysis of cell surface markers or secreted molecules from a single cell.

1 . A method of detecting cell-surface expression or secretion of a protein or peptide by a single cell, the method comprising:
 providing a sample comprising a population of cells and reagents sufficient for detection of a protein or peptide, wherein the reagents comprise rolling circle amplification reagents;   dividing the sample into subsamples, such that each subsample contains at most one of the population of cells and each subsample is encompassed a hydrophobic fluid;   maintaining the subsamples under conditions sufficient to allow detection of the protein or peptide; and   detecting the protein or peptide;   thereby detecting expression of the protein or peptide by a single cell.   
     
     
         2 . The method of  claim 1 , wherein the protein or peptide is expressed on the surface of the cell or secreted from the cell. 
     
     
         3 . The method of  claim 1 , wherein the rolling circle amplification reagents comprise phi29 DNA polymerase or circular DNA template or both. 
     
     
         4 . The method of  claim 1 , wherein the reagents comprise antibodies, e.g., antibodies that bind specifically to the protein or peptide. 
     
     
         5 . The method of  claim 1 , wherein the method further comprises sorting the cells based on expression of the protein or peptide. 
     
     
         6 . The method of  claim 5 , wherein sorting the cells comprises using a Fluorescence Activated Cell Sorter (FACS). 
     
     
         7 . The method of  claim 1 , wherein dividing the sample into subsamples comprises combining fluids from multiple inlet channels. 
     
     
         8 . The method of  claim 7 , wherein combining the fluids from multiple inlet channels comprises combining aqueous sample fluid from a first inlet channel with a hydrophobic carrier fluid, e.g., oil, from at least one second inlet channel. 
     
     
         9 . A system for detecting expression of a specific protein or peptide by individual cells of a population of cells, the system comprising:
 a fluid control device configured to divide a sample comprising the population of cells into subsamples, such that each subsample contains at most one of the population of cells and each subsample is encompassed a hydrophobic fluid;   rolling circle amplification reagents for detection of the presence of the specific protein or peptide; and   a device operable to detect the rolling circle amplification reagents in individual subsamples.   
     
     
         10 . The system of  claim 9 , further comprising a cell sorting mechanism operable to sort the subsamples based on measurement of an indicator parameter in each subsample. 
     
     
         11 . The system of  claim 10 , wherein the cell sorting mechanism comprises a fluorescence activated cell sorting mechanism. 
     
     
         12 . A kit comprising:
 a fluid control device configured to divide a sample comprising the population of cells into subsamples, such that each subsample contains at most one of the population of cells and each subsample is encompassed a hydrophobic fluid; and   rolling circle amplification reagents for detection of the presence of the specific protein or peptide.   
     
     
         13 . The kit of  claim 12 , further comprising a device operable to detect the rolling circle amplification reagents in individual subsamples. 
     
     
         14 . The kit of  claim 13 , further comprising a cell sorting mechanism operable to sort the subsamples based on measurement of an indicator parameter in each subsample. 
     
     
         15 . The kit of  claim 14 , wherein the cell sorting mechanism comprises a fluorescence activated cell sorting mechanism.