Methods for Use with Nanoreactors
The invention relates to methods of using nanoreactor technology for sample analysis in microfluidic systems.
1 . A method of washing a nanoreactor containing a particle in a microfluidic system, comprising the steps of:
a) fusing a first nanoreactor containing a particle with a second nanoreactor containing a washing solution to form a combined nanoreactor, wherein the diameter of the second nanoreactor is at least about two fold the diameter of the first nanoreactor; and wherein a molecule within the first nanoreactor is diluted in the combined nanoreactor;
b) splitting the combined nanoreactor into a plurality of nanoreactors; and
c) separating the nanoreactor containing the particle from the plurality of nanoreactors formed in step b) in a microfluidic system.
2 . The method of claim 1 , wherein the diameter of the second nanoreactor is at least about five fold of the diameter of the first nanoreactor.
3 . The method of claim 1 , wherein the diameter of the second nanoreactor is at least about ten fold of the diameter of the first nanoreactor.
4 . The method of claim 1 , wherein the particle comprises a reporter
5 . The method of claim 4 , wherein the reporter is a dye coded bead or a nano-bar code.
6 . The method of claim 1 , wherein the first nanoreactor has a cross-sectional dimension of less than about 100 microns.
7 . The method of claim 1 , wherein the first nanoreactor has a cross-sectional dimension of less than about 30 microns.
8 . The method of claim 1 , wherein the first nanoreactor has a cross-sectional dimension of less than about 10 microns.
9 . The method of claim 1 , wherein the first nanoreactor has a cross-sectional dimension of less than about 3 microns.
10 . The method of claim 1 , wherein the method is used in a washing step of a heterogeneous assay.
11 . The method of claim 1 , wherein the plurality of nanoreactors which do not contain the particle are returned to a starting pool for further analysis.
12 . A method for tracking a sample in a nanoreactor comprising the steps of a) fusing a sample nanoreactor comprising a sample and a reporter with a reagent nanoreactor comprising a particle and a reagent, wherein the reporter comprises a first reactive group, and a second reactive group and a reagent are associated with the particle; wherein the first reactive group reacts with the second reactive group so that the reporter is linked to the particle; and b) tracking the sample nanoreactor that has reacted with the reagent nanoreactor by tracking the nanoreactor containing the reporter.
13 . The method of claim 12 , wherein the reporter is covalently linked to the particle in step a).
14 . The method of claim 12 , wherein the reporter is not covalently linked to the particle in the step a).
15 . The method of claim 12 , wherein the reporter is selected from the group consisting of a dye, a fluorescent agent, an ultraviolet agent, a chemiluminescent agent, a chromophore, a radio-label, a mass spectrometry tag molecule, and a resonance raman tag molecule.
16 . A method of measuring concentration of an analyte in a sample, said method comprising:
a) compartmentalizing a sample into a plurality of nanoreactors, wherein at least about 80% of the nanoreactors contain no more than a single analyte molecule; and
b) detecting the nanoreactor containing at least an analyte molecule; wherein the number of nanoreactors containing analyte molecules indicates the concentration of the analyte in the sample.
17 . The method of claim 16 , wherein at least about 90% of the nanoreactors contain no more than a single analyte molecule.
18 . The method of claim 16 , wherein at least about 95% of the nanoreactors contain no more than a single analyte molecule.
19 . The method of claim 16 , wherein greater than 95% of the nanoreactors contain no more than a single analyte molecule.
20 . The method of claim 16 , wherein the analyte containing nanoreactors are detected by labeling the analyte with a reporter.
21 . The method of claim 16 , wherein the concentration of the analyte in the sample is about 5 aM to about 500 fM.
22 . The method of claim 16 , wherein the analyte is selected from the group consisting of a protein, a peptide, an oligonucleotide, a metabolite, a carbohydrate, a lipid, a ligand, a receptor, and a small molecule.
23 . The method of claim 16 , wherein the sample is a clinical sample selected from the group consisting of blood, plasma, serum, saliva, urine, and spinal fluid.
24 . The method of claim 16 , wherein the nanoreactors have a cross-sectional dimension of less than about 100 microns.
25 . The method of claim 16 , wherein the nanoreactors have a cross-sectional dimension of less than about 30 microns.
26 . The method of claim 16 , wherein the nanoreactors have a cross-sectional dimension of less than about 10 microns.