Substrate-mediated reactors for bioassays
The present invention provides, among other things, methods, compositions and systems for highly efficient, robust, multiplex analysis of biomolecules based on substrate-mediated compartmentalization in conjunction with controlled release of assay reagents for interference-free detection of biomolecules.
1. A method for analyzing biomolecules, comprising:
a) incubating a sample with a plurality of multifunctional substrates, wherein each multifunctional substrate comprises a target capture region bearing one or more capture moieties, each of which specifically binds a target biomolecule, and a reagent storage region bearing one or more detection reagents through a releasable means, under conditions that permit binding between the target biomolecule and the capture moieties;
b) contacting an immiscible fluid with the plurality of multifunctional substrates in a carrier fluid, thereby forming a plurality of compartments, each comprising an individual multifunctional substrate, and wherein the shape of each compartment is substantially defined by the shape of the multifunctional substrate;
c) releasing the one or more detection reagents from the reagent storage region such that the detection reagents bind to the target biomolecule bound to the capture moieties within an individual compartment; and
d) analyzing the binding between the detection reagents and the biomolecule bound to the capture moieties, thereby analyzing the presence or amount of the target biomolecule in the sample.
2. The method of claim 1 , wherein the multifunctional substrates are multifunctional microparticles.
3. The method of claim 1 , wherein the multifunctional substrates are made of hydro gel.
4. The method of any claim 2 , wherein the microparticles are non-spherical particles.
5. The method of claim 1 , wherein the plurality of multifunctional substrates comprise polydisperse microparticles between 1 μm and 500 μm in their longest dimension.
6. The method of claim 1 , wherein the one or more capture moieties are selected from antibodies, nanobodies, oligonucleotide probes, peptide nucleic acids, small molecules, aptamers, cells, bacteria, viruses, organelles, peptides, or combination thereof.
7. The method of claim 1 , wherein the one or more detection reagents are selected from detection antibodies, nano bodies, enzymes, PCR primers, proteins, oligonucleotides, peptides, aptamers, small molecules, other chemical compounds, or combination thereof.
8. The method of claim 7 , wherein the one or more detection reagents are labeled with a detection moiety selected from fluorophores, chromophores, radioisotopes, biotin, enzyme products, antibodies, quantum dots, molecular beacons, and/or aptamers.
9. The method of claim 1 , wherein the multifunctional substrates comprise one or more hydrophobic regions.
10. The method of claim 1 , wherein the immiscible fluid is an oil and the carrier fluid is an aqueous fluid.
11. The method of claim 1 , wherein the immiscible fluid is an aqueous fluid and the carrier fluid is an oil.
12. The method of claim 1 , wherein the target biomolecule is a protein, a nucleic acid, a cell, a bacteria, or a chemical compound.
13. The method of claim 1 , wherein the releasable means are selected from reversible interactions, an irreversible reaction, reversible crosslinkers, or photocleavable linkers.
14. The method of claim 13 , wherein the reversible interactions are selected from electrostatic interactions, physical interactions, magnetic interactions, chemical interactions, or nucleic acid duplex formation.
15. The method of claim 1 , wherein the one or more detection reagents are released from the reagent storage region into the compartment in a controlled manner.
16. The method of claim 15 , wherein the controlled manner comprises using heat, ultraviolet light, visible light, microwave radiation, enzymatic catalysis, pH, or a specific chemical agent as a stimulus.
17. The method of claim 1 , wherein the contacting step comprises a step of emulsification.
18. The method of claim 1 , wherein the each multifunctional substrate further comprises an encoding region.
19. The method of claim 1 , wherein the multifunctional substrates are analyzed with a flow cytometer or microarray scanner based on a fluorescent or visible identifier.