Ultra-sensitive detection of analytes
The present invention relates to screening methods, compositions, and kits for detecting for the presence or absence of one or more target analytes, e.g. biomolecules, in a sample. In particular, the present invention relates to methods that utilize nanoparticle probes in an in-solution homogeneous assay system for high-sensitivity detection of target proteins or nucleic acids based on flow analysis of single particles.
1 . A method for detecting for the presence of one or more target analytes in a sample comprising the steps of:
a) providing a plurality of nanoparticle probes conjugated to binding moieties capable of binding to a first binding site of the target analyte, wherein the nanoparticle probes comprise a metallic material and have an average diameter of less than 200 nanometers;
b) providing a capture surface comprising binding moieties capable of binding to a second binding site of the target analyte;
c) contacting the nanoparticle probes and capture surface with a sample believed to contain target analytes under conditions effective to allow for binding of the target analyte with the nanoparticle probes and the capture surface to form a complex in the presence of the target analyte;
d) optionally washing the capture surface containing the complex formed in (c) to remove all non-bound nanoparticle probes;
e) releasing the captured nanoparticle probes from the capture surface;
f) subjecting the released nanoparticle probes to confinement conditions under which individual nanoparticle probes can be detected;
g) irradiating nanoparticle probes in the confinement conditions with a light beam; and
h) measuring scatter light generated in step (g) to determine the number of released nanoparticle probes as an indicator of the presence of target analyte in the sample.
2 . A method for detecting for the presence of one or more target analytes in a sample comprising the steps of:
a) providing a first nanoparticle probe conjugated to binding moieties capable of binding to a first binding site of the target analyte, wherein the nanoparticle probes comprise a metallic material and have an average diameter of less than 200 nanometers;
b) providing a second nanoparticle probe conjugated to binding moieties capable of binding to a second binding site of the target analyte, wherein the nanoparticle probes comprise a metallic material and have an average diameter of less than 200 nanometers;
c) contacting the first and second nanoparticle probes with a sample believed to contain target analytes under conditions effective to allow for binding of the target analyte with the binding moieties on the first and second nanoparticle probes to form a complex in the presence of the target analyte;
d) subjecting the sample-nanoparticle probe mixture in (c) to confinement conditions under which individual nanoparticle probes or nanoparticle probe-target complexes can be detected;
e) irradiating the complex with light of a frequency range that covers the plasmon resonance frequency of the nanoparticles; and
f) measuring scatter light frequency to differentiate single from complexed nanoparticles,
whereby the presence of complexed particles is indicative of the presence of the target analyte in the sample.
3 . The method of claim 1 or 2 , wherein the nanoparticle probes bind to the target analyte indirectly via specific linker molecules.
4 . The method of claim 1 or 2 , wherein the binding moieties comprise oligonucleotides, antibodies, aptamers, or some combination thereof.
5 . The method of claim 1 or 2 , wherein the nanoparticle probes are about 30 to about 150 nm in diameter.
6 . The method of claim 1 or 2 , wherein the target analyte is a protein or hapten and the binding moieties are antibodies.
7 . The method of claim 6 , wherein the antibodies are polyclonal antibodies or monoclonal antibodies.
8 . The method of claim 1 or 2 , wherein the target analyte is a sequence from a genomic DNA sample and the binding moieties are oligonucleotides, the oligonucleotides having a sequence that is complementary to at least a portion of the genomic sequence.
9 . The method of claim 8 , wherein the genomic DNA is eukaryotic, bacterial, fungal or viral DNA.
10 . The method of claim 1 or 2 , wherein the target analyte is a sequence from episomal DNA sample and the binding moieties are oligonucleotides, the oligonucleotides having a sequence that is complementary to at least a portion of the episomal DNA sequence.
11 . The method of claim 1 or 2 , wherein the confinement conditions are generated by flow cytometry.
12 . The method of claim 1 or 2 , wherein the confinement conditions are generated by capillary eletrophoresis.
13 . The method of claim 1 or 2 , wherein at least one of the nanoparticle probes is further labeled with a Raman active group.
14 . The method of claim 13 , wherein more than one type of Raman active group is used in equal or different concentrations.
15 . The method of claim 1 or 2 , wherein the nanoparticle probes comprise gold, silver, copper, or platinum.
16 . The method of claim 1 or 2 , wherein the nanoparticle probes are core-shell nanoparticles.
17 . The method of claim 1 , wherein the target analyte binds to the capture surface indirectly via specific linker molecules.
18 . The method of claim 1 , wherein the capture surface is a microtiter well.
19 . The method of claim 1 , wherein the capture surface containing the complex formed in (c) is isolated from all non-bound nanoparticle probes.
20 . The method of claim 19 , wherein the capture surface is a magnetic bead.
21 . The method of claim 1 , wherein the plurality of nanoparticle probes comprises nanoparticle probes of different shapes, each differently shaped nanoparticle probe being conjugated to binding moieties that bind to a different target analyte, and wherein each differently shaped nanoparticle probe creates unique scatter light when irradiated, thereby indicating the presence of the target analyte to which it binds.
22 . The method of claim 1 , wherein the plurality of nanoparticle probes comprises nanoparticle probes of different materials, each nanoparticle probe of different material being conjugated to binding moieties that bind to a different target analyte, and wherein each nanoparticle probe of different material creates unique scatter light when irradiated, thereby indicating the presence of the target analyte to which it binds.
23 . The method of claim 1 , wherein the plurality of nanoparticle probes comprises nanoparticle probes of different sizes, each nanoparticle probe of different size being conjugated to binding moieties that bind to a different target analyte, and wherein each nanoparticle probe of different size creates unique scatter light when irradiated, thereby indicating the presence of the target analyte to which it binds.
24 . The method of claim 1 or 2 further comprising a step of providing one or more labeled microbeads that can bind to either the target analyte or to a nanoparticle probe, thereby capable of forming a complex with the nanoparticle probes and the target analyte.
25 . The method of claim 24 , wherein at least one microbead is fluorescently labeled.