Apparatuses and methods for assaying analytes using photoelectrochemical labels
(19)AUSTRALIAN PATENT OFFICE (54) TitleApparatuses and methods for assaying analytes using photoelectrochemical labels (51)6 International Patent Classification(s) G01N 033/543 CI The present invention provides apparatus, kits and methods for assaying analytes using photoelectrochemical molecules as labels. Specifically, the present invention provides a method for assaying an analyte, comprising: a) contacting a sample suspected of containing an analyte with a reactant capable of binding and/or reacting with the analyte under suitable conditions to allow binding of analyte, if present in the sample, to the reactant; and b) assessing binding and/or reacting between the analyte and the reactant to determine presence and/or amount of analyte in the sample, wherein the reactant, analyte, or additional reactant or additional analyte or analyte analog is labeled with a photoelectrochemically active molecule. The assessing step also comprises converting the photoelectrochemically active molecule with light to an excited state in the presence of an electrode, and assessing an electric current generated by an electron transfer between the excited photoelectrochemically active molecule and the electrode. What is claimed is: 1. A method for assaying an analyte, which method comprises: a) contacting a sample suspected of containing an analyte with a reactant capable of binding and/or reacting with said analyte under suitable conditions to allow binding of said analyte, if present in said sample, to said reactant;STDC0614 and b) assessing binding and/or reacting between said analyte and said reactant to determine presence and/or amount of said analyte in said sample, wherein said reactant, said analyte, or additional reactant or additional analyte or analyte analog is labeled with a photoelectrochemically active molecule and said assessing in step b) comprises converting said photoelectrochemically active molecule with light to an excited state in the presence of an electrode and assessing an electric current generated by an electron transfer between said excited photoelectrochemically active molecule and said electrode.
2. The method of claim 1, wherein the analyte is selected from the group consisting of a cell, a cellular organelle, a virus, a molecule and an aggregate or complex thereof.
3. The method of claim 2, wherein the cell is selected from the group consisting of an animal cell, a plant cell, a fungus cell, a bacterium cell, a recombinant cell and a cultured cell.
4. The method of claim 2, wherein the cellular organelle is selected from the group consisting of a nuclei, a mitochondrion, a chloroplast, a ribosome, an ER, a Golgi apparatus, a lysosome, a proteasome, a secretory vesicle, a vacuole and a microsome.
5. The method of claim 2, wherein the molecule is selected from the group consisting of an inorganic molecule, an organic molecule and a complex thereof.
6. The method of claim 5, wherein the organic molecule is selected from the group consisting of an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid and a complex thereof.
7. The method of claim 1, wherein the analyte is selected from the group consisting of a hormone, a cancer marker, a steroid, a sterol, a pharmaceutical compound, a metabolite of a pharmaceutical compound and a complex thereof. <Desc/Clms Page number 24> 8. The method of claim 1, wherein the sample is mammalian sample.
9. The method of claim 8, wherein the mammal is selected from the group consisting of bovine, goat, sheep, equine, rabbit, guinea pig, murine, human, feline, monkey, dog and porcine.
10. The method of claim 1, wherein the sample is a clinical sample.
11. The method of claim 10, wherein the clinical sample is selected from the group consisting of serum, plasma, whole blood, sputum, cerebral spinal fluid, amniotic fluid, urine, gastrointestinal contents, hair, saliva, sweat, gum scrapings and tissue from biopsies.
12. The method of claim 10, wherein the clinical sample is a human clinical sample.
13. The method of claim 1, wherein the sample is a body fluid sample.
14. The method of claim 1, wherein the reactant binds and/or reacts specifically with the analyte.
15. The method of claim 1, wherein the reactant is selected from the group consisting of a cell, a cellular organelle, a virus, a molecule and an aggregate or complex thereof.
16. The method of claim 1, wherein the reactant is an antibody.
17. The method of claim 1, wherein the reactant is a nucleic acid.
18. The method of claim 1, wherein the photoelectrochemically active molecule is a metal polypyridyl complex.
19. The method of claim 1, wherein the photoelectrochemically active molecule has the formula, M [(Ll) m (L2) n (L3) o (Pl) p (P2) q (P3-R-X)], wherein M is a metal ion, Ll, L2, L3 are mono-dentate ligands of M, P1, P2, P3 are poly-dentate ligands of M, R is a spacer, X is a reactive chemical group capable of linking the photoelectrochemically active molecule to a reactant or an analyte, m, n, o, p and q are either zero or a positive integer, and total number of bonds provided by all the ligands equal to the coordination number of M.
20. The method of claim 19, wherein the M is selected from the group consisting of osmium, ruthenium, zinc, magnesium and aluminum.
21. The method of claim 19, wherein the L1, La or L3 is a cyanide or a thiocyanide. <Desc/Clms Page number 25> 22. The method of claim 19, wherein the Ll, L2, L3 are the same or different.
23. The method of claim 19, wherein the Pi, ? 2 or P3 is a nitrogen-containing aromatic heterocycle.
24. The method of claim 23, wherein the nitrogen-containing aromatic heterocycle is selected from the group consisting of bipyridyl, bipyrazyl, terpyridyl, phenanthrolyl and phthalocyanine.
25. The method of claim 24, wherein the bipyridyl, bypyrazyl, terpyridyl and phenanthrolyl are unsubstituted or substituted.
26. The method of claim 25, wherein the substituted group is selected from the group consisting of an alkyl, an aryl, an aralkyl, a carboxylate, a carboxyaldehyde, a carboxamide, a cyano, an amino, a hydroxycarbonyl, a hydroxyamino, an aminocarbony, an amidine, an guanidium, an ureide, a sulfur-containing group, a phosphorous- containing group and a carboxylate ester of N-hydroxysuccinimide.
27. The method of claim 19, wherein the R is a C2 to C12 alkyl or poly (ethylene glycol).
28. The method of claim 19, wherein the R is a poly (ethylene glycol).
29. The method of claim 19, wherein the X is selected from the group consisting of a N-hydroxysuccinimide ester, a sulfhydryl, an epoxide, an aldehyde, a maleic anhydride, an imidoester, an amino, a carboxyl, an iosthiocyanate, a maleimide, a haloacetyl, a hydrazide and a phosphoramidite.
30. The method of claim 1, wherein the reactant or the analyte is labeled with a photoelectrochemically active molecule.
31. The method of claim 1, which is conducted in a competition assay format wherein the reactant and the analyte from the sample are not labeled and a separate analyte or analyte analog labeled with a photoelectrochemically active molecule is used.
32. The method of claim 1, which is conducted in a sandwich assay format wherein a first reactant and the analyte from the sample are not labeled and a second reactant labeled with a photoelectrochemically active molecule is used.
33. The method of claim 1, wherein the binding or reacting between the analyte and the reactant is assessed by a format selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescent assay <Desc/Clms Page number 26> (IFA), nephelometry, flow cytometry assay, chemiluminescence assay, lateral flow immunoassay, p-capture assay, inhibition assay, energy transfer assay, avidity assay, turbidometric immunoassay and time resolved amplified cryptate emission (TRACE) assay.
34. The method of claim 1, wherein the assessing in step b) comprises converting the photoelectrochemically active molecule with light to an excited state in the presence of an electrode and a regenerating agent, and assessing an electric current generated by an electron transfer between the excited photoelectrochemically active molecule and the electrode, and the oxidized or reduced photoelectrochemically active molecule at a ground state resulted from the electron transfer is reduced or oxidized by the regenerating agent to a reduced or oxidized photoelectrochemically active molecule at a ground state that can be again excited with light.
35. The method of claim 34, wherein the regenerating agent is a hydroquinone.
36. A kit for assaying an analyte, which kit comprises: a) a reactant capable of binding and/or reacting with an analyte under suitable conditions to allow binding of said analyte, if present in a sample, to said reactant; and b) means for assessing binding and/or reacting between said analyte and said reactant to determine presence and/or amount of said analyte in said sample, wherein said reactant, said analyte, or additional reactant or additional analyte or analyte analog is labeled with a photoelectrochemically active molecule and said assessing in step b) comprises converting said photoelectrochemically active molecule with light to an excited state in the presence of an electrode and assessing an electric current generated by an electron transfer between said excited photoelectrochemically active molecule and said electrode.
37. The kit of claim 36, which further comprises a regenerating agent to reduce or oxidize the oxidized or reduced photoelectrochemically active molecule at a ground state resulted from the electron transfer to a reduced or oxidized photoelectrochemically active molecule at a ground state that can be excited again with light.
38. The kit of claim 36, which further comprises an instruction for using the kit to assay the analyte. <Desc/Clms Page number 27> 39. An apparatus for assaying an analyte, which apparatus comprises: a) a reactant capable of binding and/or reacting with an analyte under suitable conditions to allow binding of said analyte, if present in a sample, to said reactant; b) a photoelectrochemically active molecule attached to a reactant, an analyte or an analyte analog; c) an electrode suitable for assessing an electric current generated by an electron transfer between an excited photoelectrochemically active molecule and said electrode ; d) a regenerating agent to convert oxidized or reduced photoelectrochemically active to a ground state that can be again excited with light; e) an electrochemical-cell having a wall transparent to light of a spectrum which will excite said photoelectrochemically active molecule;STDC0542 and f) light means which further comprise a light source having a spectrum capable of exciting said photoelectrochemically active molecule, and means for isolating said spectrum if necessary. wherein the energy level of said electrode, the redox potential of said regenerating agent and the distance from said photoelectrochemically active molecule to said electrode are adjusted to ensure measurement of an electric current generated by an electron transfer between said excited photoelectrochemically active molecule and said electrode.
40. The apparatus of claim 39, wherein the light source is selected from the group consisting of a hollow cathode lamp, a xenon lamp, a mercury-xenon lamp, a metal halide lamp, a light-emitting diode and a laser.
41. The apparatus of claim 39, which further comprises a means for distinguishing the electron transfer between the excited photoelectrochemically active molecule and the electrode from other electron transfer (s).
42. The apparatus of claim 41, wherein the means for distinguishing the electron transfer between the excited photoelectrochemically active molecule and the electrode from other electron transfer (s) comprises a light beam chopper, filters, lenses and lock-in amplifier.
43. The apparatus of claim 41, wherein the means for distinguishing the electron transfer between the excited photoelectrochemically active molecule and the <Desc/Clms Page number 28> electrode from other electron transfer (s) comprises a first working electrode exposed to said light and a second working electrode in the dark, such that the difference in current signals is the signal due to the presence of light.
44. The apparatus of claim 39, wherein the light means in f) further comprises a means for isolating the spectrum.
45. The apparatus of claim 44, wherein the means for isolating the spectrum comprises a monochromater or an optic filter.