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  Methods for the electrochemical detection of multiple target compoundsUSPTO Application #: 20060292624Title: Methods for the electrochemical detection of multiple target compounds Abstract: A method of detecting two different target molecules through a single electrode is carried out by (a) providing a conductive oxidation-reduction reaction detection electrode; (b) contacting a sample suspected of containing a first and second target molecule to the electrode under conditions in which the first and second target molecules are deposited on the electrode, wherein the first target molecule comprises a first label and the second target molecule comprises a second label; (c) contacting to the electrode a first transition metal complex that oxidizes the first preselected label in a first oxidation-reduction reaction and a second transition metal complex that oxidizes the first and second labels in a second oxidation-reduction reaction, with the first and second oxidation-reduction reactions producing different detectable signals; (d) detecting the presence of the first target molecule by detecting the first oxidation-reduction reaction; and(e) detecting the presence of the second target molecule by detecting the second oxidation-reduction reaction. Devices for carrying out the method are also described. (end of abstract)
Agent: Myers Bigel Sibley & Sajovec - Raleigh, NC, US Inventors: H. Holden Thorp, Ivana V. Yang, David H. Stewart, John W. Groelke, Veronika A. Szalai USPTO Applicaton #: 20060292624 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060292624. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application 60/324,377, filed on Sep. 24, 2001, the disclosure of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION [0002] The present invention concerns methods for the electrochemical detection of members of specific binding pairs. BACKGROUND OF THE INVENTION [0003] The detection of individual DNA sequences in heterogenous samples of DNA provides a basis for identifying genes, DNA profiling, and novel approaches to DNA sequencing. One approach to DNA hybridization detection involves the use of surface bound DNA sequences which can be assayed using an analytical response that indicates hybridization of the surface-bound oligomer to a sequence in the heterogeneous sample. These prior analytical methods generally involve laser-induced fluorescence arising from a covalently attached label on the target DNA strand, which methods are not sensitive to single-base mismatches in the surface-bound duplex. For example, U.S. Pat. Nos. 5,143,854 and 5,405,783 to Pirrung et al.; Fodor, et al., Nature 364:555 (1993); Bains, Angew. Chem. 107:356 (1995); and Noble, Analytical Chemistry 67(5):201A (1995) propose surfaces or "chips" for this application. In an alternate method, proposed by Hall, et al., Biochem. and Molec. Bio. Inter. 32(1):21 (1994), DNA hybridization is detected by an electrochemical method including observing the redox behavior of a single stranded DNA as compared to a double stranded DNA. This technique is also not sensitive to single-base mismatches in the DNA sample. [0004] U.S. Pat. Nos. 5,871,918 and 6,132,971 to Thorp et al. describe methods and apparatus for electrically detecting a target molecule by detecting a preselected base in an oxidation-reduction reaction. The methods and apparatus disclosed therein may be used in a variety of applications, including DNA sequencing, diagnostic assays, and quantitative analysis. The methods can advantageously be implemented in a variety of different assay formats and structures, including multi-well plates, with a different assay carried out in each well. However, these references do not describe how to carry out multiple assays in a single well. SUMMARY OF THE INVENTION [0005] A first aspect of the present invention is a method of detecting two different target molecules through a single common electrode. In general, the method comprises the steps of: [0006] (a) providing a conductive oxidation-reduction reaction detection electrode; [0007] (b) contacting (e.g., by affinity binding, precipitation, etc.) a sample to said electrode, (as discussed further below), said sample suspected of containing a first and second target molecule, under conditions in which the first and second target molecules are deposited on the electrode, [0008] wherein the first target molecule comprises a first preselected label, the second target molecule comprises a second preselected label, and the first and second preselected labels are different; [0009] (c) simultaneously contacting to the electrode (i) a first transition metal complex that oxidizes the first preselected label in an oxidation-reduction reaction to cause a first oxidation-reduction reaction between the first transition metal complex and the first preselected label and (ii) a second transition metal complex that oxidizes the first and second preselected labels in an oxidation-reduction reaction to cause a second oxidation-reduction reaction between the second transition metal complex and the second preselected label, from which preselected labels there is electron transfer to the corresponding transition metal complexes resulting in regeneration of the reduced form of the corresponding transition metal complex as part of a catalytic cycle, with the first and second oxidation-reduction reactions producing different detectable signals; [0010] (d) detecting the presence of the first target molecule by detecting the the first oxidation-reduction reaction; and [0011] (e) detecting the presence of the second target molecule by detecting the second oxidation-reduction reaction. [0012] The contacting step may be carried out by any suitable means, such as by sandwich assay, competitive assay, direct assay, competitive assay for immobilized target substance, or binding interaction assay, all of which are discussed in greater detail in section H below. [0013] In one embodiment of the foregoing, the sample is suspected of containing a third target molecule; the third target molecule comprises a third preselected label that is different from the first and second preselected labels; the contacting step (c) further comprises contacting to the electrode (iii) a third transition metal complex that oxidizes the first, second and third preselected labels in an oxidation-reduction reaction to cause a third oxidation-reduction reaction between the third transition metal complex and the third preselected label, with the first, second and third oxidation-reduction reactions producing different detectable signals; and the method further comprising the step of: (f) detecting the presence of the third target molecule by detecting the third oxidation-reduction reaction. [0014] In another particular embodiment of the foregoing, the sample is suspected of containing a fourth target molecule; the fourth target molecule comprises a fourth preselected label that is different from the first, second and third preselected labels; the contacting step (c) further comprises contacting to the electrode (iv) a fourth transition metal complex that oxidizes the first, second, third and fourth preselected labels in an oxidation-reduction reaction to cause a fourth oxidation-reduction reaction between the fourth transition metal complex and the fourth preselected label, with the first, second, third and fourth oxidation-reduction reactions producing different detectable signals; the method further comprising the step of: (g) detecting the presence of the fourth target molecule by detecting the fourth oxidation-reduction reaction. [0015] A second aspect of the present invention is a microelectronic device useful for the electrochemical detection of at least two different members of at least two different binding pairs. The device comprises: [0016] (a) a microelectronic substrate; [0017] (b) a conductive oxidation-reduction detection electrode on the substrate; [0018] (c) a first member of a first specific binding pair (for example, a protein, peptide or oligonucleotide probe) immobilized on a non-conductive layer, which first member binds with a second member of the first specific binding pair present in a sample, the first member of the first binding pair being adjacent (or sufficiently close to) the detection electrode so that an oxidation-reduction reaction occurring upon application of a potential to the detection electrode is detectable; and [0019] (d) a first member of a second specific binding pair (for example, a protein, peptide or oligonucleotide probe) immobilized on a non-conductive layer that binds with a second member of the second specific binding pair present in a sample, the first member of the second binding pair being adjacent the detection electrode so that an oxidation-reduction reaction occurring upon application of a potential to the detection electrode is detectable; wherein the first member of the first binding pair and the first member of the second binding pair are different. [0020] In one particular embodiment of the foregoing, the device further comprises a first member of a third specific binding pair (for example, a protein, peptide or oligonucleotide probe) immobilized on a non-conductive layer that binds with a second member of the third specific binding pair present in a sample, the first member of the third binding pair being adjacent the detection electrode so that an oxidation-reduction reaction occurring upon application of a potential to the detection electrode is detectable; wherein the first member of the first binding pair, the first member of the second binding pair, and the first member of the third binding pair are different. The device may further include a first member of a fourth binding pair adjacent the detection electrode in like manner as described with the other binding pairs. Continue reading... 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