Electrochemical sensor using intercalative, redox-active moieties
First Claim
1. A method of detecting one or more base-stacking perturbations in a target sequence comprising:
- (a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex;
(b) depositing said first complex onto an electrode or an addressable multielectorde array;
(c) adding an intercalative, redox-active moiety to said first complex to form a second complex; and
(d) measuring an electron transfer event between said electrode or addressable multielectrode array and said intercalative, redox-active moiety as an indication for the presence or absence of said base-stacking perturbations.
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Abstract
Compositions and methods for electrochemical detection and localization of genetic point mutations and other base-stacking perturbations within oligonucleotide duplexes adsorbed onto electrodes and their use in biosensing technologies are described. An intercalative, redox-active moiety (such as an intercalator or nucleic acid-binding protein) is adhered and/or crosslinked to immobilized DNA duplexes at different separations from an electrode and probed electrochemically in the presence or absence of a non-intercalative, redox-active moiety. Interruptions in DNA-mediated electron-transfer caused by base-stacking perturbations, such as mutations or binding of a protein to its recognition site are reflected in a difference in electrical current, charge and/or potential.
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Citations
36 Claims
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1. A method of detecting one or more base-stacking perturbations in a target sequence comprising:
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(a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex;
(b) depositing said first complex onto an electrode or an addressable multielectorde array;
(c) adding an intercalative, redox-active moiety to said first complex to form a second complex; and
(d) measuring an electron transfer event between said electrode or addressable multielectrode array and said intercalative, redox-active moiety as an indication for the presence or absence of said base-stacking perturbations. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method of detecting one or more base-stacking perturbations in a target sequence comprising:
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(a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex, wherein said nucleic acids are comprised of 12 to 25 nucleotides, and wherein one of said single-stranded nucleic acids is derivatized with a thiol-terminated linker comprised of 5 to 20 σ
bonds;
(b) depositing said first complex onto an addressable gold multielectrode array;
(c) adding daumomycin to said electrode-bound first complex to form a second complex; and
(d) measuring an electron transfer event between said addressable gold multielectrode array and daunomycin as an indication for the presence or absence of said base-stacking perturbations. - View Dependent Claims (17)
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18. A method of detecting one or more base-stacking perturbations in a target sequence comprising:
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(a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex, wherein said nucleic acids are comprised of 12 to 25 nucleotides, and wherein one of said single-stranded nucleic acids is derivatized with a amine-terminated linker comprised of 5 to 20 σ
bonds;
(b) depositing said first complex onto an addressable carbon multielectrode array;
(c) adding daunomycin to said electrode-bound first complex to form a second complex; and
(d) measuring an electron transfer event between said addressable carbon multielectrode array and daumonycin as an indication for the presence or absence of said base-stacking perturbations. - View Dependent Claims (19)
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20. A method of detecting one or more base-stacking perturbations in a target sequence comprising:
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(a) hybridizing a first single-stranded nucleic acid to a second single-stranded nucleic acid to form a first complex of 12 to 25 nucleotides in length, wherein said first complex contains a first single-stranded overhang of known sequence composition, and wherein said first single-stranded overhang can be the same or different;
(b) depositing said first complex onto an addressable multielectrode array, wherein said addressable multielectrode array is comprised of a monolayer of oligonucleotide duplexes of 5 to 10 base-pairs in length deposited onto said array, wherein each of said oligonucleotide duplexes is derivatized on one end with a functionalized linker and on the opposite end with a second single-stranded overhang complementary to said first single-stranded overhang;
(c) adding daumomycin to said electrode-bound first complex to form a second complex; and
(d) measuring an electron transfer event between said addressable multielectrode array and daunomycin as an indication for the presence or absence of said base-stacking perturbations. - View Dependent Claims (21)
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22. A method of detecting one or more base-stacking perturbations electrocatalytically in a target sequence comprising:
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(a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex;
(b) depositing said first complex onto an electrode or an addressable multielectrode array to form a second complex;
(c) immersing said second complex in a solution comprising an intercalative, redox-active species and a non-intercalative, redox-active species; and
(d) measuring an electron transfer event as an indication for the presence or absence of said base-stacking perturbations. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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36. A method of detecting one or more point mutations electrocatalytically within the p53 gene, comprising:
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(a) forming a set of oligonucleotide duplexes of approximately 20 base-pairs in length corresponding to the approximately 600 base pair long region within exons 5 through 8 of the p53 gene, wherein said oligonucleotide duplexes are derivatized with a thiol-terminated linker comprised of 5 to 20 σ
bonds;
(b) depositing said oligonucleotide duplexes onto an addressable gold multielectrode array;
(c) denaturing said oligonucleotide duplexes by immersing them in aqueous solution at elevated temperatures for 1 minute and removing the complementary strands to form a single-stranded monolayer;
(d) exposing said single-stranded monolayer to a first sample comprising PCR-amplified and fragmented p53 gene DNA under hybridizing conditions to form a first complex;
(e) rinsing said electrode-bound first complex to remove any unhybridized material;
(f) immersing said electrode-bound first complex into a dilute solution comprised of 1.0 μ
M methylene blue and 1.0 mM ferricyanide;
(g) measuring an electron transfer event as an indication for the presence or absence of said point mutations;
(h) denaturing said electrode-bound first complex by immersing it in an aqueous solution at elevated temperatures for 1 minute, and regenerating said single-stranded monolayer;
(i) exposing said single-stranded monolayer to a second sample containing PCR-amplified and fragmented p53 gene DNA under hybridizing conditions to form a second complex; and
(k) repeating steps (e) through (h) using several sample solutions.
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Specification