Electrochemical sensor using intercalative, redox-active moieties

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, 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.

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, 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.

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 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.

A method of detecting one or more base-stacking perturbations electrocatalytically 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 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.

A method of detecting one or more point mutations electrocatalytically within the p53 gene, comprising:

• (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 samnple 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 μM 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.