Assays and apparatus for detecting electrochemical active markers in an electric field

A method of detecting a target nucleic acid sequence in a nucleic acid solution, comprising:

• contacting the nucleic acid solution with one or more oligonucleotide probes each labeled with a metallocene moiety, wherein each oligonucleotide probe is covalently linked to a metallocene moiety;
• providing conditions at which one or more of the oligonucleotide probes hybridize with a complementary target sequence present in the nucleic acid solution and form a double stranded hybridized nucleic acid, wherein each of the one or more oligonucleotide probes is complementary along its length to the target sequence;
• degrading the double stranded hybridized nucleic acid by digestion with a duplex specific nuclease to form at least one of a mononucleotide degraded probe labeled with the metallocene moiety and a dinucleotide degraded probe labeled with the metallocene moiety, wherein the duplex specific nuclease is capable of degrading the oligonucleotide probe only when the oligonucleotide probe is present in the double stranded hybridized nucleic acid and, in the presence of the double stranded hybridized nucleic acid, the duplex specific nuclease degrades the oligonucleotide probe to form at least one of a mononucleotide degraded probe labeled with the metallocene moiety and a dinucleotide degraded probe labeled with the metallocene moiety;
• and
• electrochemically detecting a signal based on redox characteristics of the mononucleotide probe labeled with the metallocene moiety, the dinucleotide degraded probe labeled with the metallocene moiety, and any unhybridized oligonucleotide probe in the solution, wherein the signal is influenced by at least the size of the probe to which the metallocene moiety is attached;
• and wherein the signal of the mononucleotide probe or the dinucleotide probe detects the target nucleic acid sequence in the nucleic acid solution.