CPC C12Q 1/6825 (2013.01) [C12N 9/22 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6811 (2013.01); G01N 27/3272 (2013.01); G01N 27/4145 (2013.01); G01N 27/4146 (2013.01)] | 16 Claims |
1. A biosensor for assaying a target nucleic acid, the biosensor comprising:
a substrate comprising a substrate surface and at least two electrodes for passing current through the substrate, wherein the substrate surface comprises graphene;
a pyrenebutanoic acid (PBA) linker molecule comprising a pyrene ring moiety and a carboxylate moiety, wherein the linker molecule is conjugated to the substrate surface via non-covalent interaction between the pyrene ring moiety and the substrate surface;
a ribonucleoprotein (RNP) conjugated to the carboxylate moiety of the linker molecule via covalent bonding, wherein the RNP comprises Cas9 protein, wherein the linker molecule is joined to the Recombination UV C (RuvC) domain of the Cas9 protein, and wherein the RNP is immobilized to the substrate surface via the linker molecule; and
a guide ribonucleic acid (gRNA) forming a portion of the RNP and comprising a first sequence capable of binding to a protein portion of the RNP and a second sequence capable of binding to the target nucleic acid,
wherein conductivity of the substrate surface is modulated upon binding of the target nucleic acid to the gRNA, thereby enabling a detectable current change for sensing the target nucleic acid.
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