US 11,905,552 B2
Immobilized RNPs for sequence-specific nucleic acid capture and digital detection
Kiana Aran, Pasadena, CA (US); Tara deBoer, Berkeley, CA (US); and Irina Conboy, Berkeley, CA (US)
Assigned to KECK GRADUATE INSTITUTE OF APPLIED LIFE SCIENCES, Claremont, CA (US); and THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, Oakland, CA (US)
Filed by KECK GRADUATE INSTITUTE, Claremont, CA (US)
Filed on Aug. 6, 2018, as Appl. No. 15/998,353.
Claims priority of provisional application 62/597,806, filed on Dec. 12, 2017.
Claims priority of provisional application 62/541,100, filed on Aug. 4, 2017.
Prior Publication US 2019/0112643 A1, Apr. 18, 2019
Int. Cl. C12Q 1/6825 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6811 (2018.01); C12N 9/22 (2006.01); G01N 27/327 (2006.01); G01N 27/414 (2006.01)
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.