Enzymatic circuits for molecular sensors

Patent metadata

Field	Value
Publication number	US-11268123-B2
Application number	US-201916684338-A
Country	US
Kind code	B2
Filing date	Nov 14, 2019
Priority date	Apr 25, 2017
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

In various embodiments a molecular circuit is disclosed. The circuit comprises a negative electrode, a positive electrode spaced apart from the negative electrode, and an enzyme molecule conductively attached to both the positive and negative electrodes to form a circuit having a conduction pathway through the enzyme. In various examples, the enzyme is a polymerase. The circuit may further comprise molecular arms used to wire the enzyme to the electrodes. In various embodiments, the circuit functions as a sensor, wherein electrical signals, such as changes to voltage, current, impedance, conductance, or resistance in the circuit, are measured as substrates interact with the enzyme.

First claim

We claim:

1. A circuit comprising:
a first electrode; a second electrode spaced apart from the first electrode; and
an enzyme directly wired to the first electrode by a first arm molecule and directly wired to the second electrode by a second arm molecule to form a conductive pathway between the first electrode and the second electrode through the enzyme,
wherein each of the first and second arm molecules has a first end and a second end, wherein the first end of the first arm molecule is bonded to the enzyme and the second end of the first arm molecule is bonded to the first electrode, wherein the first end of the second arm molecule is bonded to the enzyme and the second end of the second arm molecule is bonded to the second. electrode, and
wherein the first end of each of the first and second arm molecules are conjugated to two different sites on the enzyme so that a portion of the enzyme between the two different sites is included in the conductive pathway.

2. The circuit of claim 1 , wherein the first electrode is a positive electrode and the second electrode is a negative electrode, or wherein the first electrode is a negative electrode and the second electrode is a positive electrode.

3. The circuit of claim 1 , further comprising an additional electrode configured as a gate or buried gate electrode.

4. The circuit of claim 1 , wherein the portion of the enzyme included. in the circuit comprises an alpha-helix or a beta-sheet.

5. The circuit of claim 4 , wherein the alpha-helix or the beta-sheet portion of the enzyme provide a conduction pathway close to an active region of the enzyme.

6. The circuit of claim 1 , wherein the conjugation between the first end of each of the first and second arm molecules and the two different sites on the enzyme comprise maleimide-cysteine conjugations.

7. A circuit comprising:
a first electrode; a second electrode spaced apart from the first electrode; and
an enzyme directly wired to the first electrode by a first arm molecule and directly wired to the second electrode by a second arm molecule to form a conductive pathway between the first electrode and the second electrode through the enzyme,
wherein each of the first and second arm molecules has a first end and a second end, wherein the first end of the first arm molecule is bonded to the enzyme and the second end of the first arm molecule is bonded to the first electrode, wherein the first end of the second arm molecule is bonded to the enzyme and the second end of the second arm molecule is bonded to the second electrode, and
wherein the first end of each of the first and second arm molecules are conjugated to two different sites on the enzyme so that a portion of the enzyme between the two different sites is included in the conductive pathway,
wherein the second end of each of the first and second arm molecules and the first and second electrodes is directly wired by a thiol-gold coupling.

8. The circuit of claim 1 , wherein at least one of the first and second arm molecules comprises a molecule having tension, twist, or torsion dependent conductivity.

9. The circuit of claim 1 , wherein the first and second arm molecules independently comprise a single-stranded DNA oligonucleotide, a double-stranded DNA oligonucleotide, a peptide nucleic acid (PNA) duplex, a PIA DNA hybrid duplex, a polypeptide, an antibody, an antibody domain, a carbon nanotube, or a graphene nanoribbon.

10. The circuit of claim 1 , wherein the enzyme comprises a polymerase, a ligase, a nuclease, a kinase, a transferase, or a reverse transcriptase.

11. The circuit of claim 1 , wherein the enzyme comprises a DNA or RNA polymerase enzyme.

12. The circuit of claim 1 , wherein the enzyme comprises E. coli DNA polymerase I (Klenow) Fragment.

13. The circuit of claim 1 , wherein the enzyme comprises a genetically modified form of an E. coli Pol I polymerase, a Bst polymerase, a Tag polymerase, a Phi29 polymerase, a T7 polymerase, or a reverse transcriptase.

14. The circuit of claim 1 , wherein the conjugation of the first and second arm molecules to two different sites on the enzyme is by thiol-metal bonds, maleimide-cysteine bonds, material binding peptides, or click chemistries.

Assignees

• Roswell Biotechnologies Inc

Inventors

• Merriman Barry L
• Govindaraj Venkatesh Alagarswamy
• Mola Paul
• Geiser Tim

Classifications

• C12Q1/001

  Enzyme electrodes · CPC title

• C12Q1/48

  involving transferase · CPC title

• C12Q2565/607

  being a sensor, e.g. electrode · CPC title

• C12Q1/485

  involving kinase · CPC title

• G01N33/48721 Primary

  Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68) · CPC title