Device, system and method for direct electrical measurement of enzyme activity
The present disclosure relates to a device, system and method for sensing functional motions of a single protein molecule via direct attachment of one or more electrodes to the molecule. The present disclosure also relates to an array, a system comprising an array and method for sequencing a biopolymer using an array.
1. A device for direct measurement of protein activity, the device comprising:
a first electrode and a second electrode, the first and second electrodes being separated by a gap; and
a protein attached to the first electrode via a first linker and to the second electrode via a second linker, wherein each of the first and the second linkers comprises at least one chemical bond and is attached to a region of the protein that is inactive.
2. The device of claim 1 , wherein the gap has a width of about 1.0 nm to about 20.0 nm.
3. The device of claim 1 , wherein the first and second electrodes are separated by a dielectric layer, wherein a passivation layer is disposed on the first and second electrodes.
4. The device of claim 1 , wherein the protein is selected from the group consisting of a polymerase, a nuclease, a proteasome, a glycopeptidase, a glycosidase, a kinase and an endonuclease.
5. The device of claim 1 , wherein the protein is a polymerase.
6. The device of claim 5 , wherein exonuclease activity of the polymerase is disabled.
7. The device of claim 1 , wherein each of the first and the second linkers comprises a covalent chemical bond.
8. The device of claim 1 , wherein the protein is biotinylated.
9. The device of claim 1 , wherein the first and/or the second linker comprises thiol-streptavidin.
10. The device of claim 1 , wherein the protein and the first and/or second electrodes are biotinylated, and wherein the first and/or the second linker comprises a streptavidin molecule comprising at least two biotin binding sites.
11. The device of claim 1 , wherein the first and/or second electrodes comprise a metal selected from the group consisting of gold, platinum, palladium, and ruthenium, or any combinations thereof.
12. The device of claim 1 , wherein the first or the second linker is attached to the N-terminal region of the protein.
13. The device of claim 5 , wherein the first or the second linker is attached to the exonuclease region of the polymerase.
14. A method for direct measurement of protein activity, the method comprising:
(a) introducing a chemical entity that is capable of interacting with the protein of the device of claim 1 ;
(b) applying a voltage bias between the first and second electrodes that is 100 mV or less; and
(c) observing fluctuations in current between the first and second electrodes that occur when the chemical entity interacts with the protein.
15. A method of sequencing a biopolymer, said method comprising:
(a) introducing a biopolymer to the device of claim 1 ;
(b) applying a voltage bias between the first and second electrodes that is 100 mV or less;
(c) detecting fluctuations in current between the first and second electrodes that occur when a monomer is removed from an end of the biopolymer; and
(d) determining the identity of each monomer removed from the biopolymer.
16. The method of claim 15 , wherein the biopolymer is a DNA molecule, an RNA molecule, a peptide, a polypeptide, or a glycan.
17. A method of sequencing a polynucleotide, said method comprising:
(a) introducing a primed polynucleotide template to the device of claim 1 , wherein the protein is a polymerase and wherein the polymerase is bound to the template;
(b) introducing a solution comprising dNTPs;
(c) applying a voltage bias between the first and second electrodes that is 100 mV or less;
(d) detecting fluctuations in current between the first and second electrodes that occur when each dNTP is incorporated as an additional nucleotide complementary to a corresponding nucleotide in the polynucleotide template; and
(e) determining the identity of each additional nucleotide being incorporated.
18. The method of claim 17 , wherein each dNTP is present in the solution at about the same concentration.
19. The method of claim 17 , wherein each dNTP is present at a concentration that is about equal to or above the saturation concentration of the template-bound polymerase.
20. The method of claim 17 , wherein step (d) comprises detecting the presence of one or more current spike(s).
21. The method of claim 20 , wherein step (e) comprises using the characteristics of each spike.
22. The method of claim 17 , wherein each additional nucleotide is introduced in turn.
23. The method of claim 17 , wherein each dNTP is introduced simultaneously, and wherein at least one dNTP is present at a lower concentration than that of the other dNTPs.