High throughput nucleic acid sequencing by expansion and related methods
A method for detecting an analyte employing a nanopore substrate positioned between first and second reservoirs by providing an indicator molecule not associated with the analyte and detecting a change in an optical signal emitted from the indicator moiety as the analyte translocates through a nanopore channel of the nanopore substrate.
1. A method of detecting an analyte, comprising:
a) providing at least one analyte;
b) providing at least one indicator moiety, wherein the indicator moiety is not associated with the analyte;
c) providing a detector construct, wherein the detector construct comprises a first and a second reservoir comprising first and second electrodes, respectively, wherein the first and second reservoirs are separated by a nanopore substrate positioned between the first and second reservoirs, and wherein the nanopore substrate has at least one nanopore channel through the substrate;
d) providing an electric potential to the first and second electrodes, wherein the electric potential is sufficient to translocate the at least one analyte and the at least one indicator moiety through the at least one nanopore channel; and
e) detecting a change in an optical signal emitted from the at least one indicator moiety at or near the at least one nanopore channel as the at least one analyte translocates through the at least one nanopore channel and thereby detecting the analyte.
2. The method of claim 1 , further comprising providing an excitation wavelength, wherein the excitation wavelength is sufficient to induce a fluorescent signal from the at least one indicator moiety.
3. The method of claim 1 , wherein the at least one analyte is a nucleic acid.
4. The method of claim 1 , wherein the at least one analyte is a surrogate polymer.
5. The method of claim 1 , wherein the at least one indicator moiety is at least one fluorophore, wherein the first reservoir comprises a high concentration of fluorophore relative to the second reservoir, and detecting a change in the optical signal further comprises detecting a change in a fluorescent signal as the at least one fluorophore translocates through the at least one nanopore channel.
6. The method of claim 5 , wherein epifluorescence microscopy is used for detecting the change in the fluorescent signal.
7. The method of claim 5 , wherein conoscopy is used for detecting the change in the fluorescent signal.
8. The method of claim 5 , wherein the nanopore substrate comprises a blocking film.
9. The method of claim 5 , wherein the at least one fluorophore is fluorescein.
10. The method of claim 5 , wherein the second reservoir comprises a fluorescence quenching agent, and wherein detecting a change in the optical signal further comprises detecting a change in the fluorescent signal as the at least one fluorophore or a quenching agent translocates through the at least one nanopore channel.
11. The method of claim 10 , wherein the quenching agent is an acceptor for fluorescence resonance energy transfer or a free radical.
12. The method of claim 1 , further comprising providing two indicator moieties, wherein a first indicator moiety is an indicator ion, and a second indicator moiety is a fluorescence indicator, wherein the first reservoir comprises the indicator ion, the second reservoir comprises the fluorescence indicator, and wherein detecting a change in the optical signal further comprises detecting a change in a fluorescence signal emitted as either the first indicator moiety or the second indicator moiety passes through the at least one nanopore channel.
13. The method of claim 12 , wherein the second reservoir further comprises a non-fluorescing absorber.
14. The method of claim 12 , wherein the at least one nanopore channel is masked to create a circular opening of about 1 μm in diameter, and wherein the opening is concentric with the nanopore channel.
15. The method of claim 12 , wherein the indicator ion is a calcium ion, a singlet hydrogen ion, a singlet oxygen ion, a potassium ion, a zinc ion, a magnesium ion, a chlorine ion, or a sodium ion.
16. The method of claim 12 , wherein the second indicator moiety is a calcium indicator moiety.
17. The method of claim 12 , wherein the second indicator moiety is fluorescence quencher.
18. The method of claim 12 , wherein the first reservoir comprises iodide ions and the second reservoir comprises fluorescein.
19. The method of claim 1 further comprising providing two indicator moieties, wherein the first reservoir comprises a first indicator moiety and the second reservoir comprises a second indicator moiety, wherein the first and second indicator moieties are capable of combining to form a third indicator moiety in an excited state, and detecting a change in the optical signal further comprises detecting photons which are emitted when the third indicator moiety relaxes to a ground state.
20. The method of claim 1 , wherein the nanopore substrate comprises a nanopore array, and wherein the nanopore array shares the first and second reservoirs.