Innovative nanopore sequencing technology
Methods and apparatus for long read, label-free, optical nanopore long chain molecule sequencing. In general, the present disclosure describes a novel sequencing technology based on the integration of nanochannels to deliver single long-chain molecules with widely spaced (>wavelength), ˜1-nm aperture “tortuous” nanopores that slow translocation sufficiently to provide massively parallel, single base resolution using optical techniques. A novel, directed self-assembly nanofabrication scheme using simple colloidal nanoparticles is used to form the nanopore arrays atop nanochannels that unfold the long chain molecules. At the surface of the nanoparticle array, strongly localized electromagnetic fields in engineered plasmonic/polaritonic structures allow for single base resolution using optical techniques.
1. A method for manipulating long chain target molecules using a nanochannel covered with a porous roof comprising tortuous sealed and unsealed nanopores, the method comprising:
introducing a buffer solution into the nanochannel;
applying a first voltage potential across the nanochannel to move the long chain target molecules in the buffer solution through the nanochannel in a first direction;
translocating the long chain target molecules through the unsealed nanopores in the porous roof of the nanochannel, wherein the porous roof further comprises a field enhancement structure aligned with the unsealed nanopores; and
detecting and sequencing individual moieties of the long chain target molecules that translocate through the unsealed nanopores using Raman scattering.
2. The method of claim 1 , further comprising:
applying a second voltage potential along the nanochannel by reversing the first voltage potential, such that the long chain target molecules move through the nanochannel in a second direction opposite to the first direction; and
detecting the long chain target molecules after applying the second voltage potential.
3. The method of claim 1 , wherein applying a first voltage potential comprises applying an alternating current (AC) voltage potential having a direct current (DC) bias.
4. The method of claim 1 , wherein translocating the long chain target molecules comprises applying a second voltage potential along the unsealed nanopores to control a translocation velocity of the long chain target molecules through the unsealed nanopores.
5. The method of claim 1 , wherein applying a first voltage potential comprises moving the long chain target molecules through the nanochannel to a barrier disposed in the nanochannel, prior to translocating the long chain target molecules through the unsealed nanopores.
6. The method of claim 1 , wherein applying a first voltage potential further comprises routinely applying multiple voltage potentials along the nanochannel.
7. The method of claim 1 , wherein applying a first voltage potential further comprises moving the long chain target molecules across the porous roof in the first direction.
8. The method of claim 1 , wherein:
the long chain target molecules comprise single-stranded or double stranded nucleic acids; and
detecting the long chain target molecules comprises detecting a base sequence of the long chain target molecules.
9. The method of claim 1 , wherein introducing a buffer solution comprises applying the buffer solution to the porous roof, such that the buffer solution passes through the unsealed nanopores and enters the nanochannel.
10. The method of claim 1 , wherein:
the long chain target molecules comprise single-stranded (SS) nucleic acids;
introducing a buffer solution comprises introducing a buffer solution comprising double-stranded (DS) nucleic acid molecules into the nanochannel; and
the method further comprises introducing an exonuclease into the nanochannel to digest the DS nucleic acid molecules and form the long chain target molecules.
11. The method of claim 1 , wherein:
the long chain target molecules comprise single-stranded (SS) nucleic acids; and
introducing a buffer solution comprises introducing a buffer solution comprising the SS nucleic acids.
12. The method of claim 1 , wherein:
the long chain target molecules comprise single-stranded (SS) nucleic acids; and
the buffer solution comprises a formamide buffer.
13. The method of claim 1 , wherein the detecting comprises optically detecting by an optical detector.
14. The method of claim 1 , wherein the detecting comprises detecting target individual moieties of the long chain target molecules.
15. The method of claim 1 , wherein the nanochannel is formed by stacking a plurality of nanoparticles on each other.
16. The method of claim 1 , wherein the Raman scattering comprises surface-enhanced coherent anti-Stokes Raman scattering (SECARS) or surface enhanced Raman scattering (SERS).