Innovative nanopore sequencing technology

Abstract

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.

What is claimed is: 
     
         1 . A method for manipulating 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 long chain target molecules in the buffer solution through the nanochannel in a first direction;   translocating the target molecules through the unsealed nanopores and onto the roof; and   detecting the target molecules that translocate through the unsealed nanopores.   
     
     
         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 target molecules move through the nanochannel in a second direction opposite to the first direction; and   detecting the 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 target molecules comprises applying a second voltage potential along the unsealed nanopores to control a translocation velocity of the target molecules through the unsealed nanopores. 
     
     
         5 . The method of  claim 1 , wherein:
 the roof further comprises a field enhancement structure aligned with the unsealed nanopores; and   detecting the target molecules comprises detecting individual moieties of the target molecules using surface-enhanced coherent anti-Stokes Raman scattering (SECARS).   
     
     
         6 . The method of  claim 1 , wherein applying a first voltage potential comprises moving the target molecules through the nanochannel to a barrier disposed in the nanochannel, prior to translocating the target molecules through the unsealed nanopores. 
     
     
         7 . The method of  claim 1 , wherein applying a first voltage potential further comprises routinely applying multiple voltage potentials along the nanochannel. 
     
     
         8 . The method of  claim 1 , wherein applying a first voltage potential further comprises moving the target molecules across the roof in the first direction. 
     
     
         9 . The method of  claim 1 , wherein:
 the target molecules comprise single-stranded or double stranded nucleic acids; and   detecting the target molecules comprises detecting a base sequence of the target molecules.   
     
     
         10 . The method of  claim 1 , wherein introducing a buffer solution comprises applying the buffer solution to the roof, such that the buffer solution passes through the unsealed nanopores and enters the nanochannel. 
     
     
         11 . The method of  claim 1 , wherein:
 the 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 target molecules.   
     
     
         12 . The method of  claim 1 , wherein:
 the target molecules comprise single-stranded (SS) nucleic acids; and   introducing a buffer solution comprises introducing a buffer solution comprising the SS nucleic acids.   
     
     
         13 . The method of  claim 1 , wherein:
 the target molecules comprise single-stranded (SS) nucleic acids; and   the buffer solution comprises a formamide buffer.   
     
     
         14 . A fluidic chip for manipulating target molecules, comprising:
 at least one nanochannel;   a porous roof covering the nanochannel, the porous roof comprising tortuous sealed and unsealed nanopores; and   first and second electrodes disposed on opposing ends of the nanochannel, the first and second electrodes configured to apply a first voltage potential along the nanochannel to move the target molecules through a buffer solution disposed in the nanochannel,   wherein,   each of the unsealed nanopores are configured such that only one of the target molecules can be translocated through each unsealed nanopore at a time, and   each of the unsealed nanopores have a density configured such that light emitted from the target molecules translocating through each unsealed nanopore can be individually detected.   
     
     
         15 . The fluidic chip of  claim 14 , further comprising a transparent third electrode spaced apart from the porous roof by a space configured to receive a fluid volume. 
     
     
         16 . The fluidic chip of  claim 15 , wherein the third electrode comprises a transparent conductive material or a grid. 
     
     
         17 . The fluidic chip of  claim 15 , further comprising a field enhancement structure aligned with the unsealed nanopores. 
     
     
         18 . The fluidic chip of  claim 17 , wherein field enhancement structure and the third electrode are configured to apply a second voltage potential across the unsealed nanopores to control a translocation velocity of the target molecules through the unsealed nanopores. 
     
     
         19 . The fluidic chip of  claim 18 , wherein field enhancement structure comprises a metal-insulator-metal structure. 
     
     
         20 . The fluidic chip of  claim 14 , further comprising a barrier structure disposed in the nanochannel and configured to direct at least some of the target molecules toward the unsealed nanopores.