High speed molecular sensing with nanopores

Abstract

Described herein are methods and devices for capturing and determining the identity of molecules using nanopores. The molecules can be counted, sorted and/or binned rapidly in a parallel manner using a large number of nanopores (e.g., 132,000 nanopores reading 180 million molecules in 1 hour). This fast capture and reading of a molecule can be used to capture probe molecules or other molecules that have been generated to represent an original, hard to detect molecule or portions of an original molecule. Precise counting of sample molecules or surrogates for sample molecules can occur. The methods and devices described herein can, among other things, replace flow cytometers and other counting instruments (e.g., while providing increased precision and throughput relative to a flow cytometer). In some cases, the devices and methods capture and hold particular molecules or surrogates of molecules in the nanopores and then eject them into clean solution to perform a capture, sorting, and binning function similar to flow cytometers.

What is claimed is: 
     
         1 . A method for molecular counting and/or sorting, comprising:
 a. providing an array of nanopores, wherein an individual nanopore of said array is individually addressable by an adjacent sensing electrode;   b. providing a plurality of markers that each comprise nucleotides, wherein at least two of the nucleotides hybridize with a nucleic acid sample, and wherein the markers are capable of being captured by the individual nanopore and identified using the sensing electrode; and   c. capturing and identifying the markers with the array of nanopores at a rate of at least about 1 marker per second per nanopore.   
     
     
         2 . The method of  claim 1 , further comprising releasing the captured markers from the nanopore. 
     
     
         3 . The method of  claim 1 , wherein the plurality of markers comprise markers to be sorted, wherein the markers to be sorted are captured, identified and held in the nanopores, and wherein markers other than the markers to be sorted are captured, identified and released from the nanopores. 
     
     
         4 . The method of  claim 3 , wherein the markers to be sorted are released as a group and collected. 
     
     
         5 . The method of  claim 3 , wherein the markers to be sorted are released as a group when the ratio of the number of markers to be sorted divided by a remaining number of markers that are captured and identified by the nanopores increases above a threshold. 
     
     
         6 . The method of  claim 1 , wherein the array of nanopores is configured to have a plurality of regions capable of performing the method on different samples. 
     
     
         7 . The method of  claim 1 , wherein the markers are identified based on a current that flows through the individual nanopore and/or a voltage at which the marker leaves the nanopore. 
     
     
         8 . The method of  claim 1 , wherein the markers each comprise a single stranded nucleic acid molecule attached to a bead. 
     
     
         9 . The method of  claim 1 , wherein the markers are generated by:
 a. hybridizing a first probe to a nucleic acid sample;   b. hybridizing a second probe to the nucleic acid sample adjacent to the first probe;   c. ligating the first probe to the second probe to produce a combined probe; and   d. capturing the combined probe with a bead attached to an oligonucleotide, wherein the olignonucleotide hybridizes with the combined probe.   
     
     
         10 . The method of  claim 9 , further comprising determining copy number variation of a nucleic acid sequence in the nucleic acid sample. 
     
     
         11 . The method of  claim 9 , further comprising quantifying relative RNA expression levels in the nucleic acid sample. 
     
     
         12 . The method of  claim 9 , further comprising performing an ELISA assay on the nucleic acid sample. 
     
     
         13 . The method of  claim 9 , wherein the first probe comprises biotin. 
     
     
         14 . The method of  claim 9 , wherein the bead is magnetic. 
     
     
         15 . The method of  claim 14 , further comprising concentrating the markers adjacent or in proximity to the array of nanopores with a magnetic field. 
     
     
         16 . A method for molecular counting and/or sorting, comprising:
 a. providing an array of nanopores, wherein an individual nanopore of said array is individually addressable by an adjacent sensing electrode operated in non-faradaic mode;   b. providing a plurality of markers capable of being captured by the individual nanopore and identified using the sensing electrode; and   c. capturing and identifying the markers with the array of nanopores at a rate of at least about 1 marker per second per nanopore.   
     
     
         17 . A method for sequencing, counting, and/or sorting molecules, comprising:
 a. providing an array of nanopores, wherein an individual nanopore of said array is individually addressable by an adjacent sensing electrode operated in non-faradic mode or faradaic mode;   b. providing a plurality of magnetically attractable beads each coupled to a molecule among a plurality of molecules to be sequenced, counted and/or sorted using the array of nanopores;   c. concentrating the magnetically attractable beads in the vicinity of the array of nanopores with a magnet; and   d. sequencing, counting and/or sorting the molecules with the array of nanopores.   
     
     
         18 . The method of  claim 17 , wherein the magnetically attractable beads comprise metal. 
     
     
         19 . The method of  claim 17 , wherein the magnetically attractable beads comprise a permanent magnetic material. 
     
     
         20 . The method of  claim 17 , wherein the concentration of the magnetically attractable beads near the array of nanopores is increased by at least 100-fold by said concentrating.