Translocation of a polymer through a nanopore

Patent metadata

Field	Value
Publication number	US-2016018384-A1
Application number	US-201414773072-A
Country	US
Kind code	A1
Filing date	Mar 5, 2014
Priority date	Mar 5, 2013
Publication date	Jan 21, 2016
Grant date	—

Abstract

Embodiments of the present disclosure are directed to methods, systems and devices, for analyzing the molecules. For example, in some embodiments, a system is provided which includes a first volume of conducting fluid, a second volume of conducting fluid, an orifice in communication with said first and second volumes of fluid, and means for applying an electric potential difference between said first and second volumes of fluid. In some such embodiments, a conjugate product is provided which comprises charged polymers each having attached thereto at least one first molecule for analysis, where the product carries a predetermined charge greater than the charge on the first molecule, and upon dissolving a product in the first volume of fluid, the product is directed into the orifice.

First claim

1 . A device for analyzing molecules comprising:
a first volume of conducting fluid; a second volume of conducting fluid; an orifice in communication with said first and second volumes of fluid; and means for applying an electric potential difference between said first and second volumes of fluid, wherein: a product comprising charged polymers is provided, each charged polymer having attached thereto at least one first molecule for analysis, the first molecule having an amine terminus to which the charged polymer is attached, the product carries a predetermined charge greater than the charge on the first molecule, and upon dissolving the product in the first volume of fluid, the product is directed into the orifice.

2 . A device for directing a peptide molecule for analysis into an orifice of an analysis device, the system comprising:
a first chamber and a second chamber, wherein each compartment includes an electrolytic solution; a membrane separating the second chamber from the first chamber; an orifice provided in the membrane configured to receive and/or pass the product between the first and the second chambers; a first electrode provided in the first chamber; and a second electrode provided in the second chamber; wherein a product is provided comprising a plurality of peptide fragments for analyzing, each fragment being coupled at a corresponding N-termini to a polymeric ion via a cross linker, and upon the product being dissolved in the electrolyte solution in the first chamber and a bias being applied between the first and second electrodes, the product is at least pulled into the nanopore upon the bias of the second electrode being configured to attract the polymeric ion of the product.

3 . The device of claim 2 , wherein upon the polymeric ion comprising repeated negative charges, and upon the first electrode being biased negative and the second electrode being biased positive, the negatively charged polymeric ions are pulled into the nanopore.

4 . The device of claim 2 , wherein a first electric field of a first predetermined amount is established on a first side of the nanopore facing the first chamber, the first electric field extending a distance λ from the nanopore, and a second electric field of a second predetermined amount is established on a second side of the nanopore facing the second chamber, the second electric field extending the distance λ, wherein the first electric field pulls the product into the nanopore from the first chamber, and the second electric field pulls the product out of the nanopore into the second chamber.

5 . The device of claim 2 , wherein the bias is configured such that the product does not fold.

6 . The device of claim 2 , wherein the plurality of peptide fragments comprise between about 2 and about 60 peptides.

7 . The device of claim 2 , further comprising a mass spectrometer configured to identify the peptide fragments.

8 . The device of claim 2 , wherein the plurality of peptides are functionalized by modifying the N-terminus with succinic anhydride.

9 . The device of claim 2 , further comprising a pair of orifice electrodes in communication with the orifice, wherein an AC voltage of at least about 1 kHz in frequency is applied between the orifice electrodes.

10 . The device of claim 9 , wherein the presence of a molecule in the orifice is detected by means of non-linear processing of the AC current signal.

11 . The device of claim 9 , further comprising an electronic circuit for controlling the values of bias applied between the first electrode and the orifice electrodes and the second electrode and the orifice electrodes, wherein the circuit receives input from a signal generated by the orifice electrodes.

12 . The device of claim 9 , wherein the voltage applied between the orifice electrodes comprises an AC and a DC component.

13 . The device of claim 2 , further comprising the electrolytic solution in the first chamber, wherein the electrolytic solution comprises a salt solution of less than about 1M concentration.

14 . The device of claim 13 , further comprising a salt solution in the second chamber.

15 . The device of claim 2 , wherein the plurality of peptides are functionalized by modifying the N-terminus with azidoacetic anhydride.

16 . (canceled)

17 . A method for directing peptide fragments of a protein into an orifice of a protein sequencing apparatus/device, the method comprising:
providing the system of claim 2 ; digesting a protein for sequencing with endoproteinase Lys-C to produce a plurality of peptide fragments; reacting the fragments with 3-(2-propynyl)succinic anhydride at a predetermined pH in a buffered solution to produce alkyne-modified peptides; reacting a polyion polymer terminated with an azide with the alkyne-modified peptides in the presence of Cu(I), resulting in a conjugate product of polymer and peptide; purifying the conjugate product on a size exclusion column; placing the conjugate in the first chamber of the system of claim 2 , wherein the second chamber is biased positively to draw the conjugate product through the orifice.

18 . (canceled)

19 . (canceled)

20 . (canceled)

21 . (canceled)

22 . (canceled)

23 . (canceled)

24 . (canceled)

25 . (canceled)

26 . (canceled)

27 . A method for directing peptide fragments of a protein into an orifice of a protein sequencing apparatus/device, the method comprising:
providing the system of claim 2 ; digesting a protein for sequencing with endoproteinase Lys-C, trypsin, or Lys-C/trypsin to produce a plurality of peptide fragments; reacting the fragments with azidoacetic anhydride at a predetermined pH in a sodium acetate or other buffered solution to produce alkyne-modified peptides; reacting a polyion polymer terminated with a cyclooctyne or its derivative with the azide-modified peptides in the absence of Cu(I), resulting in a conjugate product of polymer and peptide; purifying the conjugate product on a size exclusion column; placing the conjugate in the first chamber of the system of claim 2 , wherein the second chamber is biased positively to draw the conjugate product through the orifice.

28 . (canceled)

29 . (canceled)

Assignees

• Univ Arizona

Inventors

• Lindsay Stuart
• Zhang Peiming

Classifications

• G01N33/5438

  Electrodes · CPC title

• Y10T436/255

  including use of a solid sorbent, semipermeable membrane, or liquid extraction · CPC title

• G01N33/48721 Primary

  Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68) · CPC title

• G01N15/12

  by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle · CPC title

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title