Systems and methods for high efficiency electronic sequencing and detection

• US 10,093,975 B2
• Filed: 12/03/2012
• Issued: 10/09/2018
• Est. Priority Date: 12/01/2011
• Status: Active Grant

• Current Assignee: Sequencing Health Incorporated
• Original Assignee: GenapSys, Inc.
• Inventors: Esfandyarpour, Hesaam
• Primary Examiner(s): Bertagna, Angela Marie
• Assistant Examiner(s): Oyeyemi, Olayinka A
• Application Number:
  US14/361,902
• Publication Number:
  US 20140329699A1
• Time in Patent Office: 2,136 Days
• Field of Search: None
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B03C 1/286 disposed at the inner circumference of a recipient, e.g. magnetic drain bolt
• CPC Class Codes: B03C 1/288 disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 2201/26 for use in medical applications
• CPC Class Codes: B03C 5/005 Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength B03C5/02 takes precedence; electrophoresis B01D57/02
• CPC Class Codes: B03C 5/02 Separators
• CPC Class Codes: B03C 5/022 Non-uniform field separators
• CPC Class Codes: B03C 5/026 using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/116 : electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/607 : being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: G01N 27/3278 : involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745

Abstract

The present disclosure relates to systems and methods for high efficiency electronic sequencing of nucleic acids and molecular detection. In an example embodiment of the instant disclosure, the NanoNeedle may be utilized to detect a change in impedance resulting from the modulation of the counter ion concentration or Debye length associated with a biomolecule of interest, such as DNA or protein, for an application of interest, such as DNA sequencing, DNA hybridization, or protein detection.

Claims

1. A method for sequencing a nucleic acid molecule, comprising:

(a) providing a plurality of particles adjacent to a sensor array, wherein an individual particle of said plurality of particles is positioned adjacent to an individual sensor of said sensor array, wherein said nucleic acid molecule is attached to said individual particle and has a primer hybridized thereto;

(b) bringing said nucleic acid molecule having said primer hybridized thereto in contact with nucleotides under conditions sufficient to conduct a polymerization reaction to yield a nucleic acid strand complementary to said nucleic acid molecule;

(c) using said individual sensor to measure steady state signals indicative of impedance, charge, or conductivity change within a Debye length of said individual particle or said nucleic acid molecule, to identify individual incorporation events associated with said nucleotides during said polymerization reaction; and

(d) using said steady state signals to identify a sequence of said nucleic acid strand, thereby sequencing said nucleic acid molecule.

2. The method of claim 1, further comprising conducting said polymerization reaction in the presence of a reaction buffer that is suitable for the completion of said polymerization reaction, and measuring said steady state signals in the presence of a measurement buffer that is suitable for detecting said steady state signals.

3. The method of claim 2, wherein said reaction buffer and said measurement buffer are different buffers.

4. The method of claim 2, wherein the conductivity of said measurement buffer is lower than the conductivity of said reaction buffer.

5. The method of claim 1, wherein (b) is performed by bringing different types of nucleotides in contact with said nucleic acid molecule having said primer hybridized thereto, one type of nucleotide at a time.

6. The method of claim 1, wherein said individual sensor comprises at least two electrodes that are electrically coupled to the Debye length of said individual particle or said nucleic acid molecule.

7. The method of claim 1, wherein said individual particle is magnetically immobilized to said sensor array.

8. The method of claim 1, further comprising obtaining said steady state signals using differential measurement.

9. The method of claim 8, wherein said differential measurement utilizes (i) at least one particle that does not comprise a nucleic acid as a reference, (ii) a sensor that is not positioned adjacent to a particle as a reference, (iii) another individual sensor of said sensor array that is not subject to a nucleotide incorporation event as a reference, (v) another nucleic acid molecule of known sequence that is coupled to said nucleic acid molecule as a reference, or (vi) a cross-talk deconvolution function matrix.

10. The method of claim 1, wherein measuring said steady state signals does not include detecting a transient pH signal accompanying said individual incorporation events.

11. The method of claim 1, wherein said individual sensor comprises a well.

12. The method of claim 11, wherein said individual particle is held in said well.

13. The method of claim 1, wherein said sensor array includes a flat surface.

14. The method of claim 13, wherein said individual particle is held adjacent to said individual sensor on said flat surface.

15. The method of claim 1, wherein said individual particle is electrically immobilized to said sensor array.