Systems and methods for automated reusable parallel biological reactions

• US 9,187,783 B2
• Filed: 10/04/2011
• Issued: 11/17/2015
• Est. Priority Date: 10/04/2010
• Status: Active Grant

• Current Assignee: Sequencing Health Incorporated
• Original Assignee: GenapSys, Inc.
• Inventors: Esfandyarpour, Hesaam, Oldham, Mark F., Nordman, Eric S., Parizi, Kosar Baghbani
• Primary Examiner(s): Switzer, Juliet
• Assistant Examiner(s): KAUP, SAHANA S
• Application Number:
  US13/824,129
• Publication Number:
  US 20140045701A1
• Time in Patent Office: 1,505 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0418 electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0478 pistons
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• 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: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• 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 1/30 : Combinations with other devices, not otherwise provided for
• 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 5/022 : 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 1/6848 : characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 : using modified primers or templates
• 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 2523/307 : Denaturation or renaturation by electric current/voltage
• CPC Class Codes: C12Q 2563/116 : electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2563/143 : Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2565/543 : characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: C12Q 2565/607 : being a sensor, e.g. electrode
• CPC Class Codes: C40B 60/10 : for identifying library members
• CPC Class Codes: G01N 1/40 : Concentrating samples
• CPC Class Codes: G01N 2001/4038 : electric methods, e.g. electromigration, electrophoresis, ionisation
• CPC Class Codes: G01N 27/4145 : specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
• CPC Class Codes: G01N 27/4146 : involving nanosized elements, e.g. nanotubes, nanowires
• CPC Class Codes: G01N 33/5438 : Electrodes
• CPC Class Codes: H01L 29/00 : Semiconductor devices specially adapted for rectifying, amplifying, oscillating or switching and having potential barriers; Capacitors or resistors having potential barriers, e.g. a PN-junction depletion layer or carrier concentration layer; Details of semiconductor bodies or of electrodes thereof ; Multistep manufacturing processes therefor H01L31/00 - H01L33/00, H10K10/00, H10N take precedence; details other than of semiconductor bodies or of electrodes thereof H01L23/00; devices consisting of a plurality of solid state components formed in or on a common substrate H01L27/00

Abstract

A method comprises magnetically holding a bead carrying biological material (e.g., nucleic acid, which may be in the form of DNA fragments or amplified DNA) in a specific location of a substrate, and applying an electric field local to the bead to isolate the biological material or products or byproducts of reactions of the biological material. For example, the bead is isolated from other beads having associated biological material. The electric field in various embodiments concentrates reagents for an amplification or sequencing reaction, and/or concentrates and isolates detectable reaction by-products. For example, by isolating nucleic acids around individual beads, the electric field can allow for clonal amplification, as an alternative to emulsion PCR. In other embodiments, the electric field isolates a nanosensor proximate to the bead, to facilitate detection of at least one of local pH change, local conductivity change, local charge concentration change and local heat. The beads may be trapped in the form of an array of localized magnetic field regions.

Claims

1. A method for sequencing, comprising:

(a) providing a plurality of particles, wherein each particle of said plurality is coupled to on average at most one nucleic acid molecule;

(b) performing simultaneous nucleic acid amplification reactions on said nucleic acid molecules coupled to said particles, wherein said nucleic acid amplification reactions are performed while subjecting the particles to an electric field, thereby producing isolated, clonally-amplified nucleic acid molecules coupled to said plurality of particles;

(c) performing nucleic acid extension reactions on said clonally-amplified nucleic acid molecules using primers that hybridize to said clonally-amplified nucleic acid molecules; and

(d) detecting nucleic acid incorporation events from said extension reactions using a sensor array to determine a sequence of each of said clonally-amplified nucleic acid molecules, wherein each particle of said plurality of particles is adjacent to a sensor of said sensor array, and wherein each said sensor of said sensor array comprises two electrodes that are within a Debye length of their respective individual particle or within a Debye length of a nucleic acid molecule on said individual particle.

2. The method of claim 1, wherein said nucleic acid amplification reactions are emulsion free.

3. The method of claim 1, further comprising, subsequent to (b), separating particles carrying clonally-amplified nucleic acid molecules from particles not carrying clonally-amplified nucleic acid molecules.

4. The method of claim 1, wherein (b) is performed with the plurality of particles held adjacent to the sensor array.

5. The method of claim 1, wherein, in (c), the particles are at least one of electrically and magnetically immobilized.

6. The method of claim 1, further comprising releasing one or more of said plurality of particles.

7. The method of claim 1, wherein, in (d), detecting said nucleic acid incorporation events further comprises using an individual sensor among the plurality of sensors to detect at least one of local impedance change and local conductivity change.

8. The method of claim 1, wherein the electric field isolates or concentrates one or more of template DNA fragments, primers, polymerase, and dNTPs around each of the particles.

9. The method of claim 1, wherein the electric field isolates or concentrates products of nucleic acid amplification reactions.

14. The method of claim 1, wherein an individual electrode of said two electrodes has a Debye length.

15. The method of claim 14, wherein said Debye length of said individual electrode is within said Debye length of said individual particle or said Debye length of said nucleic acid molecule on said individual particle.

10. A method for nucleic acid sequencing, comprising:

(a) directing a plurality of particles onto an array of sensors, wherein an individual particle comprises a nucleic acid molecule, and wherein an individual sensor of said array comprises a pair of electrodes that are electrically coupled to a Debye length of said individual particle during nucleic acid sequencing;

(b) positioning said individual particle at said individual sensor;

(c) directing a plurality of primers onto said array;

(d) performing a primer extension reaction at said individual sensor; and

(e) during or subsequent to performing said primer extension reaction, measuring a change in impedance of said individual particle with the aid of said pair of electrodes that are each electrically coupled to said Debye length of said individual particle.

11. The method of claim 10, further comprising amplifying said nucleic acid molecule prior to (d).

12. The method of claim 11, wherein said nucleic acid molecule is amplified while subjecting said individual particle to an electric field.

13. The method of claim 11, wherein said nucleic acid molecule is amplified while said individual particle is held at said given sensor.

16. The method of claim 10, wherein, in (e), the electrodes of said pair of electrodes are within said Debye length of said individual particle.

17. The method of claim 16, wherein an individual electrode of said pair of electrodes has a Debye length.

18. The method of claim 17, wherein said Debye length of said individual electrode is within said Debye length of said individual particle.