Active-source-pixel, integrated device for rapid analysis of biological and chemical specimens

• US 9,983,135 B2
• Filed: 11/17/2014
• Issued: 05/29/2018
• Est. Priority Date: 11/17/2013
• Status: Active Grant

• Current Assignee: Quantum-Si Incorporated
• Original Assignee: Quantum-Si Incorporated
• Inventors: Rothberg, Jonathan M., Kabiri, Ali, Sickler, Jason W., Gyarfas, Brett J., Lackey, Jeremy, Schmid, Gerard
• Primary Examiner(s): Priest, Aaron A
• Application Number:
  US14/543,888
• Publication Number:
  US 20150141268A1
• Time in Patent Office: 1,289 Days
• Field of Search: None
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 2300/168 Specific optical properties, e.g. reflective coatings
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• 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 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6478 Special lenses
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6454 using an integrated detector array
• CPC Class Codes: G01N 21/648 : using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/6486 : Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 21/7743 : the reagent-coated grating coupling light in or out of the waveguide
• CPC Class Codes: G01N 21/7746 : the waveguide coupled to a cavity resonator
• CPC Class Codes: G01N 2201/02 : Mechanical
• CPC Class Codes: G01N 2201/06113 : Coherent sources; lasers
• CPC Class Codes: G01N 2201/0612 : Laser diodes
• CPC Class Codes: G01N 2201/062 : LED's
• CPC Class Codes: G01N 2201/068 : Optics, miscellaneous
• CPC Class Codes: G01N 2201/125 : Digital circuitry
• CPC Class Codes: Y10T 29/49016 : Antenna or wave energy "plumbing" making

Abstract

An active-source-pixel, integrated device capable of performing biomolecule detection and/or analysis, such as single-molecule nucleic acid sequencing, is described. An active pixel of the integrated device includes a sample well into which a sample to be analyzed may diffuse, an excitation source for providing excitation energy to the sample well, and a sensor configured to detect emission from the sample. The sensor may comprise two or more segments that produce a set of signals that are analyzed to differentiate between and identify tags that are attached to, or associated with, the sample. Tag differentiation may be spectral and/or temporal based. Identification of the tags may be used to detect, analyze, and/or sequence the biomolecule.

Claims

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

providing excitation energy to a sample well formed at a first pixel on a substrate;

receiving, at a sensor formed at the first pixel, a first emission from the sample well, wherein the first emission is associated with a type of nucleic acid subunit from among different types of nucleic acid subunits;

producing, by the sensor, a first signal and a second signal representative of the received first emission;

acquiring the first signal at a first time and the second signal at a second time during charge-accumulation at the sensor for the first emission;

analyzing the first signal and the second signal; and

identifying the type of the nucleic acid subunit based upon the analysis of the first signal and the second signal.

2. The method of claim 1, wherein the providing excitation energy comprises activating an excitation source formed at the first pixel.

3. The method of claim 2, wherein the excitation source comprises an organic light-emitting diode.

4. The method of claim 2, wherein the excitation source comprises a nano-scale excitation source that is self-aligned to the sample well.

5. The method of claim 4, wherein the nano-scale excitation source comprises a light-emitting diode, a laser diode, or a vertical-cavity surface-emitting laser.

6. The method of claim 4, wherein the nano-scale excitation source comprises a semiconductor diode.

7. The method of claim 1, wherein the sample well comprises a sub-cutoff nanoaperture.

8. The method of claim 1, wherein the first emission determines a non-zero signal level of both the first signal and the second signal.

9. The method of claim 1, wherein the sensor comprises at least two spatially separated photodetector segments, and the first signal and the second signal are representative of a spatial distribution pattern for a first spectral band of the first emission.

10. The method of claim 1, wherein the analyzing comprises evaluating a ratio of signal levels for the first signal and the second signal.

11. The method of claim 1, wherein the excitation source, sample well, and sensor are located within a first pixel of a plurality of pixels formed on the substrate, and each of the plurality of pixels comprises a respective excitation source, sample well, and sensor.

12. A method for sequencing a target nucleic acid molecule, the method comprising:

providing an integrated device that includes (i) a sample well containing said target nucleic acid molecule, a polymerizing enzyme and a plurality of types of nucleotides or nucleotide analogs, and (ii) at least one excitation source that directs excitation energy to said sample well;

performing an extension reaction at a priming location of said target nucleic acid molecule in the presence of said polymerizing enzyme to sequentially incorporate said nucleotides or nucleotide analogs into a growing strand that is complementary to said target nucleic acid molecule, wherein upon incorporation and excitation by excitation energy from said excitation source, said nucleotides or nucleotide analogs produce emissions from said sample well;

detecting said emissions at a sensor that is configured to receive said emissions from said sample well;

receiving signal sets from the sensor for each detected emission, wherein the signal sets are representative of spatial and/or temporal distributions of said detected emissions and distinguish types of nucleotides or nucleotide analogs and wherein a signal set of the signal sets comprises a first signal acquired at a first time and a second signal acquired at a second time during charge-accumulation at the sensor; and

identifying the types of nucleotides or nucleotide analogs based on said received signal sets, thereby sequencing said target nucleic acid molecule.

13. The method of claim 12, wherein said nucleotides or nucleotides analogs include tags that produce said emissions upon incorporation in said growing strand.

14. The method of claim 13, wherein said tags are luminescent tags.

15. The method of claim 12, wherein said nucleotides or nucleotide analogs are identified subsequent to detecting said signal sets.

16. The method of claim 12, wherein said plurality of types of nucleotides or nucleotide analogs include four different types of nucleotides or nucleotide analogs, and wherein said signal sets are associated with said four different types of nucleotides or nucleotide analogs and are distinguishable from one another.

17. The method of claim 12, wherein said spatial distribution patterns are distinguishable based on wavelengths of said emissions.

18. The method of claim 12, wherein said priming location includes a primer that is complementary to said target nucleic acid molecule.

19. The method of claim 18, wherein performing an extension reaction comprises performing a primer extension reaction using said primer hybridized to said target nucleic acid molecule to yield said growing strand.

20. The method of claim 12, wherein said target nucleic acid molecule is double stranded.

21. The method of claim 20, wherein said priming location is a gap or nick in said target nucleic acid molecule.

22. The method of claim 12, wherein said polymerizing enzyme is immobilized in said well.

23. The method of claim 22, wherein said polymerizing enzyme is immobilized at a bottom portion of said well.

24. The method of claim 22, wherein said polymerizing enzyme is immobilized using a linker that is attached to a surface of said well.

25. The method of claim 12, wherein said polymerizing enzyme exhibits strand displacement activity.

26. The method of claim 12, wherein said sample well is among a plurality of sample wells in said integrated device.

27. The method of claim 26, wherein said integrated device includes a plurality of excitation sources operatively coupled to said plurality of sample wells.

28. The method of claim 27, wherein said integrated device includes a plurality of sensors configured to receive emissions from said plurality of sample wells.