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

• US 10,712,274 B2
• Filed: 05/09/2018
• Issued: 07/14/2020
• 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:
  US15/974,826
• Publication Number:
  US 20180348133A1
• Time in Patent Office: 797 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. An integrated device for analyzing a plurality of samples in parallel, the device comprising:

a plurality of pixels formed on a substrate;

a sample well formed at each pixel of the plurality of pixels; and

a sensor formed at each pixel of the plurality of pixels, wherein the sensor is arranged to receive a first emission from the sample well, and wherein the sensor is further configured to acquire a first signal and a second signal during a charge-accumulation cycle of the sensor for the first emission and to output the first signal and the second signal as representative of the received first emission.

2. The integrated device of claim 1, further comprising integrated circuitry formed on the substrate with the sensor and arranged to receive the first signal and the second signal from the sensor.

3. The integrated device of claim 2, wherein the integrated circuitry includes a communication interface for transmitting data to a computing device external to the integrated device.

4. The integrated device of claim 1, further comprising an excitation source arranged to provide excitation energy to the sample well that excites the first emission.

5. The integrated device of claim 4, wherein the excitation source provides optical radiation having a characteristic wavelength between approximately 350 nm and approximately 1000 nm.

6. The integrated device of claim 4, wherein the excitation source is located between the sample well and the sensor.

7. The integrated device of claim 1, wherein the sample well is adapted to retain nucleotides or nucleotide analogs.

8. The integrated device 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.

9. The integrated device of claim 1, wherein each pixel of the plurality of pixels comprises a sample well and a sensor arranged to receive optical emission from the sample well.

10. The integrated device of claim 1, further comprising a micro-optical or nano-optical structure that increases an intensity of excitation energy provided to the sample well.

11. The integrated device of claim 1, further comprising a walled chamber formed around the plurality of pixels that is configured to hold a specimen.

12. The integrated device of claim 11, wherein the integrated device and walled chamber are packaged in a single module having exterior electrical contacts that are arranged for electrical connection with contacts of a receiving dock of an instrument.

13. The integrated device of claim 1, further comprising a divot extending from the sample well into an optically-transparent layer of material.

14. The integrated device of claim 1, wherein the integrated device and sample well are arranged to receive samples in a fluid suspension.

15. The integrated device of claim 1, wherein a minimum diameter of the sample well is between approximately 30 nanometers and approximately 250 nanometers.

16. The integrated device of claim 1, further comprising read-out circuitry formed on the substrate and arranged to read out signals from sensors in each of the plurality of pixels.

17. The integrated device of claim 16, wherein the sensor and read-out circuitry comprise CMOS circuit elements.

18. A method of analyzing a plurality of samples in parallel, the method comprising:

receiving, at a first sensor formed at a first pixel of a plurality of pixels formed on a substrate of an integrated device, a first emission from a first sample well formed at the first pixel;

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

acquiring the first signal and the second signal during a charge-accumulation cycle of the first sensor for the first emission; and

transmitting the first signal and the second signal from the first sensor for analysis.

19. The method of claim 18, further comprising providing excitation energy to the sample well to excite the first emission.

20. The method of claim 18, further comprising evaluating a ratio of the first signal and the second signal to determine a property of a sample in the sample well.

21. The method of claim 18, further comprising performing an extension reaction at a priming location of a target nucleic acid molecule in the sample well and in the presence of a polymerizing enzyme to sequentially incorporate nucleotides or nucleotide analogs into a growing strand that is complementary to the target nucleic acid molecule.

22. The method of claim 21, further comprising:

receiving signal sets from the sensor following excitation of the sample well; and

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