Integrated device with external light source for probing detecting and analyzing molecules

• US 10,288,565 B2
• Filed: 07/05/2017
• Issued: 05/14/2019
• Est. Priority Date: 08/08/2014
• 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, West, Lawrence C., Fife, Keith G., Cipriany, Benjamin, Ghasemi, Farshid
• Primary Examiner(s): Ayub, Hina F
• Application Number:
  US15/641,949
• Publication Number:
  US 20170299518A1
• Time in Patent Office: 678 Days
• 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: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6463 Optics
• CPC Class Codes: G01N 2021/6471 Special filters, filter wheel
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• 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 2201/064 Stray light conditioning
• CPC Class Codes: G01N 2201/0696 Pulsed
• CPC Class Codes: G01N 2201/08 Optical fibres; light guides
• CPC Class Codes: G01N 2201/12 Circuits of general importance; Signal processing

Abstract

System and methods for analyzing single molecules and performing nucleic acid sequencing. An integrated device includes multiple pixels with sample wells configured to receive a sample, which when excited, emits radiation. The integrated device includes at least one waveguide configured to propagate excitation energy to the sample wells from a region of the integrated device configured to couple with an excitation energy source. A pixel may also include at least one element for directing the emission energy towards a sensor within the pixel. The system also includes an instrument that interfaces with the integrated device. The instrument may include an excitation energy source for providing excitation energy to the integrated device by coupling to an excitation energy coupling region of the integrated device. One of multiple markers distinguishable by temporal parameters of the emission energy may label the sample and configuration of the sensor within a pixel may allow for detection of a temporal parameter associated with the marker labeling the sample.

Claims

1. A method of identifying a sample, the method comprising:

delivering a plurality of pulses of optical excitation energy to a sample well of an integrated device by propagating the plurality of pulses of optical excitation energy along a waveguide of the integrated device, wherein the sample well is positioned to receive the plurality of pulses of optical excitation energy from the waveguide and the sample is located in the sample well;

detecting, using a sensor of the integrated device positioned to receive light from the sample well, a signal indicative of lifetime of luminescence emitted by the sample, wherein the sensor is configured to aggregate, into at least two time bins, charge carriers produced by received photons of the luminescence emitted from the sample in response to the plurality of pulses of optical excitation energy; and

identifying the sample based on the signal.

2. The method of claim 1, wherein the sample is a biological sample identifiable by a lifetime of luminescence emitted from the biological sample in response to the optical excitation energy, and the sample well is configured to retain the biological sample.

3. The method of claim 1, wherein the sample is a biological sample, and the method further comprises detecting a plurality of luminescently labeled subunits of the biological sample.

4. The method of claim 3, wherein the biological sample comprises a target nucleic acid molecule, and wherein identifying the sample comprises identifying a sequence of the target nucleic acid molecule.

5. The method of claim 4, further comprising identifying the sequence of the target nucleic acid molecule by analyzing a time sequence of incorporation of luminescently labeled nucleotides and/or nucleotide analogs incorporated sequentially into a nucleic acid molecule complementary to the target nucleic acid molecule.

6. The method of claim 1, further comprising obtaining the signal by reading out the signal from the sensor after delivering a portion of the plurality of pulses of optical excitation energy to the sample well.

7. The method of claim 1, wherein the sensor is configured to generate a signal indicative of an intensity of the luminescence emitted by the sample in response to the optical excitation energy, and identifying the sample comprises identifying the sample based on the intensity of the luminescence emitted by the sample.

8. The method of claim 1, wherein the sensor is further configured to selectively direct the charge carriers into at least two storage regions of the sensor based upon times at which the charge carriers are produced, and wherein the at least two storage regions correspond to the at least two time bins.

9. The method of claim 1, wherein the method further comprises:

generating, using at least one first monitoring sensor, a signal indicative of alignment of a beam of optical excitation energy to a grating coupler of the integrated device that is optically coupled to the waveguide; and

aligning the beam of optical excitation energy to the grating coupler based, at least in part, on the signal indicative of alignment of the beam of optical excitation energy to the grating coupler.

10. The method of claim 1, wherein the method further comprises:

generating, using at least one second monitoring sensor, a signal indicative of power of optical excitation energy coupled out of the waveguide; and

aligning the beam of optical excitation energy to the grating coupler based, at least in part, on the signal indicative of power of optical excitation energy coupled out of the waveguide.

11. A method for nucleic acid sequencing comprising:

delivering a plurality of pulses of optical excitation energy to a sample well of an integrated device by propagating the plurality of pulses of optical excitation energy along a waveguide of the integrated device, wherein the sample well is positioned to receive the plurality of pulses of optical excitation energy from the waveguide;

exposing a complex having a template nucleic acid molecule, a primer, and a polymerizing enzyme to a plurality of types of luminescently labeled nucleotides and/or nucleotide analogs, wherein the complex is located in the sample well and each type of luminescently labeled nucleotide and/or nucleotide analog has a different luminescent lifetime and/or luminescent intensity;

detecting, using the sensor, a signal indicative of lifetime of luminesce emitted from the sample well in response to the plurality of pulses of excitation energy, wherein the signal is generated by receiving photons of the luminescence at the sensor and aggregating charge carriers produced by the received photons into at least two time bins; and

determining, based on the signal, an incorporation event of a nucleotide and/or a nucleotide analog of the plurality of types of luminescently labeled nucleotides and/or nucleotide analogs into a nucleic acid molecule complementary to the template nucleic acid molecule.

12. The method of claim 11, wherein the signal is further indicative of intensity of luminescence emitted from the sample well.

13. The method of claim 12, wherein determining the incorporation event comprises identifying one type of the plurality types of luminescently labeled nucleotides and/or nucleotide analogs based on the intensity of the luminescence.

14. The method of claim 11, wherein the sensor is configured to generate a plurality of signals unique to the different types of the plurality of types of luminescently labeled nucleotides and/or nucleotide analogs.

15. The method of claim 11, wherein each type of luminescently labeled nucleotide and/or nucleotide analog emits a different combination of luminescent lifetime and luminescent intensity.

16. The method of claim 11, further comprising identifying a sequence of the target nucleic acid molecule by analyzing a time sequence of incorporation of luminescently labeled nucleotides and/or nucleotide analogs incorporated sequentially into a nucleic acid molecule complementary to the target nucleic acid molecule.

17. The method of claim 11, further comprising obtaining the signal by reading out the signal from the sensor after delivering a portion of the plurality of pulses of optical excitation energy to the sample well.

18. The method of claim 11, wherein the sensor is further configured to selectively direct the charge carriers into at least two storage regions of the sensor based upon times at which the charge carriers are produced, and wherein the at least two storage regions correspond to the at least two time bins.

19. The method of claim 11, wherein the method further comprises:

generating, using at least one first monitoring sensor, a signal indicative of alignment of a beam of optical excitation energy to a grating coupler of the integrated device that is optically coupled to the waveguide; and

aligning the beam of optical excitation energy to the grating coupler based, at least in part, on the signal indicative of alignment of the beam of optical excitation energy to the grating coupler.

20. The method of claim 11, wherein the method further comprises:

generating, using at least one second monitoring sensor, a signal indicative of power of optical excitation energy coupled out of the waveguide; and

aligning the beam of optical excitation energy to the grating coupler based, at least in part, on the signal indicative of power of optical excitation energy coupled out of the waveguide.