OPTICAL SYSTEM AND ASSAY CHIP FOR PROBING, DETECTING AND ANALYZING MOLECULE

• US 20160041095A1
• Filed: 08/07/2015
• Published: 02/11/2016
• 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, Glenn, Paul E., West, Lawrence C., Cipriany, Benjamin, Fife, Keith G.
• Granted Patent: US 9,921,157 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 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 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 2201/0446 Multicell plate, sequential
• CPC Class Codes: G01N 2201/062 LED's

Abstract

Apparatus and methods for analyzing single molecule and performing nucleic acid sequencing. An apparatus can include an assay chip that includes multiple pixels with sample wells configured to receive a sample, which, when excited, emits emission energy; at least one element for directing the emission energy in a particular direction; and a light path along which the emission energy travels from the sample well toward a sensor. The apparatus also includes an instrument that interfaces with the assay chip. The instrument includes an excitation light source for exciting the sample in each sample well; a plurality of sensors corresponding the sample wells. Each sensor may detect emission energy from a sample in a respective sample well. The instrument includes at least one optical element that directs the emission energy from each sample well towards a respective sensor of the plurality of sensors.

Claims

1. An instrument configured to interface with an assay chip comprising a plurality of sample wells, each sample well of the plurality of sample wells configured to receive a sample, the instrument comprising:

at least one pulsed excitation light source configured to excite the sample of at least a portion of the plurality of sample wells;

a plurality of sensors, each sensor of the plurality of sensors corresponding to a sample well of the plurality of sample wells, wherein each sensor of the plurality of sensors is configured to detect emission energy from the sample in a respective sample well, wherein each sensor of the plurality of sensors is capable of detecting the detection time of the emission energy; and

at least one optical element configured to direct the emission energy from each sample well of the plurality of sample wells towards a respective sensor of the plurality of sensors.

2. The instrument of claim 1, further comprising:

a polychroic mirror configured to reflect excitation light from the at least one excitation light source towards the assay chip and transmit the emission energy from the plurality of samples towards the plurality of sensors.

3. The instrument of claim 1, wherein the at least one optical element comprises a relay lens.

4. The instrument of claim 1, wherein the at least one excitation light source comprises a plurality of light sources, each light source of the plurality of light sources emitting excitation light at one or more of a plurality of wavelengths.

5. The instrument of claim 4, further comprising a wavelength combiner for spatially overlapping the light emitted from each of the plurality of light sources.

7. The instrument of claim 1, further comprising at least one spectral filter configured to transmit the emission energy and absorb and/or reflect excitation light from the at least one excitation light source.

8. The instrument of claim 1, further comprising at least one spectral sorting element for spatially separating emission energy of a first wavelength from emission energy of a second wavelength.

9. The instrument of claim 8, wherein the at least one spectral sorting element comprises a diffractive optical element.

10. The instrument of claim 9, wherein the diffractive optical element both chromatically disperses the emission energy and focuses the emission energy.

11. The instrument of claim 9, wherein the diffractive optical element comprises an offset Fresnel lens.

12. The instrument of claim 8, wherein the at least one spectral sorting element is a light-filtering element.

13. The instrument of claim 1, further comprising a control system that is programmed to (i) direct excitation light to said plurality of sample wells, (ii) detect signals at said plurality of sensors from said plurality of wells, and (iii) use a time distribution of said signals to identify said sample or subunit thereof.

14. An apparatus comprising:

an assay chip comprising a plurality of pixels, each of the plurality of pixels comprising;

a sample well configured to receive a sample, which, when excited, emits emission energy;

at least one element for directing the emission energy in a particular direction, wherein the at least one element is selected from the group consisting of a refractive element, a diffractive element, a plasmonic element and a resonator; and

a light path along which the emission energy travels from the sample well toward a sensor;

and

an instrument configured to interface with the assay chip, the instrument comprising;

at least one pulsed excitation light source configured to excite the sample in each sample well;

a plurality of sensors, each sensor of the plurality of sensors corresponding to a respective sample well, wherein each sensor of the plurality of sensors is configured to detect emission energy from the sample in the respective sample well, wherein each sensor of the plurality of sensors is capable of detecting the detection time of the emission energy; and

at least one optical element configured to direct the emission energy from each sample well towards a respective sensor of the plurality of sensors.

15. The apparatus of claim 14, wherein the assay chip is configured to be connected to and removed from the instrument.

16. The apparatus of claim 15, wherein, when the assay chip is connected to the instrument, the optical distance between a sample well of the plurality of sample wells and the corresponding sensor of the plurality of sensors is less than 30 cm.

17. The apparatus of claim 15, wherein, when the assay chip is connected to the instrument, the optical distance between a sample well of the plurality of sample wells and the corresponding sensor of the plurality of sensors is less than 5 cm.

18. The apparatus of claim 15, wherein, when the assay chip is connected to the instrument, the optical distance between a sample well of the plurality of sample wells and the corresponding sensor of the plurality of sensors is less than 1 cm.

19. The apparatus of claim 14, wherein:

each sample comprises a luminescent tag that emits the emission energy within one wavelength band of a plurality of wavelength bands; and

each sensor of the plurality of sensors comprises a sub-sensor configured to detect the emission energy at each of the plurality of wavelength bands.

20. The apparatus of claim 19, wherein each sensor of the plurality of sensors comprises at least two sub-sensors.

21. The apparatus of claim 20, wherein each sensor of the plurality of sensors comprises at least four sub-sensors.

23. The apparatus of claim 20, wherein the instrument further comprises at least one wavelength dependent element that directs emission energy of a first wavelength towards a first sub-sensor of the at least two sub-sensors and directs emission energy of a second wavelength towards a second sub-sensor of the at least two sub-sensors.

26. The apparatus of claim 14, wherein each sample comprises a luminescent tag with a lifetime that is different from a lifetime of at least one other luminescent tag of another sample.

27. The apparatus of claim 19, wherein:

a first luminescent tag associated with a first sample is excited by light of a first wavelength but is not excited by light of a second wavelength; and

a second luminescent tag associated with a second sample is excited by light of the second wavelength but is not excited by light of the first wavelength.

24. The apparatus of claim 22, wherein the at least one wavelength dependent element is a diffractive optical element.

25. The apparatus of claim 22, wherein the at least one wavelength dependent element is a spectral filter.

28. A method of analyzing a specimen, the method comprising:

providing the specimen on the top surface of an assay chip comprising a plurality of sample wells;

aligning the chip with an instrument comprising at least one excitation light source and at least one sensor;

exciting a sample from the specimen in at least one of the plurality of sample wells with pulsed excitation light from the at least one pulsed excitation light source; and

detecting, with the at least one sensor, emission energy generated by the sample in the at least one sample well in response to excitation by the excitation light, wherein the at least one sensor is capable of determining the lifetime of the emission energy generated by the sample.

29. The method of claim 28, further comprising:

determining, based on the detection of the emission energy, a type of the molecule that emitted the emission energy.

30. The method of claim 29, wherein determining the type of molecule comprises:

measuring a spectral property and/or a temporal property of the emission energy.

31. The method of claim 29, wherein determining the type of molecule further comprises:

determining the wavelength of the excitation light that excited the sample.

32. The method of claim 28, wherein the at least one excitation light source comprises a plurality of excitation light sources, each excitation light source of the plurality of excitation light sources emitting light of a different wavelength.

33. The method of claim 28, wherein the sample comprises at least one nucleotide attached to a fluorophore.

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

(a) providing a chip adjacent to an instrument that includes a pulsed excitation source and a sensor capable of detecting at least one temporal property of light, wherein said chip includes at least one well that is operatively coupled to said excitation source and said sensor when said chip is at a sensing position of said instrument, and wherein said well contains said target nucleic acid molecule, a polymerizing enzyme and a plurality of types of nucleotides or nucleotide analogs;

(b) with said chip at said sensing position, 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 nucleotides analogs emit signals in said well;

(c) using said sensor to detect spatial and/or temporal distribution patterns of said signals that are distinguishable for said plurality of types of nucleotides or nucleotide analogs; and

(d) identifying said nucleotides or nucleotide analogs based on said spatial and/or temporal distribution patterns of said signals, thereby sequencing said target nucleic acid molecule.

35. The method of claim 34, wherein said nucleotides or nucleotides analogs include tags that emit said signals upon incorporation in said growing strand.

36. The method of claim 35, wherein said tags are luminescent tags.

37. The method of claim 34, wherein said nucleotides or nucleotide analogs are identified subsequent to detecting said spatial and/or temporal distribution patterns of said signals.

38. The method of claim 34, wherein said plurality of types of nucleotides or nucleotide analogs include four different types of nucleotides or nucleotide analogs, and wherein said spatial and/or temporal distribution patterns of said signals associated with said four different types of nucleotides or nucleotide analogs are distinguishable from one another.

39. The method of claim 34, wherein said spatial and/or temporal distribution patterns of said signals associated with said four different types of nucleotides or nucleotide analogs are detected separately from one another.

40. The method of claim 34, wherein said spatial and/or temporal distribution patterns of said signals are distinguishable from one another based on a shape and/or intensity distribution of each of said spatial and/or temporal distribution patterns.

41. The method of claim 34, wherein said priming location includes a primer that is complementary to said target nucleic acid molecule.

42. The method of claim 34, wherein (b) comprises performing a primer extension reaction using said primer hybridized to said target nucleic acid molecule to yield said growing strand.

43. The method of claim 42, wherein said target nucleic acid molecule is double stranded.

44. The method of claim 43, wherein said priming location is a gap or nick in said target nucleic acid molecule.

45. The method of claim 34, wherein said polymerizing enzyme is immobilized in said well.

46. The method of claim 45, wherein said polymerizing enzyme is immobilized at a bottom portion of said well.

47. The method of claim 46, wherein said polymerizing enzyme is immobilized using a linker that is attached to a surface of said well.

48. The method of claim 34, wherein said polymerizing enzyme exhibits strand displacement activity.

49. The method of claim 34, wherein said well is among a plurality of wells in said chip.

50. The method of claim 34, wherein said instrument includes a plurality of excitation sources operatively coupled to said plurality of wells.

51. A method for nucleic acid sequencing, comprising:

(a) providing a chip adjacent to an instrument, wherein said chip includes a plurality of wells that are each operatively coupled to (i) a pulsed excitation source and (ii) a sensor of said instrument when said chip is at a sensing position of said instrument, and wherein an individual well of said plurality contains said target nucleic acid molecule, a polymerizing enzyme and a plurality of types of nucleotides or nucleotide analogs;

(b) with said chip at said sensing position, subjecting said target nucleic acid molecule to a polymerization reaction to yield a growing strand that is complementary to said target nucleic acid molecule in the presence of said nucleotides or nucleotide analogs and said polymerizing enzyme, wherein said nucleotides or nucleotides analogs emit signals in said individual well upon excitation by excitation energy from said excitation source during incorporation;

(c) using said sensor to detect temporal distribution patterns of said signals that are distinguishable for said plurality of types of nucleotides or nucleotide analogs; and

(d) identifying a sequence of said target nucleic acid molecule based on said spatial and/or temporal distribution patterns of said signals.

52. The method of claim 51, wherein said nucleotides or nucleotides analogs include tags that emit said signals upon incorporation in said growing strand.

53. The method of claim 52, wherein said tags are luminescent tags.

54. The method of claim 51, wherein said sequence is identified subsequent to detecting said spatial and/or temporal distribution patterns of said signals.

55. The method of claim 51, wherein said plurality of types of nucleotides or nucleotide analogs include four different types of nucleotides or nucleotide analogs, and wherein said spatial and/or temporal distribution patterns of said signals associated with said four different types of nucleotides or nucleotide analogs are distinguishable from one another.

56. The method of claim 55, wherein said spatial and/or temporal distribution patterns of said signals associated with said four different types of nucleotides or nucleotide analogs are detected separately from one another.

57. The method of claim 51, wherein (b) comprises performing a primer extension reaction using a primer hybridized to said target nucleic acid molecule to yield said growing strand.

58. The method of claim 51, wherein said target nucleic acid molecule is single-stranded.

59. The method of claim 51, wherein said polymerizing enzyme is immobilized in said well.

60. The method of claim 51, wherein said excitation source is operatively coupled to said plurality of wells.

61. The method of claim 51, wherein said instrument includes a plurality of excitation sources that are operatively coupled to said plurality of wells.

62. The method of claim 51, wherein said instrument includes a plurality of sensors that are operatively coupled to said plurality of wells.