Real Time Microarrays

• US 20080039339A1
• Filed: 06/05/2007
• Published: 02/14/2008
• Est. Priority Date: 06/05/2006
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Riechmann, Jose, Vikalo, Haris, Hassibi, Babak, Hassibi, Arjang
• Granted Patent: US 9,133,504 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

This invention provides methods and systems for measuring the binding of analytes in solution to probes bound to surfaces in real-time.

Claims

1. A method comprising measuring binding of analytes to probes on a microarray in real-time.

2. A method of claim comprising the steps of:

(a) contacting a fluid volume comprising a plurality of different analytes with a solid substrate comprising a plurality of different probes, wherein the probes are capable of specifically binding to the analytes; and

(b) measuring signals at multiple time points while the fluid volume is in contact with the substrate, wherein the signals measured at multiple time points can be correlated with the amount of binding of the analytes with the probes.

3. The method of claim 2 further comprising (c) using the signals measured at multiple time points to determine the concentration of an analyte in the fluid volume.

4. The method of claim 2 wherein a change in the signals with time correlates with the amount of the analytes bound to the probes.

5. The method of claim 2 wherein the signals comprise electrochemical, electrical, mechanical, magnetic, acoustic, or electromagnetic signals.

6. The method of claim 2 wherein the signals are electromagnetic signals comprising fluorescence, absorption, or luminescence.

7. The method of claim 6 wherein the analytes comprise quenching moieties, and the electromagnetic signals measured at multiple time points correlates, at least in part to the quenching of fluorescence.

8. The method of claim 7 wherein the fluorescence that is quenched is due at least in part to fluorescent moieties bound to the probes.

9. The method of claim 7 wherein the fluorescence that is quenched is due at least in part to fluorescent moieties bound to the solid substrate, wherein such fluorescent moieties are not covalently bound to the probes.

10. The method of claim 2 wherein a the optical signals measured at multiple time points are due, at least in part, to fluorescent resonance energy transfer (FRET).

11. The method of claim 2 wherein the different probes are located on the solid substrate at different addressable locations.

12. The method of claim 2 comprising at least about 3, 4, 5 10, 50, 100, 100, 1000, 10,000, 100,000, or 1,000,000 probes.

13. The method of claim 2 wherein the solid substrate comprises one or more beads.

14. The method of claim 2 wherein the analytes and/or the probes comprise the chemical species of nucleic acids or nucleic acid analogs, proteins, carbohydrates, or lipids.

15. The method of claim 14 wherein the analytes and the probes are the same type of chemical species.

16. The method of claim 14 wherein the analyte and the probe are each a different type of chemical species.

17. The method of claim 2 wherein the analyte and the probe comprise nucleic acids or nucleic acid analogs.

18. The method of claim 2 wherein the probes comprise proteins.

19. The method of claim 14 wherein the probes comprise antibodies or enzymes.

20. The method of claim 2 wherein the at least two time points are measured at a time when the amount of binding of an analyte to a probe corresponding to that signal is less than 50% of saturation.

21. The method of claim 2 wherein two or more time points are measured when the fluid volume is at one temperature, and two or more time points are measured when the fluid volume is at a second temperature.

22. The method of claim 2 wherein two or more time points are measured when the analyte in the fluid volume is at one concentration, and two or more time points are measured when the fluid volume is at a second concentration.

23. The method of claim 22 wherein the concentration of the analyte is enriched between the sets of time points.

24. The method of claim 22 wherein the concentration of the analyte is diluted between the sets of time points.

25. The method of claim 11 wherein the solid substrate also comprises control spots.

26. The method of claim 6 comprising multiple fluorescent species, with different excitation and/or emission spectra.

27. The method of claim 2 further comprising determining binding of a probe with two or more analytes comprising subjecting binding data corresponding to signal and time to an algorithm which determines the contribution of each of the two or more analytes to the binding data.

28. The method of claim 7 wherein the probes comprise a fluorescent moiety and the initial surface concentration of probes is determined by measuring fluorescence.

29. The method of claim 2 wherein:

(a) the analytes comprise nucleic acid molecules labeled with a quencher;

(b) the probes comprise nucleic acid molecules;

(c) the substrate is substantially planar and comprises an array of discrete locations at different addresses on the substrate, wherein the locations have attached thereto different probes and a fluorescent label that produces the signal, wherein the fluorescent label is in sufficient proximity to the probe whereby hybridization between an analyte and probe at the location causes FRET and/or quenching of the signal.

30. The method of claim 29 wherein the fluorescent label is attached to the surface through the probe.

31. The method of claim 30 wherein the fluorescent label is non-covalently attached to the probe.

32. The method of claim 29 wherein the fluorescent label is not attached to the surface through the probe.

33. The method of claim 29 further comprising determining the concentration of the analyte in the fluid based on the kinetics of change of the signal over time.

34. The method of claim 2 wherein the fluid volume further comprises competitor molecules labeled with a quencher, wherein the competitor molecules compete with the analytes for binding with the probes.

35. A method of detecting binding between analyte molecules and an array of probe molecules comprising:

(a) incubating the analyte molecules with the array of probe molecules, wherein binding between a analyte molecule and a probe molecule results in a change in a signal from the array; and

(b) measuring the signal from the array over time during incubation.

36. The method of claim 35 wherein binding between a target molecule and a probe molecule results in a decrease in a signal from the array.

37. The method of claim 35 wherein binding between a target molecule and a probe molecule results in an increase in a signal from the array.

38. A system comprising:

(a) a device with (i) a solid support having a surface and (ii) a plurality of different probes, wherein the different probes are immobilized to the surface;

(b) a fluid volume comprising an analyte wherein the fluid volume is in contact with the solid support, and

(c) a detector assembly comprising means to detect signals measured at multiple time points from each of a plurality of spots on the microarray while the fluid volume is in contact with the solid support.

39. The system of claim 38 wherein the signals measured at multiple time points detected by the detectors can be correlated with the binding of analyte to the probes.

40. The system of claim 38 further comprising:

(d) an assembly that controls temperature of the solid support and/or the fluid volume.

41. The system of claim 38 wherein the different probes are immobilized at different addressable locations.

42. The system of claim 40, further comprising:

(e) a data acquisition system for acquiring and storing the data, and

(f) a computing system for analyzing the signals.

43. The system of claim 38 wherein the plurality of detectors comprises an array of transducers.

44. The system of claim 38 wherein the array of transducers is capable of measuring an electrochemical, electrical, mechanical, magnetic, acoustic, or optical signal.

45. The system of claim 43 wherein the solid substrate is in contact with the array of transducers.

46. The system of claim 43 wherein the transducer array spaced away from the solid substrate.

47. The system of claim 43 wherein one or more transducers corresponds to an addressable location.

48. The system of claim 44 wherein the transducer array is an optical transducer array which is optically coupled to the solid substrate.

49. The system of claim 48 wherein the optical transducer array is optically coupled to the solid substrate via one or more lenses.

50. The system of claim 38 that is capable of measuring a plurality of binding rates simultaneously.

51. The system of claim 50 wherein the plurality of binding rates is used to determine the concentration of a plurality of analytes.

52. The system of claim 38, wherein the signal detected by the detector comprises an electrochemical, electrical, mechanical, magnetic, acoustic, or optical signal.

53. The system of claim 52 wherein the signal us used for cyclic voltammetry, impedance spectroscopy, or surface plasmon resonance.

54. The system of claim 52 wherein the signal is an optical signal.

55. The system of claim 54 wherein the signal is from fluorescence, absorption, or luminescence.

56. The system of claim 55 wherein the signals measured at multiple time points, correlating to the binding of analyte is due, at least in part, to fluorescent resonance energy transfer (FRET).

57. The system of claim 55 wherein the signals measured at multiple time points, correlating to the binding of analyte is due, at least in part, to quenching of a fluorescent signal.

58. The system of claim 57 wherein the signals measured at multiple time points are due, at least in part, to the interaction between an analyte comprising a quenching moiety, and probe comprising a fluorescent moeity.

59. The system of claim 58 wherein the moiety comprising the optical signal is bound to the probe molecule.

60. The system of claim 58 wherein the moeity comprising the optical signal is bound to the substrate.

61. A system comprising:

(a) an assay assembly comprising means to engage a microarray and means to perform an assay on a surface of the microarray; and

(b) a detector assembly comprising means to detect signals measured at multiple time points from each of a plurality of spots on the microarray during the performance of the assay.

62. The system of claim 61 wherein the means to perform the assay comprise means to provide a compartment wherein the surface of the microarray comprises a floor of the compartment and means to deliver reagents and analytes into the compartment.

63. A device comprising:

a solid substrate having a surface and a plurality of different probes, wherein

(a) the different probes are immobilized to the surface at different addressable locations,

(b) the addressable locations comprise optical signal moieties bound to the surface,

(c) the optical signal moieties are not bound directly to the probes, and

(d) the optical signal from the optical signal moieties is capable of changing upon binding of an analyte to the probes.

64. The device of claim 63 wherein the optical signal moiety comprises a dye, a luminescent moiety, or a fluorescent moiety.

65. Software comprising:

(a) a computer executable code that accesses information about signals measured at multiple time points at each of a plurality of known locations on a microarray, wherein the signal intensity is a function of the number of binding events between analyte molecules and probe molecules attached to the microarray at known locations.

66. The software of claim 65 comprising:

(b) code that executes an algorithm that uses the information to determine the expected number of binding events when binding has reached saturation.