Apparatus and method for performing nucleic acid analysis

• US 9,588,051 B2
• Filed: 09/11/2015
• Issued: 03/07/2017
• Est. Priority Date: 09/17/2004
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Korlach, Jonas
• Primary Examiner(s): Chowdhury, Tarifur
• Assistant Examiner(s): AKANBI, ISIAKA O
• Application Number:
  US14/852,045
• Publication Number:
  US 20160061738A1
• Time in Patent Office: 543 Days
• Field of Search: 356/446, 356/432, 250/251, 250/459.1, 359/358, 359/850, 359/538
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6463 Optics
• CPC Class Codes: G01N 2021/7786 Fluorescence
• CPC Class Codes: G01N 21/00 Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light G01N3/00 - G01N19/00 take precedence
• 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 21/645 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 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/77 by observing the effect on a chemical indicator
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers

Abstract

The present invention relates to optical confinements, methods of preparing and methods of using them for analyzing molecules and/or monitoring chemical reactions. The apparatus and methods embodied in the present invention are particularly useful for high-throughput and low-cost single-molecular analysis.

Claims

1. An analytical system comprising:

a) a transparent substrate having disposed on its surface a transparent layer, the index of refraction of the substrate and the transparent layer selected to provide for total internal reflection when illuminated through the transparent substrate;

b) nanoscale holes in the transparent layer, wherein each of the nanoscale holes provides a degree of lateral confinement given by its diameter;

c) a solution comprising an analyte, the analyte disposed on top of the transparent layer and extending into the nanoscale holes; and

d) an optical system operatively coupled to the nanoscale holes that detects signals from the nanoscale holes.

2. The analytical system of claim 1, wherein the transparent layer has an index of refraction substantially identical to the index of refraction of the solution.

3. The analytical system of claim 1, wherein the transparent layer comprises a polymer.

4. The analytical system of claim 3, wherein the transparent layer comprises a polymethyl methacrylate coating.

5. The analytical system of claim 1, wherein the transparent layer comprises a resist resin.

6. The analytical system of claim 1, wherein the transparent substrate is a high index transparent substrate.

7. The analytical system of claim 6, wherein the high index transparent substrate is a sapphire substrate.

8. The analytical system of claim 1, wherein the nanoscale holes are a result of exposure of the transparent layer to electron beam lithography.

9. The analytical system of claim 1, wherein the optical system comprises a photon detector.

10. The analytical system of claim 9, wherein the photon detector is selected from the group consisting of optical readers, high-efficiency photon detection systems, photodiodes, cameras, charge coupled devices, electron-multiplying charge-coupled devices, intensified charge coupled devices, and confocal microscopes equipped with any of the foregoing photon detectors.

11. The analytical system of claim 9, wherein the optical system comprises an optical transmission element that directs optical signals from the nanoholes to the photon detector.

12. The analytical system of claim 11, wherein the optical transmission element also modifies wavelengths of the emitted optical signals.

13. The analytical system of claim 11, wherein the optical transmission element is selected from the group consisting of diffraction gratings, arrayed waveguide gratings, optic fibers, optical switches, mirrors, lenses, collimators, optical attenuators, polarization filters, wavelength filters, wave-plates, prisms, and delay lines.

14. A method of optically analyzing an analyte in a solution comprising:

a) providing a transparent substrate having disposed on its surface a transparent layer, the index of refraction of the transparent substrate and transparent layer selected to provide for the total internal reflection when illuminated through the transparent substrate, further wherein the transparent layer comprises nanoscale holes;

b) disposing a solution comprising the analyte upon the transparent layer and into the nanoscale holes, wherein each of the nanoscale holes laterally confines the solution to a degree given by its diameter;

c) generating and transmitting incident light to the nanoscale holes, thereby optically exciting any of the analyte within the nanoscale holes; and

d) detecting a signal emitted from said any of the analyte within the nanoscale holes, thereby optically analyzing the analyte.

15. The method of claim 14, wherein the transparent layer has an index of refraction substantially identical to the index of refraction of the solution.

16. The method of claim 14, wherein the transparent layer comprises a polymer.

17. The method of claim 16, wherein the transparent layer comprises a polymethyl methacrylate coating.

18. The method of claim 14, wherein the transparent layer comprises a resist resin.

19. The method of claim 14, wherein the transparent substrate is a high index transparent substrate.

20. The method of claim 19, wherein the high index transparent substrate is a sapphire substrate.

21. The method of claim 14, wherein the nanoscale holes are a result of exposure of the transparent layer to electron beam lithography.

22. The method of claim 14, wherein the detecting is performed using an optical system comprising a photon detector.

23. The method of claim 22, wherein the photon detector is selected from the group consisting of optical readers, high-efficiency photon detection systems, photodiodes, cameras, charge coupled devices, electron-multiplying charge-coupled devices, intensified charge coupled devices, and confocal microscopes equipped with any of the foregoing photon detectors.

24. The method of claim 22, wherein the optical system comprises an optical transmission element that directs optical signals from the nanoholes to the photon detector.

25. The method of claim 24, wherein the optical transmission element is selected from the group consisting of diffraction gratings, arrayed waveguide gratings, optic fibers, optical switches, mirrors, lenses, collimators, optical attenuators, polarization filters, wavelength filters, wave-plates, prisms, and delay lines.