Illumination of integrated analytical systems

• US 10,138,515 B2
• Filed: 09/03/2015
• Issued: 11/27/2018
• Est. Priority Date: 02/19/2010
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Fehr, Adrian, McCaffrey, Nathaniel Joseph, Turner, Stephen
• Primary Examiner(s): Forman, Betty J
• Application Number:
  US14/844,492
• Publication Number:
  US 20160069806A1
• Time in Patent Office: 1,181 Days
• Field of Search: None
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0663 Whole sensors
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/168 Specific optical properties, e.g. reflective coatings
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B82Y 20/00 Nanooptics, e.g. quantum optics or photonic crystals
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• 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 2021/0346 Capillary cells; Microcells
• CPC Class Codes: G01N 2021/6434 Optrodes
• 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 2021/757 using immobilised reagents
• CPC Class Codes: G01N 2021/7786 Fluorescence
• CPC Class Codes: G01N 21/03 Cuvette constructions
• CPC Class Codes: G01N 21/0303 Optical path conditioning in cuvettes, e.g. windows; adapted optical elements or systems; path modifying or adjustment G01N21/031 - G01N21/15 take precedence
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/64 : Fluorescence; Phosphorescence
• 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 21/6454 : using an integrated detector array
• CPC Class Codes: G01N 21/6456 : Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/648 : using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/75 : Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated systems in which material is burnt in a flame or plasma G01N21/72, G01N21/73
• CPC Class Codes: G01N 21/77 : by observing the effect on a chemical indicator
• CPC Class Codes: G01N 2201/067 : Electro-optic, magneto-optic, acousto-optic elements
• CPC Class Codes: G01N 2201/068 : Optics, miscellaneous
• CPC Class Codes: G01N 2201/08 : Optical fibres; light guides
• CPC Class Codes: G01N 33/54373 : involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G02B 6/1226 : involving surface plasmon interaction
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

An analytical device including an optically opaque cladding, a sequencing layer including a substrate disposed below the cladding, and a waveguide assembly for receiving optical illumination and introducing illumination into the device. The illumination may be received from a top, a side edge, and a bottom of the device. The waveguide assembly may include a nanoscale aperture disposed in the substrate and extending through the cladding. The aperture defines a reaction cell for receiving a set of reactants. In various aspects, the device includes a sensor element and the illumination pathway is through the sensor element. Waveguides and illumination devices, such as plasmonic illumination devices, are also disclosed. Methods for forming and operating the devices are also disclosed.

Claims

1. A method for sequencing a nucleic acid comprising:

providing an integrated analytical device comprising

an optically opaque cladding layer having a plurality of nanoscale apertures extending through the cladding layer into a transparent substrate below the cladding layer, the nanoscale apertures separated by regions of the substrate from any other nanoscale apertures, wherein the nanoscale apertures each define a reaction cell for receiving a set of sequencing reagents, the sequencing reagents comprising a plurality of fluorescently labeled nucleotides each having a different excitation spectrum;

a waveguide layer below the reaction cells comprising a plurality of waveguides that direct excitation illumination to the reaction cells; and

a detection layer below the waveguide layer comprising a plurality of sensor elements in optical communication with the reaction cells, wherein each reaction cell has at least one sensor element associated with it, wherein each sensor element comprises a pixel having multiple integrated storage elements;

carrying out nucleic acid sequencing reactions in the reaction cells while illuminating the reaction cells with a pulsed excitation light comprising pulses of at least two different excitation wavelengths iteratively over time, wherein each respective excitation wavelength in the at least two different excitation wavelengths of the pulsed excitation light corresponds to the excitation spectrum of a corresponding fluorescently labeled nucleotide in the plurality of fluorescently labeled nucleotides, and the excitation light at each respective excitation wavelength excites the corresponding fluorescently labeled nucleotide to emit a signal;

measuring emitted signals over time from each reaction cell while carrying out the sequencing reactions; and

correlating said emitted signals with the timing of the pulses of the pulsed excitation light to identify the fluorescently labeled nucleotides excited by the excitation light at each respective excitation wavelength in the at least two different excitation wavelengths, thereby sequencing the nucleic acid.

2. The method of claim 1 wherein the plurality of fluorescently labeled nucleotides comprise four fluorescently labeled nucleotides.

3. The method of claim 1 wherein a single sensor element is associated with each reaction cell.

4. The method of claim 1 wherein the pixel has four integrated storage elements.

5. The method of claim 4 wherein each of the storage elements are electronically gated by the activation of a separate excitation source.

6. The method of claim 4 wherein the analytical device further comprises a modulated controller element coupled to both the detector layer and the waveguide layer to synchronize illumination and storage.

7. The method of claim 1 wherein a delay between an excitation event and emission for each label is preprogrammed into the sensor elements.

8. The method of claim 1 wherein the sensor elements comprise a CMOS, NMOS, or PMOS sensors.

9. The method of claim 1 wherein the analytical device comprises between about 1,000,000 and 10,000,000 sensor elements.

10. The method of claim 1 wherein the reaction cells comprise zero mode waveguides.

11. The method of claim 1 wherein the analytical device further comprises a fluidic conduit that extends across multiple reaction cells.

12. The method of claim 1 wherein the analytical device further comprises a transmission layer between the waveguide layer and the detector layer that transmits emitted light from the reaction cells to the detector layer.

13. The method of claim 12 wherein the transmission layer comprises an optical tunnel.

14. The method of claim 12 wherein the transmission layer comprises optical filters.

15. The method of claim 1, wherein each integrated storage element is an integrating node, wherein the integrating nodes are independently reset to clear previous charges from their circuits prior to transfer operations.