Illumination of integrated analytical systems
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.
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.