Substrates and optical systems and methods of use thereof having a single optically resolvable immobilized reaction component disposed within a nanometer-scale aperture
This invention provides substrates for use in various applications, including single-molecule analytical reactions. Methods for propagating optical energy within a substrate are provided. Devices comprising waveguide substrates and dielectric omnidirectional reflectors are provided. Waveguide substrates with improved uniformity of optical energy intensity across one or more waveguides and enhanced waveguide illumination efficiency within an analytic detection region of the arrays are provided.
1. A method of detecting a signal from an analyte, comprising:
a) providing a waveguide substrate comprising:
i) two or more optical waveguides disposed upon or within the waveguide substrate; and
ii) an analyte region disposed sufficiently proximal to a core of one of the two or more optical waveguides to be illuminated by an evanescent field emanating from the core when optical energy is passed through the core, wherein the analyte region comprises a single optically resolvable immobilized reaction component, and further wherein the analyte region is disposed within a nanometer-scale aperture that penetrates into a first surface on a first side of the waveguide substrate and extends toward the core,
wherein the first surface is fluidically linked to the analyte region;
b) exposing the first surface to an analyte comprising a fluorescent or fluorogenic moiety that emits a signal in response to the evanescent field;
c) directing optical energy through the core, thereby generating the evanescent field and further illuminating the single optically resolvable immobilized reaction component; and
d) detecting a signal from the analyte upon interaction of the analyte with the single optically resolvable immobilized reaction component.
2. The method of claim 1 , wherein the two or more optical waveguides are configured to receive the optical energy at a portion of the two or more waveguides comprising an optical grating, and wherein the optical grating normalizes an intensity of the optical energy among the two or more waveguides.
3. The method of claim 1 , wherein said directing optical energy comprises directing a single beam of optical energy to a diffractive optical element that splits the single beam of optical energy into two or more beams of optical energy.
4. The method of claim 1 , wherein the analyte region is disposed within a nanohole.
5. The method of claim 1 , wherein the analyte region is disposed within a zero-mode waveguide.
6. The method of claim 1 , wherein the nanometer-scale aperture descends into the waveguide cladding.
7. The method of claim 1 , wherein the detecting a signal from the analyte comprises disposing a detector proximal to the waveguide substrate on a second surface, the second surface on a second side of the waveguide substrate opposite the first side; and during the directing optical energy of c), detecting fluorescent or fluorogenic emissions from the analyte.
8. The method of claim 1 , further comprising providing at least one relay lens and at least one microscope objective for focusing one of the two or more beams of optical energy at the two or more optical waveguides.
9. The method of claim 1 , wherein the single optically resolvable immobilized reaction component comprises an enzyme.
10. The method of claim 9 , wherein the enzyme is a polymerase.
11. The method of claim 10 , wherein the analyte comprises a nucleotide or nucleotide analog.
12. The method of claim 1 , wherein the waveguide substrate comprises a plurality of analyte regions, each of which is disposed sufficiently proximal to a core of one of the two or more optical waveguides to be illuminated by an evanescent field emanating from the core when optical energy is passed through the core, and further wherein each of the analyte regions comprises a single optically resolvable immobilized reaction component.
13. The method of claim 1 , wherein the waveguide substrate further comprises a reflective or absorptive layer disposed over the first surface.
14. The method of claim 13 , wherein the reflective or absorptive comprises a metal.
15. The method of claim 14 , wherein the metal comprises Al.
16. The method of claim 13 , wherein the waveguide substrate further comprises a mask layer disposed between the first surface of the waveguide substrate and the reflective or absorptive layer, wherein the nanometer-scale aperture extends through the mask layer.
17. The method of claim 1 , wherein the reaction component comprises an enzyme and the analyte is used by the enzyme in an enzymatic reaction.