Optical detection of label-free biomolecular interactions using microreplicated plastic
View Patent ↗Methods and compositions are provided for detecting biomolecular interactions. The use of labels is not required and the methods can be performed in a high-throughput manner. The invention also provides optical devices useful as narrow band filters.
1. A method of determining the amount of one or more binding partners in a test sample comprising:
(a) illuminating a biosensor with light, wherein the biosensor comprises an optical grating; a substrate layer that supports the optical grating; the optical grating being replicated from a master grating structure by causing a liquid to harden after dispensing the liquid between the master grating structure and the substrate layer, wherein the optical grating comprises the hardened liquid and wherein the optical grating comprises features having a depth and a cross-sectional period; and one or more specific binding substances immobilized on a surface of the optical grating; wherein, when the biosensor is illuminated a resonant grating effect is produced on the reflected radiation spectrum, and wherein the depth and cross-sectional period of the optical grating are less than the wavelength of the resonant grating effect;
(b) applying a test sample comprising one or more binding partners to the biosensor;
(c) illuminating the biosensor with light; and
(d) detecting reflected wavelength of light from the biosensor; and
(e) (i) comparing the reflected wavelength of light from the biosensor to positive and negative controls; or (ii) comparing the reflected wavelength of light from the biosensor to a reflected wavelength of light detected from the biosensor prior to the applying the test sample to the biosensor, wherein the amount of one or more binding partners in a test sample is detected.
2. The method of claim 1 , wherein a narrow band of optical wavelengths is reflected from the biosensor when the biosensor is illuminated with a broad band of optical wavelengths.
3. The method of claim 1 , wherein the substrate layer comprises plastic.
4. The method of claim 1 , wherein the hardened liquid comprises hardened optical cement.
5. The method of claim 1 , wherein the surface of the optical grating opposite the substrate layer is coated with a material having a refractive index higher than the substrate layer, and wherein the one or more specific binding substances are immobilized on the surface of the material having a refractive index higher than the substrate layer opposite the optical grating.
6. The method of claim 5 , wherein the material having a refractive index higher than the substrate layer is selected from the group consisting of zinc sulfide, titanium dioxide, tantalum oxide, and silicon nitride.
7. The method of claim 1 further comprising a cover layer on the surface of the optical grating opposite the substrate layer, wherein the one or more specific binding substances are immobilized on the surface of the cover layer opposite the optical grating.
8. The method of claim 7 , wherein the cover layer comprises a material that has a lower refractive index than the optical grating.
9. The method of claim 8 , wherein the cover layer comprises a material selected from the group consisting of glass, epoxy, and plastic.
10. The method of claim 1 , wherein the optical grating comprises a repeating pattern having a cross-sectional period of about 0.01 microns to about 1 micron and a depth of about 0.01 microns to about 1 micron.
11. The method of claim 1 , wherein the one or more specific binding substances are arranged in an array of distinct locations.
12. The method of claim 1 , wherein the one or more specific binding substances are immobilized on the optical grating by physical adsorption or by chemical binding.
13. The method of claim 1 , wherein the one or more specific binding substances are bound to their binding partners.
14. The method of claim 13 , wherein the binding partners are selected from the group consisting of proteins, peptides, single strand DNA, double strand DNA, RNA, chemical molecules in solution, nucleic acids, polypeptides, antigens, polyclonal antibodies, monoclonal antibodies, single chain antibodies (scFv), F(ab) fragments, F(ab′) 2 fragments, Fv fragments, small organic molecules, cells, viruses, bacteria, and biological samples.
15. The method of claim 1 , wherein the one or more specific binding substances are selected from the group consisting of protein solutions, peptide solutions, DNA solutions, RNA solutions, solutions of combinatorial chemical libraries, nucleic acids, polypeptides, antigens, polyclonal antibodies, monoclonal antibodies, single chain antibodies (scFv), F(ab) fragments, F(ab′) 2 fragments, Fv fragments, small organic molecules, cells, viruses, bacteria, and biological samples.
16. The method of claim 1 , further comprising an antireflective dielectric coating on the surface of the substrate opposite of the optical grating.
17. The method of claim 1 , wherein the biosensor is attached to a bottomless microtiter plate.
18. The method of claim 1 , wherein the optical grating is comprised of a repeating pattern of shapes selected from the group consisting of continuous parallel lines, squares, circles, ellipses, triangles, ovals, trapezoids, sinusoidal waves, rectangles, and hexagons.
19. The method of claim 1 , wherein the optical grating comprises an optically transparent material that conducts electricity, wherein the surface of the optical grating is coated with an electrical insulator.
20. The method of claim 1 , wherein the surface of the optical grating is amine activated, aldehyde activated, or nickel activated and the one or more specific binding substances are immobilized on the surface of the amine activated, aldehyde activated, or nickel activated optical grating.
21. The method of claim 20 , wherein the one or more specific binding substances are immobilized on the surface of the amine activated, aldehyde activated, or nickel activated optical grating via a chemical linker.