# US 5,344,784 A · Fluorescent assay and sensor therefor > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,344,784 A - Document ID: US5344784A - Status: Granted - Kind: A - Application number: 07/689,850 - Filed: 1991-05-21 - Granted: 1994-09-06 - Earliest claimed: 1988-11-29 - Assignees: Applied Research Systems ARS Holding N.V. - Inventors: John W. Attridge - Web page: https://patentgrep.com/patent/US5344784A ## CPC - G01N 21/648: {using evanescent coupling or surface plasmon coupling for the excitation of fluorescence} - 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)} - G01N 21/7703: {using reagent-clad optical fibres or optical waveguides (using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B)} - G01N 33/54373: {involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings} - G01N 2021/7786: {Fluorescence} - Y10S 435/808: Optical sensing apparatus - Y10S 436/805: Optical property - Y10S 436/807: Apparatus included in process claim, e.g. physical support structures - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract The invention relates to a device for use in optical methods of assay. The device comprises an optical structure comprising a dielectric substrate (2) and a thin film waveguide (5), between which is interposed a buffer layer (4). The waveguide carries a layer (10) of reagent suitable for a desired assay. The invention also relates to methods of using the device in an assay. ## Claims ### Claim 1 A method of assaying for a ligand in a sample which comprises: (A.) incubating, simultaneously or in any desired sequence, (i) the sample, and (ii) a specific binding partner to the ligand, wherein reagent (ii) is labelled with a fluorophore and is immobilised directly or indirectly on a waveguide surface of an optical structure comprising: (a) a dielectric substrate transparent at least at wavelengths of radiation involved in the assay; (b) a thin-film waveguide of dielectric material having a refractive index higher than that of the substrate; and (c) interposed between (a) and (b), a buffer layer of dielectric material having a refractive index lower than that of the substrate, wherein the buffer layer and the waveguide each are of a thickness such that, when said optical structure is in use, one or more guided modes may be propagated within the waveguide; (B.) irradiating a surface of the optical structure's substrate with light of an appropriate wavelength at a suitable angle to the normal such that total internal reflection occurs at the interface between the substrate and the buffer layer of said optical structure and whereby an evanescent field associated therewith gives rise to propagation of one or more guided modes in the waveguide of said optical structure, and such that floroescence arising from excitation of bound fluorophore within said evanescent field and which couples back into the substrate leaves the interface between the buffer layer and the substrate at angles to the normal which are less than the critical angle at the interface; and (C.) determining whether an optical property of floroescence emerging from said optical structure is altered by formation of immunocomplexes on the waveguide, whereby the alteration in optical property of fluorescence is related to the presence of ligand in the sample. ### Claim 2 (depends on 1) A method as claimed in claim 1 wherein the extent to which or the rate at which, or both, the optical property of fluorescence emerging from said optical structure is altered by formation of immunocomplexes on the waveguide is determined. ### Claim 3 A method of assaying for a ligand in a sample which comprises: (A.) incubating simultaneously or in any desired sequence, (i) the sample, (ii) a specific binding partner to the ligand, and (iii) a reagent selected from the group consisting of a specific binding partner to the ligand and a ligand analogue, wherein at least one of the above reagents (ii) or (iii) is labelled with a fluorophore and one of the above reagents (ii) and (iii) is immobilised directly or indirectly on a waveguide surface of an optical structure comprising: (a) a dielectric substrate transparent at least at wavelengths of radiation used in the assay; (b) a thin-film waveguide of dielectric material having a refractive index higher than that of the substrate; and (c) interposed between (a) and (b), a buffer layer of dielectric material having a refractive index lower than that of the substrate, wherein the buffer layer and the waveguide each are of a thickness such that, when said optical structure is in use, one or more guided modes may be propagated within the waveguide; (B.) irradiating a surface of the optical structure's substrate with light of an appropriate wavelength at a suitable angle to the normal such that total interest reflection occurs at the interface between the substrate and the buffer layer of said optical structure and whereby an evanescent field associated therewith gives rise to propagation of one or more guided modes in the waveguide of said optical structure, and such that fluorescence arising form excitation of bound fluorophore within said evanescent field and which couples back into the substrate leaves the interface between the buffer layer and the substrate at angles to the normal which are less than the critical angle at the interface; and (C.) determining whether an optical property of fluorescence emerging from said optical structure is altered by formation of immunocomplexes on the waveguide, whereby the alteration in optical property of fluorescence is related to the presence of ligand in the sample. ### Claim 4 (depends on 3) A method as claimed in claim 3 wherein the extent to which or the rate at which, or both, the optical property of fluorescence emerging from said optical structure is altered by formation of immunocomplexes on the waveguide is determined. ### Claim 5 (depends on 3) A method as claimed in claim 3 wherein reagent (iii) is a ligand analogous possessing a fluorophore as a label. ### Claim 6 (depends on 5) A method as claimed in claim 5 wherein the extent to which or the rate at which, or both, the optical property of fluorescence emerging from said optical structure is altered by formation of immunocomplexes on the waveguide is determined. ### Claim 7 (depends on 1, 3) A method as claimed in claims 1 or 3 wherein the ligand is an antigen and the specific binding partner comprises an antibody to said antigen. ### Claim 8 (depends on 7) A method as claimed in claim 7 wherein the extent to which or the rate at which, or both, the optical property of fluorescence emerging from said optical structure is altered by formation of immunocomplexes on the waveguide is determined. ### Claim 9 An assay sensor which comprises an optical structure comprising: (a) a dielectric substrate; (b) a thin-film waveguide of dielectric material having a refractive index higher than that of the substrate; and (c) interposed between (a) and (b), a buffer layer of dielectric material having a refractive index lower than that of the substrate, said optical structure having immobilised directly or indirectly on a surface of the waveguide remote from the substrate a continuous or discontinuous layer of a reagent appropriate to an assay to be carried out using said optical structure, wherein the buffer layer and the waveguide each are of a thickness such that, when said optical structure is in use, one or more guided modes may be propagated within the waveguide, and such that fluorescence arising from excitation of any fluorophore which, in use, is within an evanescent field associated with the optical structure and which fluorescence couples back into the substrate leaves the interface between the buffer layer and the substrate at angles to the normal which are less than the critical angle at the interface. ### Claim 10 (depends on 9) A sensor as claimed in claim 9 wherein the thicknesses of the buffer layer and the waveguide are such that, in use, a single transverse magnetic (TM) and/or transverse electric (TE) guided mode may be propagated within the waveguide. ### Claim 11 (depends on 9) A sensor as claimed in claim 9 wherein the buffer layer has a thickness between 0.1 and 2.0 microns and the waveguide has a thickness of between 0.16 and 1.0 microns. ### Claim 12 (depends on 11) A sensor as claimed in claim 11 wherein the buffer layer is silicon dioxide and has a thickness of about 0.6 microns and the waveguide is tin oxide and has a thickness of about 0.3 microns. ### Claim 13 (depends on 11) A sensor as claimed in claim 11 wherein the buffer layer is magnesium fluoride and has a thickness of about 0.7 microns and the waveguide is zinc sulphide and has a thickness of about 0.2 microns. ### Claim 14 (depends on 9) A specifically reactive sample collecting and testing device possessing a cavity or cavities each having a dimension small enough to enable sample liquid to be drawn into the cavity by capillary action, and wherein at least one part of a wall of said cavity comprises a sensor as claimed in claim 9.