Fluorescent assay and sensor therefor

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.

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.