Planar integrated optical methods for examining thin films and their surface adlayers.

Thin film integrated optical waveguides (IOWs) have gained acceptance as a method for characterizing ultrathin dielectrical films and adlayers bound to the film surface. Here, we present the expressions that govern IOW methods as well as describe the common experimental configurations used in attenuated total reflection, fluorescence and Raman applications. The applications of these techniques to the study of adsorbed or surface-bound proteins to polymer and glass waveguides are reviewed.

Femtomolar sensitivity using a channel-etched thin film waveguide fluoroimmunosensor.

A dual channel, evanescent fluoroimmunoassay format is used to detect femtomolar analyte concentrations (i.e. less than 1 part per trillion [w/w]) on an etched channel siliconoxynitride thin film integrated optical waveguide. Two assays are used to demonstrate the dose-response behaviour of the sensor: (1) a direct assay of a fluorescently-labeled protein ligand binding to an immobilized protein receptor, and (2) an indirect sandwich assay of a non-fluorescent protein ligand binding to an immobilized protein receptor, as detected by the binding of a fluorescently-labeled secondary receptor protein. A red-emitting cyanine dye (Cy-5), which minimized background fluorescence and scatter losses of the waveguide, was used in both assays. To our knowledge, this is the first report of femtomolar sensitivity in an immunosensing instrument.

Multiple-analyte fluoroimmunoassay using an integrated optical waveguide sensor.

A silicon oxynitride integrated optical waveguide was used to evanescently excite fluorescence from a multianalyte sensor surface in a rapid, sandwich immunoassay format. Multiple analyte immunoassay (MAIA) results for two sets of three different analytes, one employing polyclonal and the other monoclonal capture antibodies, were compared with results for identical analytes performed in a single-analyte immunoassay (SAIA) format. The MAIA protocol was applied in both phosphate-buffered saline and simulated serum solutions. Point-to-point correlation values between the MAIA and SAIA results varied widely for the polyclonal antibodies (R2 = 0.42-0.98) and were acceptable for the monoclonal antibodies (R2 = 0.93-0.99). Differences in calculated receptor affinities were also evident with polyclonal antibodies, but not so with monoclonal antibodies. Polyclonal antibody capture layers tended to demonstrate departure from ideal receptor-ligand binding while monoclonal antibodies generally displayed monovalent binding. A third set of three antibodies, specific for three cardiac proteins routinely used to categorize myocardial infarction, were also evaluated with the two assay protocols. MAIA responses, over clinically significant ranges for creatin kinase MB, cardiac troponin I, and myoglobin agreed well with responses generated with SAIA protocols (R2 = 0.97-0.99).