Optical biosensor utilizing the evanescent wave originating from a planar waveguide

Abstract

The development and use of a biosensor utilizing the evanescent wave to excite fluorophores bound, via an antigen-antibody interaction, to the surface of a planar optical waveguide are described in this work. Comprehensive theoretical discussions of the physical, which are stressed, and chemical processes of importance are included. Total internal reflection and the evanescent wave phenomenon are explained; kinetics is treated and the characteristics of the utilized assays are discussed. A measurement series using an assay with fluorescently labeled streptavidin and biotin- ylated bovine serum albumin was performed. Disposable microscope slides were used for these experiments, since the chemical properties of the custom-made waveguides that we intended to use deteriorated after they were reused several times. The detection limit with this assay was 􏰃 10 ng streptavidin per mL. Unfortunately significant inherent system instability, probably due to variations of the laser’s output, prevented lower concentrations to be sensed. Starting from these experiments a survey was made seeking the answer to the question: Can we measure the initial slope of the signal response curve and then predict the amplitude rather than wait for reaction equilibrium? The outcome was positive since it showed a linear relationship between the slope and the amplitude. A few experiments investigating the ability to detect prostate specific antigen (PSA) were carried out. A waveguide made of poly-methyl–meth-acrylate (PMMA) had to be used, because the PSA assay did not function as desired on the disposable microscope slides. The detection limit of PSA was 􏰃 0.5 ng/mL. However, the potential resolution limit using the same experimental setup, estimated to be < 0.05 ng/mL, is much lower. The PMMA waveguide provided a high offset level, which greatly restrained the detector’s amplification. The presented experimental setup shows sufficiently good performance for a sensor to be used for point-of-care testing, at least regarding detection of PSA. Extraordinary sen- sitivity seems possible to achieve. Either a waveguide with excellent optical qualities must be implemented, or the use of time-resolved fluorometry can be taken advantage of. The sensor can be enhanced in many aspects, e.g. by increasing the irradiance of the evanescent field and gather the fluorescent emission more efficiently.