Waveguide-based biosensors for pathogen detection.

Optical phenomena such as fluorescence, phosphorescence, polarization, interference and non-linearity have been extensively used for biosensing applications. Optical waveguides (both planar and fiber-optic) are comprised of a material with high permittivity/high refractive index surrounded on all sides by materials with lower refractive indices, such as a substrate and the media to be sensed. This arrangement allows coupled light to propagate through the high refractive index waveguide by total internal reflection and generates an electromagnetic wave-the evanescent field-whose amplitude decreases exponentially as the distance from the surface increases. Excitation of fluorophores within the evanescent wave allows for sensitive detection while minimizing background fluorescence from complex, "dirty" biological samples. In this review, we will describe the basic principles, advantages and disadvantages of planar optical waveguide-based biodetection technologies. This discussion will include already commercialized technologies (e.g., Corning's EPIC(®) Ô, SRU Biosystems' BIND(™), Zeptosense(®), etc.) and new technologies that are under research and development. We will also review differing assay approaches for the detection of various biomolecules, as well as the thin-film coatings that are often required for waveguide functionalization and effective detection. Finally, we will discuss reverse-symmetry waveguides, resonant waveguide grating sensors and metal-clad leaky waveguides as alternative signal transducers in optical biosensing.

Functional PEG-modified thin films for biological detection.

We report a general procedure to prepare functional organic thin films for biological assays on oxide surfaces. Silica surfaces were functionalized by self-assembly of an amine-terminated silane film using both vapor- and solution-phase deposition of 3'-aminopropylmethyldiethoxysilane (APMDES). We found that vapor-phase deposition of APMDES under reduced pressure produced the highest quality monolayer films with uniform surface coverage, as determined by atomic force microscopy (AFM), ellipsometry, and contact angle measurements. The amine-terminated films were chemically modified with a mixture of carboxylic acid-terminated poly(ethylene glycol) (PEG) chains of varying functionality. A fraction of the PEG chains (0.1-10 mol %) terminated in biotin, which produced a surface with an affinity toward streptavidin. When used in pseudo-sandwich assays on waveguide platforms for the detection of Bacillus anthracis protective antigen (PA), these functional PEG surfaces significantly reduced nonspecific binding to the waveguide surface while allowing for highly specific binding. Detection of PA was used to validate these films for sensing applications in both buffer and complex media. Ultimately, these results represent a step toward the realization of a robust, reusable, and autonomous biosensor.

Microsphere-based duplexed immunoassay for influenza virus typing by flow cytometry.

We have developed a rapid, duplexed microsphere-based immunoassay for the characterization of influenza virus types that has the potential to overcome many of the limitations of current detection methods. The assay uses microspheres of two sizes, each coupled to an influenza type A- or type B-specific monoclonal antibody (MAb), to capture influenza viruses in the sample. A cocktail of fluorescently labeled, influenza-specific polyclonal antibodies then binds the captured viruses. The sandwich complexes are measured using a multiparameter flow cytometer. The assay can distinguish between influenza types A and B in a single reaction with good reproducibility and high sensitivity. Detection sensitivity is much higher than that of commercially available influenza diagnosis quick kits: the FLU OIA (Thermo Biostar) kit and the Directigen Flu A+B kit (Becton Dickinson). The multiplexing capabilities of the current assay, which are not possible with enzyme-linked immunosorbent assay (ELISA) and the commercially available kits, reduce sample handling and consume fewer costly reagents. This assay represents a more efficient and sensitive method of characterizing influenza types. With inclusion of influenza subtype-specific antibodies as capture antibodies, this microsphere-based immunoassay can be expanded to differentiate among influenza types and subtypes in a single reaction to improve world-wide influenza surveillance.