W. Kevin Grace

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.

Optimizing a Waveguide-Based Sandwich Immunoassay for Tumor Biomarkers: Evaluating Fluorescent Labels and Functional Surfaces

The sensor team at the Los Alamos National Laboratory has developed a waveguide-based optical biosensor for the detection of biomarkers associated with disease. We have previously demonstrated the application of this technology to the sensitive detection of carcinoembryonic antigen in serum and nipple aspirate fluid from breast cancer patients. In this publication, we report improvements to this technology that will facilitate transition to a point-of-care diagnostic system and/or robust research tool. The first improvement involved replacing phospholipid bilayers used for waveguide functionalization with self-assembled monolayers. These thin films are stable, specific, and robust silane-based surfaces that reduce nonspecific binding and enhance the signal to background ratio. Second, we have explored four different fluorescent labeling paradigms to determine the optimal procedure for use in the assay. Labeling the detector antibody with an organic dye (AlexaFluor 647) in the hinge region allows for unusual signal enhancement with repeat excitation (at 635 nm) in our assay format, thereby facilitating a better signal resolution at lower concentrations of the antigen. We have also labeled the detector antibody with photostable quantum dots through either the amine groups of lysine (Fc, NH) or using a histidine tag in the hinge region of the antibody (Hinge, H). Both labeling strategies allow for acceptable signal resolution, but quantum dots show much greater resistance to photobleaching than organic dyes.

Pathogen detection using single mode planar optical waveguides

We utilize an optical waveguide-based biosensor recently developed at Los Alamos National Laboratory for the sensitive and specific detection of protein markers. Our planar optical waveguides are based on single mode structures that provide high optical field intensity at the active surface while providing discrimination from sample background fluorescence through spatial filtering. We have incorporated sandwich immunoassays where the capture antibody is immobilized on a biocompatible film attached to the surface of an optical waveguide. The approach can be adapted to any marker protein and is faster (<10 min), more sensitive, and as specific as conventional enzyme-linked immunosorbent assays (ELISA, the current lab standard). Results are presented for the detection of several analytes of Bacillus anthracis including protective antigen, a virulence marker for B. anthracis, and the bacterial cell or cellular debris.

Identifying moa gastroliths using a video light scattering instrument

When not found with fossil bone, gastroliths (fossil gizzard stones) may be hard to identify. One attribute that is potentially useful is their high degree of surface polish, presumably caused by abrasion in the animals gizzard. A novel video laser light scattering instrument is used to characterize the surface roughness of suspected moa gastroliths, as well as similar (non-gastrolith) quartz rocks that were polished by ocean waves. The instrument is fairly successful at distinguishing between the two types of samples.

Flow Cytometric Sizing of DNA Fragments

We have developed the capability to detect and identify single fluorescent molecules as they transit a focused laser beam. Single molecule detection allows one to measure properties of individual molecules that would be difficult or impossible with bulk measurements where properties of individual molecules are hidden in ensemble averages. This attribute is particularly important for analyzing the components of a heterogeneous mixture without separating the sample into its individual components. The use of this technology for sizing individual DNA fragments in a sample containing a mixture of DNA fragments of different sizes is an excellent example of the power of this approach. In this article, we summarize our approach to single molecule detection and the application of this technology to DNA fragment sizing.

Robust Sensing Films for Pathogen Detection and Medical Diagnostics

Our team has developed polyethylene glycol (PEG)-modified, self-assembled monolayers (SAMs) for biological detection on either planar or spherical substrates, which resist non-specific binding while facilitating specific ligand attachment. The preparation and characterization of these thin films, their validation against B. anthracis protective antigen (PA) in a sandwich assay format, and the application of these thin films for quantitative analysis of several medically interesting targets (breast cancer, tuberculosis, and influenza) will be shown.

Reagentless optical biosensor

Critical to our ability to respond effectively to a biothreat attack is the development of sensitive and specific sensor systems that can easily be used for rapid screening of potential victims for infection due to biothreat agents and detection of pathogens in the environment. To help address these needs, we have developed a Reagentless Optical Biosensor (ROB) based on protein specific assays and waveguide-based evanescent fluorescence excitation. Modeled on host pathogen interactions, the sensors membrane based assay provides rapid, sensitive detection without the addition of reagents. We report here the development of two waveguide based detection systems: a laboratory sensor test-bed system and a handheld, battery operated, prototype. Evanescent fluorescence excitation using planar optical waveguides provides spatial filtering of background auto-fluorescence found in many natural samples, thereby permitting direct analysis of complex environmental and medical samples. The waveguide based assay is fully self-contained in a small, exchangeable cartridge that is optically coupled to the sensor detection system making ROB simple to use and offering the possibility of inexpensive, disposable sensor elements. Using assays for cholera toxin we compare results using flourimetry of vesicle solutions against results for our waveguide based test-bed and prototype sensor systems.

Single Molecule Nucleic Acid Analysis by Fluorescence Flow Cytometry

During the past decade the sensitivity and selectivity of laser-induced fluorescence detection methods in liquids have been refined to the point where a molecule labeled with a single fluorophore dissolved in solution can be detected and identified as it flows through a focused excitation laser beam. Progress in this field is reviewed in a number of recent papers. [1-5]

Multiparameter Light Scattering for Rapid Virus Identification

Multiparameter light scattering (MLS) is a term we have used to describe the simultaneous measurement of multiple elements of the Mueller matrix at specific wavelengths and scattering angles. This 4 Χ 4 matrix describes the polarization sensitive transformation of an incident beam of light into a scattered beam of light by a scattering object such as a virus or suspension of virus particles. The Mueller matrix contains a great deal of information about the internal structure and shape of the virus particle. This information is sufficient in many cases to enable discrimination among a wide variety of different viruses of clinical significance.