Chris A. Rowe-Taitt

Comparison of chemical cleaning methods of glass in preparation for silanization

The uniform deposition of a silane monolayer upon glass has been shown to require small amounts of water along with hydroxyl groups in an isolated or geminal configuration on the substrate surface. In order to expose these groups, organic compounds at the surface must be removed. We present a qualitative evaluation of eight chemical methods commonly used to clean glass microscope slides in preparation for silanization. Mean contact angle of deionized water was measured before covalent attachment of (3-mercaptopropyl)triethoxysilane to assess the efficacy of each procedure. Contact angles were also measured after silanization as a means of determining the uniformity and reproducibility of the silane monolayer. The results indicate that a 1:1 methanol/HC1 wash followed by a bath in concentrated H2SO4 removes surface contaminants most effectively and allows for a very even silanization of the glass surface.

Array biosensor for detection of biohazards

A fluorescence-based biosensor has been developed for simultaneous analysis of multiple samples for multiple biohazardous agents. A patterned array of antibodies immobilized on the surface of a planar waveguide is used to capture antigen present in samples; bound analyte is then quantified by means of fluorescent tracer antibodies. Upon excitation of the fluorophore by a small diode laser, a CCD camera detects the pattern of fluorescent antibody:antigen complexes on the waveguide surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This array biosensor has been used to detect toxins, toxoids, and killed or non-pathogenic (vaccine) strains of pathogenic bacteria. Limits of detection in the mid-ng/ml range (toxins and toxoids) and in the 10(3)-10(6) cfu/ml range (bacterial analytes) were achieved with a facile 14-min off-line assay. In addition, a fluidics and imaging system has been developed which allows automated detection of staphylococcal enterotoxin B (SEB) in the low ng/ml range.

Simultaneous detection of six biohazardous agents using a planar waveguide array biosensor

Recently, we demonstrated that an array biosensor could be used with cocktails of fluorescent antibodies to perform three assays simultaneously on a single substrate, and that multiple samples could be analyzed in parallel. We extend this technology to demonstrate the simultaneous analysis of six samples for six different hazardous analytes, including both bacteria and protein toxins. The level of antibody cross-reactivity is explored, revealing a possible common epitope in two of the toxins. A panel of environmental interferents was added to the samples; these interferents neither prevented the detection of the analytes nor caused false-positive responses.

Water quality monitoring using an automated portable fiber optic biosensor: RAPTOR

The RAPTOR is a portable, automated biosensor capable of performing rapid, ten-minute assays on a sample for four target analytes simultaneously. Samples are analyzed using a fluorescent sandwich immunoassay on the surface of short polystyrene optical probes with capture antibody adsorbed to the probe surface. Target analytes bound to the fiber by capture antibodies are detected with fluorescently labeled tracer antibodies, which are held in a separate reservoir. Since target recognition is a two-step process, selectivity is enhanced, and the optical probes can be reused up to forty times, or until a positive result is obtained. This greatly reduces the logistical burden for field operations. Numerous assays for toxins, such as SEB and ricin, and bacteria, such as Bacillus anthracis and Francisella tularensis, have been developed for the RAPTOR. An assay of particular interest for water quality monitoring and the screening of fruits and vegetables is detection of Giardia cysts. Giardia lamblia is a parasitic protozoan common in the developing world that causes severe intestinal infections. Thus, a simple field assay for screening water supplies would be highly useful. Such an assay has been developed using the RAPTOR. The detection limit for Giardia cysts was 5x104/ml for a 10-minute assay.

Array biosensor for multi-analyte sensing

As biosensors become more sophisticated and commercially available, the general appreciation for their capabilities also increases. We now focus on multi-analyte sensors and address the problems inherent in discriminating multiple simultaneous signals without loss in assay speed, specificity or sensitivity. Furthermore, the goals of portability, simplicity and low cost have not diminished in importance. NRL is developing a multi-analyte sensor designed to be portable, inexpensive, and easy to use. To achieve these goals, we use a room temperature CCD, a diode laser, and a disposable waveguide. While our goals of using automated fluidics and automated image processing are not yet completely realized, we have fabricated a prototype biosensor which fits into a tackle box with a associated portable computer. Simple microscope slides are used as waveguides and precoated with arrays of immobilized antibodies. Fluorescence immunoassays are performed on these waveguides using as many as 6 samples at a time and assaying for up to 5 different analytes in each samples Sensitivities in the 1-10 ng/ml range have been achieved in 10-minute assays. Initial studies in clinical fluids indicate that assays can be run on samples such as whole blood, plasma, urine, saliva and nasal secretions.

Array biosensor for simultaneous detection of multiple analytes

The array biosensor has been developed for simultaneous analysis of multiple samples for multiple analytes. A patterned array of capture antibodies is immobilized on the surface of a planar waveguide and a sandwich immunoassay conducted using a cocktail of fluorescent tracer antibodies. Upon excitation of the fluorescent label using a 635 nm diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. The assays are fast, sensitive, and specific.

An automated portable array biosensor

Array biosensors provide the capability of immobilizing multiple capture biomolecules onto a single surface and therefore offer the exciting prospect of multi-analyte detection. A miniaturized, fully automated, stand-alone biosensor is reported which can simultaneously test multiple samples for multiple analytes. This portable system (< 10 lbs) is particularly appropriate for on-site monitoring for food safety, infectious disease detection, and biological warfare defense. The surface-selective nature of this technology allows determination of binding constants and tracking of both specific and non-specific binding events as they occur. Thus, it provides an exciting new research tool for characterizing the interactions of biomolecules with surfaces or immobilized receptors in real time. This capability has important implications for development of new materials and sensors.

Array biosensor for environmental monitoring

The array biosensor is capable of detecting and identifying multiple analytes in multiple samples simultaneously. Using fluorescence immunoassays on a planar waveguide and miniaturized fluidics, the sensor is automated and portable. Assays are sensitive and require 12 minutes to perform. Environmental contaminants in the sample fail to generate false positive or false negative results in tests performed to date. Measurements can be conducted in real time using spots as small as 80 micrometers . The waveguide can be coated with indium tin oxide (ITO) to create a charged field at the surface to further regulate the interaction of sample components with the surface.