Lisa C. Shriver-Lake

Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces

A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional cross-linkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.

Detection of Clostridium botulinum toxin A using a fiber optic-based biosensor

A rapid, sensitive, analytical method for the detection of Clostridium botulinum toxin has been developed. The fiber optic-based biosensor utilizes the evanescent wave of a tapered optical fiber for signal discrimination. A 50 mW argon-ion laser, which generates laser light at 514 nm, is used in conjunction with an optical fiber probe that is tapered at the distal end. Antibodies specific for C. botulinum are covalently attached to the surface of the tapered fiber. The principle of the system is a sandwich immunoassay using rhodamine-labeled polyclonal anti-toxin A immunoglobin G (IgG) antibodies for generation of the specific fluorescent signal. Various anti-toxin antibodies were immobilized to the fibers. Affinity-purified polyclonal horse anti-toxin A antibodies performed better than the IgG fraction from the same horse serum or than the monoclonal anti-toxin A antibody BA11-3. Botulinum toxin could be detected within a minute, at concentrations as low as 5 ng/ml. The reaction was highly specific and no response was observed against tetanus toxin.

Antibody immobilization using heterobifunctional crosslinkers

Covalent attachment of functional proteins to a solid support is important for biosensors. One method employs thiol-terminal silanes and heterobifunctional crosslinkers such as N-succinimidyl 4-maleimidobutyrate (GMBS) to immobilize proteins through amino groups onto glass, silica, silicon or platinum surfaces. In this report, several heterobifunctional crosslinkers are compared to GMBS for their ability to immobilize active antibodies onto glass cover slips at a high density. Antibodies were immobilized at densities of 74-220 ng/cm2 with high levels of specific antigen binding. Carbohydrate-reactive crosslinkers were also compared to GMBS using a fiber optic biosensor to detect fluorescently-labeled antigen. At the concentrations tested, the antibodies immobilized with carbohydrate-reactive crosslinkers bound more antigen than GMBS immobilized antibodies as indicated by the fluorescence signal.

A Portable Array Biosensor for Detecting Multiple Analytes in Complex Samples

The Multi-Analyte Array Biosensor (MAAB) has been developed at the Naval Research Laboratory (NRL) with the goal of simultaneously detecting and identifying multiple target agents in complex samples with minimal user manipulation. This paper will focus on recent improvements in the biochemical and engineering aspects of this instrument. These improvements have enabled the expansion of the repertoire of analytes detected to include Salmonella typhimurium and Listeria monocytogenes, and also expanded the different sample matrices tested. Furthermore, all components of the biochemical assays could be prepared well in advance of sample testing, resulting in a “plug-and-play” methodology. Simultaneous detection of three toxins (ricin, staphylococcal enterotoxin B, and cholera toxin) was demonstrated using a novel fluidics cube module that limits the number of manipulations to only the initial sample loading. This work demonstrates the utility of the MAAB for rapid analysis of complex samples with multianalyte capability, with a minimum of operator manipulations required for either sample preparation or final analysis.

Multi-analyte explosive detection using a fiber optic biosensor

A fiber optic immunosensor has been developed for simultaneous detection of the most common explosives, 2,4,6,-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). It employs a competitive immunoassay on the antibody-coated fiber optic probes, in which a fluorescent antigen competes with free antigen of unknown concentration for binding sites on the fiber surface. To achieve dual explosive detection, two α-TNT fiber probes and two α-RDX fiber probes are connected in series. The sample is mixed with fluorescent analogs, Cy5-ethylenediamine-trinitrobenzene (Cy5-EDA-TNB) and Cy5-ethylenediamine-RDX hapten (Cy5-EDA-RDH). Inhibition of the maximum signal in the presence of the sample is proportional to the concentration of the explosive. The detection limits for the multi-analyte assays are equivalent (6 ng/ml for both TNT and RDX) to those of the individual assays (5 ng/ml for both TNT and RDX). The standard curves for TNT and RDX represent a linear relationship between percent signal inhibition and the natural logarithm of analyte concentration in the multi-analyte format, as well as in single analyte assays, thus allowing a simple and precise method of quantification. There is minimal cross-reactivity for the two antigens in the multi-analyte immunosensor, so it is also an effective means in analyzing samples containing mixtures of RDX and TNT.

Fluorometer and tapered fiber optic probes for sensing in the evanscent wave

To develop an improved fiber optic biosensor both the optical component selection and the signal coupling efficiency were investigated. The emission filter and fiber connectors were carefully chosen to reduce their contribution to noise in the system. We used long, fused silica fibers that had several centimeters of cladding removed along the distal end. This exposed core is coated with the recognition molecules that bind analyte-fluorophore complexes from the sample solution. A fluorescent signal generated in the evanescent wave region of the unclad, immersed portion of the probe is lost as it enters the cladded portion of the fiber because of a V-number mismatch. To minimize the mismatch, the core radius is reduced along the uncladded region to form a continuous taper. An assay using the tapered fiber and the described optical configuration is presented that demonstrates instantaneous signal generation in response to nanogram amounts of a toxic material.

Detection of Staphylococcal Enterotoxin B in Spiked Food Samples

Contamination of food with infectious agents, intentional or not, is a global concern with far-reaching economic and social impact. Staphylococcal enterotoxins are a major cause of food poisoning, but most methods for the identification of these agents in food require extensive pretreatment or concentration of the sample prior to analysis. The array biosensor was developed as a portable device for the simultaneous analysis of multiple complex samples for multiple targets with minimal sample preparation. In this study, we use an array biosensor to expand and improve on a staphylococcal enterotoxin B (SEB) assay with the ultimate intent of incorporating testing for SEB into a battery of sensitive and convenient assays for food safety validation. In addition to buffer studies, six different types of food samples, including beverages, homogenates of fruit and meat, and carcass washings, were spiked with SEB, incubated for at least 2 h to permit antigen sequestration, and assayed. For all samples, there were differences in fluorescence intensity, but 0.5 ng of SEB per ml could be detected in <20 min with little if any pretreatment and no sample preconcentration.

Fluorescence‐based array biosensors for detection of biohazards

Total internal reflection fluorescence (TIRF) is a process whereby fluorophores that are either attached to or are in close proximity with the surface of a waveguide are selectively excited via an evanescent wave. Planar waveguides provide the possibility of immobilizing multiple capture biomolecules onto a single surface and therefore, offer the exciting prospect of multi-analyte detection. The production of arrays and the results of various groups which use TIRF to interrogate such surfaces is reviewed, along with a look at how far the field has advanced toward the production of an automated, portable, multi-analyte array biosensor for real-time biohazard detection. In particular, a miniaturized, fully automated, stand-alone array biosensor developed at the Naval Research Laboratory is reported that monitors interactions between binding partners either as the final image or in real-time. A variety of analytes including toxins, bacteria and viruses have been detected both in buffer and complex matrices, such as blood and soil suspensions, with comparable detection limits. A number of developments have led to a TIRF array biosensor weighing only 5.5 kg which is automated for environmental, clinical and food monitoring or for detection of bioterrorist agents.

A portable automated multianalyte biosensor.

The array biosensor employs an array of capture molecules on a planar optical waveguide to interrogate multiple samples simultaneously for multiple targets. In assay development and demonstration studies published previously, we have quantified this biosensors capability for rapid identification of a wide variety of targets in complex sample media. This paper describes the miniaturization and automation of the array biosensor for portability and on-site use. The fluidics have been redesigned and constructed with reliability and commercial production of disposable components in mind. To demonstrate the automated operation, simultaneous assays were automatically conducted on samples containing both ovalbumin and staphylococcal enterotoxin B. Results demonstrate the capability of the biosensor for detection and quantification.

Portable multichannel fiber optic biosensor for field detection

A compact, portable fiber optic biosensor is developed that enables monitoring of up to four fiber optic probes simultaneously. The sensor employs a novel optical fiber bundle jumper for exciting and col- lecting fluorescence emission from the evanescent wave fiber optic probes. A single fiber in the center of the bundle couples laser excitation into the sensor probe, while the surrounding fibers collect the returning fluorescent emission light. This design requires no beamsplitter, enabling the detection optics and control circuitry to be reduced to a 436 in. circuit card. Four of these cards are integrated into a single portable system. Results from detection assays for hazardous biological agents and an environmental pollutant are shown.