Chris R. Taitt

Detection of Salmonella enterica Serovar Typhimurium by Using a Rapid, Array-Based Immunosensor

ABSTRACT The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 × 104 CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 × 103 CFU/g.

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.

Biosensor Detection of Botulinum Toxoid A and Staphylococcal Enterotoxin B in Food

ABSTRACT Immunoassays were developed for the simultaneous detection of staphylococcal enterotoxin B and botulinum toxoid A in buffer, with limits of detection of 0.1 ng/ml and 20 ng/ml, respectively. The toxins were also spiked and measured in a variety of food samples, including canned tomatoes, sweet corn, green beans, mushrooms, and tuna.

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.

Nonantibody-based recognition: alternative molecules for detection of pathogens.

Immunoassays have been well established for many years as the cornerstone of detection technologies. These assays are sensitive, selective and, in general, highly resistant to interference from complex sample matrices when compared with nucleic acid-based tests. However, both antibody- and nucleic acid-based detection systems require a priori knowledge of the target and development of specific reagents; multiplexed assays can become increasingly problematic when attempting to detect a plethora of different targets, the identities of which are unknown. In an effort to circumvent many of the limitations inherent in these conventional assays, other recognition reagents are being explored as alternatives, or indeed as adjuncts, to antibodies for pathogen and toxin detection. This article will review a number of different recognition systems ranging in complexity from small molecules, such as nucleic-acid aptamers, carbohydrates and peptides, to systems as highly complicated as whole cells and organisms. All of these alternative systems have tremendous potential to achieve superior sensitivity, selectivity, and stability, but are also subject to their own limitations, which are also discussed. In short, while in its infancy, this field holds great promise for the development of rapid, fieldable assays that are highly complementary to existing antibody- and nucleic acid-based technologies.

Reduction of Non-Specific Protein Adsorption Using Poly(ethylene) Glycol (PEG) Modified Polyacrylate Hydrogels In Immunoassays for Staphylococcal Enterotoxin B Detection

Three PEG molecules (PEG-methacrylate, -diacrylate and -dimethacrylate) were incorporated into galactose-based polyacrylate hydrogels and their relative abilities to reduce non-specific protein adsorption in immunoassays were determined. Highly crosslinked hydrogels containing amine-terminated functionalities were formed and used to covalently attach antibodies specific for staphylococcal enterotoxin B (SEB). Patterned arrays of immobilized antibodies in the PEG-modified hydrogels were created with a PDMS template containing micro-channels for use in sandwich immunoassays to detect SEB. Different concentrations of the toxin were applied to the hydrogel arrays, followed with a Cy3-labeled tracer antibody specific for the two toxins. Fluorescence laser scanning confocal microscopy of the tracer molecules provided both qualitative and quantitative measurements on the detection sensitivity and the reduction in non-specific binding as a result of PEG incorporation. Results showed the PEG-modified hydrogel significantly reduced non-specific protein binding with a detection limit for SEB of 1 ng/mL. Fluorescence signals showed a 10-fold decrease in the non-specific binding and a 6-fold increase in specific binding of SEB.

Array Biosensor for Toxin Detection: Continued Advances

The following review focuses on progress made in the last five years with the NRL Array Biosensor, a portable instrument for rapid and simultaneous detection of multiple targets. Since 2003, the Array Biosensor has been automated and miniaturized for operation at the point-of-use. The Array Biosensor has also been used to demonstrate (1) quantitative immunoassays against an expanded number of toxins and toxin indicators in food and clinical fluids, and (2) the efficacy of semi-selective molecules as alternative recognition moieties. Blind trials, with unknown samples in a variety of matrices, have demonstrated the versatility, sensitivity, and reliability of the automated system.

Multiplexed Detection of Mycotoxins in Foods with a Regenerable Array

The occurrence of different mycotoxins in cereal products calls for the development of a rapid, sensitive, and reliable detection method that is capable of analyzing samples for multiple toxins simultaneously. In this study, we report the development and application of a multiplexed competitive assay for the simultaneous detection of ochratoxin A (OTA) and deoxynivalenol (DON) in spiked barley, cornmeal, and wheat, as well as in naturally contaminated maize samples. Fluoroimmunoassays were performed with the Naval Research Laboratory array biosensor, by both a manual and an automated version of the system. This system employs evanescent-wave fluorescence excitation to probe binding events as they occur on the surface of a waveguide. Methanolic extracts of the samples were diluted threefold with buffer containing a mixture of fluorescent antibodies and were then passed over the arrays of mycotoxins immobilized on a waveguide. Fluorescent signals of the surface-bound antibody-antigen complexes decreased with increasing concentrations of free mycotoxins in the extract. After sample analysis was completed, surfaces were regenerated with 6 M guanidine hydrochloride in 50 mM glycine, pH 2.0. The limits of detection determined by the manual biosensor system were as follows: 1, 180, and 65 ng/g for DON and 1, 60, and 85 ng/g for OTA in cornmeal, wheat, and barley, respectively. The limits of detection in cornmeal determined with the automated array biosensor were 15 and 150 ng/g for OTA and DON, respectively.

Plasma-Based Surface Modification of Polystyrene Microtiter Plates for Covalent Immobilization of Biomolecules

In recent years, polymer surfaces have become increasingly popular for biomolecule attachment because of their relatively low cost and desirable bulk physicochemical characteristics. However, the chemical inertness of some polymer surfaces poses an obstacle to more expansive implementation of polymer materials in bioanalytical applications. We describe use of argon plasma to generate reactive hydroxyl moieties at the surface of polystyrene microtiter plates. The plates are then selectively functionalized with silanes and cross-linkers suitable for the covalent immobilization of biomolecules. This plasma-based method for microtiter plate functionalization was evaluated after each step by X-ray photoelectron spectroscopy, water contact angle analysis, atomic force microscopy, and bioimmobilization efficacy. We further demonstrate that the plasma treatment followed by silane derivatization supports direct, covalent immobilization of biomolecules on microtiter plates and thus overcomes challenging issues typically associated with simple physisorption. Importantly, biomolecules covalently immobilized onto microtiter plates using this plasma-based method retained functionality and demonstrated attachment efficiency comparable to commercial preactivated microtiter plates.