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Biomedical subjects

Chris R Taitt

Publications and source records attributed to Chris R Taitt.

15 recordsLinked to original sources
PubMed2007

The array biosensor: portable, automated systems.

With recent advances in surface chemistry, microfluidics, and data analysis, there are ever increasing reports of array-based methods for detecting and quantifying multiple targets. However, only a few systems have been described that require minimal preparation of complex samples and possess a means of quantitatively assessing matrix effects. The NRL Array Biosensor has been developed with the goal of rapid and sensitive detection of multiple targets from multiple samples analyzed simultaneously. A key characteristic of this system is its two-dimensional configuration, which allows controls and standards to be analyzed in parallel with unknowns. Although the majority of our work has focused on instrument automation and immunoassay development, we have recently initiated efforts to utilize alternative recognition molecules, such as peptides and sugars, for detection of a wider variety of targets. The array biosensor has demonstrated utility for a variety of applications, including food safety, disease diagnosis, monitoring immune response, and homeland security, and is presently being transitioned to the commercial sector for manufacturing.

Automation↗
PubMed2006

TNT detection using multiplexed liquid array displacement immunoassays.

The presence of trace contamination of soil and groundwater with explosives is an ongoing concern, for which improved methods are required to facilitate their detection and quantification. This is true both for the monitoring of remediation and for site characterization. Immunosensors have been found effective for solution-phase detection of environmental contaminants. Our work utilized the Luminex100 (flow cytometer) to detect TNT in a multiplexed displacement immunoassay format. The Luminex100 can perform a multiplexed assay by discriminating between up to 100 different bead sets. We used this capability to evaluate four different TNT monoclonal antibodies, two recombinant TNT antibodies, and a control antibody simultaneously for the rapid detection of TNT and other nitroaromatics. TNT could be detected at 0.1 ppb and quantified over the range of 1.0 ppb to 10 ppm. In addition, the assay was shown to be effective in various matrixes such as lake water, seawater, and acetone extracts of soil. Seawater required dilution with two parts buffer to avoid loss of microspheres, while the acetone extracts were diluted 100-fold or more to minimize solvent affects.

Antibodies, Monoclonal↗
PubMed2006

Detection of deoxynivalenol in foods and indoor air using an array biosensor.

Deoxynivalenol (DON), a mycotoxin produced by several Fusaruim species, is a worldwide contaminant of foods and feeds. Because of the potential dangers due to accidental or intentional contamination of foods with DON, there is a need to develop a rapid and highly sensitive method for easy identification and quantification of DON. In this study, we have developed and utilized a competitive immunoassay technique to detect DON in various food matrixes and indoor air samples using an array biosensor. A DON-biotin conjugate, immobilized on a NeutrAvidin-coated optical waveguide, competed with the DON in the sample for binding to fluorescently labeled DON monoclonal antibodies. To demonstrate a simple procedure amenable for on-site use, DON-spiked cornmeal, cornflakes, wheat, barley, and oats were extracted with methanol-water (3:1) and assayed without cleanup or preconcentration. The limits of detection ranged from 0.2 ng/mL in buffer to 50 ng/g in oats. The detection limit of DON spiked into an aqueous effluent from an air sampler was 4 ng/mL.

Air Pollution, Indoor↗
PubMed2006

Indirect competitive immunoassay for detection of aflatoxin B1 in corn and nut products using the array biosensor.

Because of the potential health risks of aflatoxin B1 (AFB1), it is essential to monitor the level of this mycotoxin in a variety of foods. An indirect competitive immunoassay has been developed using the NRL array biosensor, offering rapid, sensitive detection and quantification of AFB1 in buffer, corn and nut products. AFB1-spiked foods were extracted with methanol and Cy5-anti-AFB1 added to the resulting sample. The extracted sample/antibody mix was passed over a waveguide surface patterned with immobilized AFB1. The resulting fluorescence signal decreased as the concentration of AFB1 in the sample increased. The limit of detection for AFB1 in buffer, 0.3 ng/ml, was found to increase to between 1.5 and 5.1 ng/g and 0.6 and 1.4 ng/g when measured in various corn and nut products, respectively.

Aflatoxin B1↗
PubMed2006

Simultaneous determination of kinetic parameters for the binding of cholera toxin to immobilized sialic acid and monoclonal antibody using an array biosensor.

Interactions between protein toxins and carbohydrate receptors are often semi-selective processes and the kinetic parameters that define the binding of a receptor to different toxins may vary with each interaction. In this study, we have determined the affinity constants for binding of cholera toxin (CT) to immobilized sialic acid and to anti-CT antibody (as a simultaneous reference) by measuring real-time binding processes using an array biosensor. N-Acetylneuraminic acid (Neu5Ac), a member of the sialic acid family, was covalently immobilized onto maleimide-activated planar waveguides via a thiol-terminated linker attached to the anomeric carbon of the sugar. Control antibodies were immobilized using two different approaches: covalent attachment onto maleimide-activated slides via the thiol on cysteine residues and non-covalent attachment using a biotin-NeutrAvidin linkage. Cy5-labeled CT was flowed over the immobilized receptors and the fluorescent intensity of the bound CT-receptor complex was recorded as a function of time. The association constants for CT binding to covalently attached Neu5Ac, to covalently attached anti-CT monoclonal antibody, and to antibody tethered by biotin-NeutrAvidin interactions were determined to be 1.3 x 10(8), 2.1 x 10(8) and 5.7 x 10(8)M(-1), respectively.

Antibodies, Monoclonal↗
PubMed2006

An array biosensor for detection of bacterial and toxic contaminants of foods.

The Naval Research Laboratory has developed an array-based biosensor system capable of detecting multiple pathogenic and toxic species in complex matrices. Sandwich fluoroimmunoassays are performed on the surface of a patterned microscope slide that acts as an optical waveguide. Fluorescence from immunocomplexes formed on the slide surface is excited using the evanescent field, an electromagnetic component of light, and the pattern of fluorescence is imaged using a charge-coupled device camera. Using the evanescent wave for excitation allows real-time imaging. Alternatively, a confocal scanner can also be used to detect and quantify fluorescent spots. A method for immobilizing capture antibodies, performing assays, and detecting bound targets is presented.

Animals↗
PubMed2006

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.

Bacteria↗
PubMed2006

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.

Biosensing Techniques↗
PubMed2005

Detection of bacterial toxins with monosaccharide arrays.

A large number of bacterial toxins, viruses and bacteria target carbohydrate derivatives on the cell surface to attach and gain entry into the cell. We report here the use of a monosaccharide-based array to detect protein toxins. The array-based technique provides the capability to perform simultaneous multianalyte analyses. Arrays of N-acetyl galactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) derivatives were immobilized on the surface of a planar waveguide and were used as receptors for protein toxins. These arrays were probed with fluorescently labeled bacterial cells and protein toxins. While Salmonella typhimurium, Listeria monocytogenes, Escherichia coli and staphylococcal enterotoxin B (SEB) did not bind to either of the monosaccharides, both cholera toxin and tetanus toxin bound to GalNAc and Neu5Ac. The results show that the binding of the toxins to the carbohydrates is density dependent and semi-selective. Both toxins were detectable at 100 ng/ml.

Adsorption↗
PubMed2005

Array biosensor for detection of ochratoxin A in cereals and beverages.

Contamination of food by mycotoxins occurs in minute quantities, and therefore, there is a need for a highly sensitive and selective device that can detect and quantify these organic toxins. We report the development of a rapid and highly sensitive array biosensor for the detection and quantitation of ochratoxin A (OTA). The array biosensor utilizes a competitive immunoassay format. Immobilized OTA derivatives compete with toxin in solution for binding to fluorescent anti-OTA antibody spiked into the sample. This competition is quantified by measuring the formation of the fluorescent immunocomplex on the waveguide surface. The fluorescent signal is inversely proportional to the concentration of OTA in the sample. Analyses for OTA in buffer and a variety of food and beverage samples were performed. Samples were extracted with methanol, without any sample cleanup or preconcentration step prior to analysis. The limit of detection for OTA in several cereals ranged from 3.8 to 100 ng/g, while in coffee and wine, detection limits were 7 and 38 ng/g, respectively.

Beverages↗
PubMed2005

Biosensor detection of botulinum toxoid A and staphylococcal enterotoxin B in food.

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.

Animals↗
PubMed2004

Detection of proteins cross-linked within galactoside polyacrylate-based hydrogels by means of a quantum dot fluororeagent.

Protein toxins have been immobilized in a galactoside polyacrylate hydrogel in a microarray format. The large pore size and solution-like environment of these novel hydrogels allow for easy penetration of large proteins and detection reagents. Confocal microscopy provided three-dimensional visualization of dye-labeled toxins cross-linked within the gel and of streptavidin-coated quantum dot (QD) fluorophores used to visualize the toxins after incubation with biotinylated anti-toxin antibodies. Fluorescence microscopy was utilized to visualize arrays of toxins detected by a biotinylated antibody and then exposure to streptavidin-conjugated QDs. The intensity of the QD fluorescence was quantified, and binding to two toxins on three types of hydrogels was examined.

Acrylic Resins↗
PubMed2004

Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications.

We report the fabrication, characterization and evaluation of three-dimensional (3D) hydrogel thin films used to measure protein binding (antigenicity) and antibody functionality in a microarray format. Protein antigenicity was evaluated using the protein toxin, staphylococcal enterotoxin B (SEB), as a model on highly crosslinked hydrogel thin films of polyacrylamide and on two-dimensional (2D) glass surfaces. Covalent crosslinking conditions were optimized and quantified. Interrogation of the modified 3D hydrogel was measured both by direct coupling of a Cy5-labeled SEB molecule and Cy5-anti-SEB antibody binding to immobilized unlabeled SEB. Antibody functionality experiments were conducted using three chemically modified surfaces (highly crosslinked polyacrylamide hydrogels, commercially available hydrogels and 2D glass surfaces). Cy3-labeled anti-mouse IgG (capture antibody) was microarrayed onto the hydrogel surfaces and interrogated with the corresponding Cy5-labeled mouse IgG (antigen). Five different concentrations of Cy5-labeled mouse IgG were applied to each microarrayed surface and the fluorescence quantified by scanning laser confocal microscopy. Experimental results showed fluorescence intensities 3-10-fold higher for the 3D films compared to analogous 2D surfaces with attomole level sensitivity measured in direct capture immunoassays. However, 2D surfaces reported equal or greater sensitivity on a per-molecule basis. Reported also are the immobilization efficiencies, inter-and intra-slide variability and detection limits.

Antigen-Antibody Complex↗
PubMed2004

Detection of campylobacter and Shigella species in food samples using an array biosensor.

Campylobacter and Shigella bacteria are common causes of food- and water-borne illness worldwide. There is a current need in food, medical, environmental, and military markets for a rapid and user-friendly method of detecting such pathogens. The array biosensor developed at the NRL encompasses these qualities. In this study, 25-min, sandwich immunoassays were developed for the detection of Campylobacter and Shigella species in both buffer and a variety of food and beverage samples. The limit of detection for Shigella dysenteriae in buffer and chicken carcass wash was 4.9 x 10(4) cfu mL(-)(1), whereas Campylobacter jejuni could be measured at concentrations as low as 9.7 x 10(2) cfu mL(-)(1). The limits of detection and dynamic range were found to vary depending on the sample matrix, but could be improved by running the sample over the waveguide surface for longer periods of time. Samples were run with no preconcentration or enrichment steps and little-to-no sample pretreatment prior to analysis.

Journal Article↗