Biomedical subjects

Kim E Sapsford

Publications and source records attributed to Kim E Sapsford.

12 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

Materials for fluorescence resonance energy transfer analysis: beyond traditional donor-acceptor combinations.

The use of Förster or fluorescence resonance energy transfer (FRET) as a spectroscopic technique has been in practice for over 50 years. A search of ISI Web of Science with just the acronym "FRET" returns more than 2300 citations from various areas such as structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting and metallic nanomaterials. The advent of new classes of fluorophores including nanocrystals, nanoparticles, polymers, and genetically encoded proteins, in conjunction with ever more sophisticated equipment, has been vital in this development. This review gives a critical overview of the major classes of fluorophore materials that may act as donor, acceptor, or both in a FRET configuration. We focus in particular on the benefits and limitations of these materials and their combinations, as well as the available methods of bioconjugation.

Fluorescence Resonance Energy Transfer↗

PubMed2006

Designer variable repeat length polypeptides as scaffolds for surface immobilization of quantum dots.

We demonstrate the use of a series of engineered, variable-length de novo polypeptides to discretely immobilize luminescent semiconductor nanocrystals or quantum dots (QDs) onto functional surfaces. The polypeptides express N-terminal dicysteine and C-terminal hexahistidine residues that flank a variable number (1, 3, 5, 7, 14, 21, 28, or 35) of core beta-strand repeats, with tyrosine, glutamic acid, histidine, and lysine residues located at the turns. Polypeptides have molecular weights ranging from 4 to 83 kDa and retain a rigid structure based on the antiparallel beta-sheet motif. We first use a series of dye-labeled polypeptides to test and characterize their self-assembly onto hydrophilic CdSe-ZnS QDs using fluorescence resonance energy transfer (FRET). Results indicate that peptides maintain their beta-sheet conformation after self-assembly onto the QD surfaces, regardless of their length. We then immobilize biotinylated derivatives of these polypeptides on a NeutrAvidin-functionalized substrate and use them to capture QDs via specific interactions between the peptides' polyhistidine residues and the nanocrystal surface. We found that each of the polypeptides was able to efficiently capture QDs, with a clear correlation between the density of the surface-tethered peptide and the capacity for nanocrystal capture. The versatility of this capture strategy is highlighted by the creation of a variety of one- and two-dimensional polypeptide-QD structures as well as a self-assembled surface-immobilized FRET-based nutrient sensor.

Amino Acid Sequence↗

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↗

PubMed2005

A cowpea mosaic virus nanoscaffold for multiplexed antibody conjugation: application as an immunoassay tracer.

Cowpea mosaic virus (CPMV), an icosahedral 30 nm virus, offers a uniquely programmable biological nanoscaffold. This study reports initial optimization of the simultaneous modification of two CPMV mutants with AlexaFluor 647 fluorescent dyes and either IgG proteins or antibodies at specific sites on the virus scaffold. The capacity of CPMV as a simultaneous carrier for different types of molecules was demonstrated, specifically, when applied as a tracer in direct and sandwich immunoassays. The ability to label the virus capsid with antibody and up to 60 fluorescent dyes resulted in an improved limit of detection in SEB sandwich immunoassays, when used as a tracer, relative to a mole equivalent of dye-labeled antibody.

Antigens↗

PubMed2005

Decoration of discretely immobilized cowpea mosaic virus with luminescent quantum dots.

This report describes two related methods for decorating cowpea mosaic virus (CPMV) with luminescent semiconductor nanocrystals (quantum dots, QDs). Variants of CPMV are immobilized on a substrate functionalized with NeutrAvidin using modifications of biotin-avidin binding chemistry in combination with metal affinity coordination. For example, using CPMV mutants expressing available 6-histidine sequences inserted at loops on the viral coat protein, we show that these virus particles can be specifically immobilized on NeutrAvidin functionalized substrates in a controlled fashion via metal-affinity coordination. To accomplish this, a hetero-bifunctional biotin-NTA moiety, activated with nickel, is used as the linker for surface immobilization of CPMV (bridging the CPMVs' histidines to the NeutrAvidin). Two linking chemistries are then employed to achieve CPMV decoration with hydrophilic CdSe-ZnS core-shell QDs; they target the histidine or lysine residues on the exterior virus surface and utilize biotin-avidin interactions. In the first scheme, QDs are immobilized on the surface-tethered CPMV via electrostatic attachment to avidin previously bound to the virus particle. In the second strategy, the lysine residues common to each viral surface asymmetric unit are chemically functionalized with biotin groups and the biotinylated CPMV is discretely immobilized onto the substrate via NeutrAvidin-biotin interactions. The biotin units on the upper exposed surface of the immobilized CPMV then serve as capture sites for QDs conjugated with a mixture of avidin and a second protein, maltose binding protein, which is also used for QD-protein conjugate purification. Characterization of the assembled CPMV and QD structures is presented, and the potential uses for protein-coated QDs functionalized onto this symmetrical virion nanoscaffold are discussed.

Chromogenic Compounds↗

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

Surface-immobilized self-assembled protein-based quantum dot nanoassemblies.

Luminescent semiconductor quantum dot (QD)-based optical biosensors have the potential to overcome many of the limitations associated with using conventional organic dyes for biodetection. We have previously demonstrated a hybrid QD-protein-based fluorescence resonance energy transfer (FRET) sensor. Although the QD acted as an energy donor and a protein scaffold in the sensor, recognition and specificity were derived from the proteins. Transitioning this hybrid prototype sensor into flow cells and integrated devices will require a surface-immobilization strategy that allows the QD-based sensor to sample the environment and still maintain a distinct protein-covered QD architecture. We demonstrate a self-assembled strategy designed to accomplish this. Using glass slides coated with a monolayer of neutravidin (NA) as the template, QDs with maltose binding protein (MBP) and avidin coordinated to their surface were attached to the glass slides in discrete patterns using an intermediary bridge of biotinylated MBP or antibody linkers. Control of the surface location and concentration of the QD-protein-based structures is demonstrated. The utility of this self-assembly strategy is further demonstrated by assembling a QD-protein structure that allows the QDs to engage in FRET with a dye located on the surface-covering protein.

Avidin↗

PubMed2004

Real-time analysis of protein adsorption to a variety of thin films.

The ability of a fluorescence-based array biosensor to screen surfaces for the adsorption of biomolecules in real-time is demonstrated. Glass microscope slides were coated with silanes, including 3-mercaptopropyl-triethoxysilane, 3-glycidyloxypropyltrimethoxysilane, 3-aminopropyltrimethoxy-silane, octadecyl-trichlorosilane, and 2-methoxy((polyethylenoxy)propyl)tri-methoxysilane, or with polymer thin films, including polystyrene, polyimide, sol-gel, poly(dimethylsiloxane), and agarose. The adsorption of Cy5-labeled proteins, bovine serum albumin, fibrinogen, and lysozyme onto these surfaces was measured using total internal reflection spectroscopy over a period of 50 min. The majority of the modified surfaces, apart from notable exceptions including the thiol silane and PDMS, behaved as expected upon protein adsorption, and the observations could be related to the properties of both the individual surfaces and proteins. This study highlights the complex nature of the mechanisms involved when a protein interacts at a solid-liquid interface. However, it also demonstrates a comparatively generic method with which to screen surfaces for their protein resistant properties and to measure surface interactions in real time. Furthermore, since the array biosensor can perform multiple measurements simultaneously, the interactions of a variety of proteins with a single surface can be monitored.

Adsorption↗

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↗

PubMed2003

Array biosensor for detection of toxins.

The array biosensor is capable of detecting multiple targets rapidly and simultaneously on the surface of a single waveguide. Sandwich and competitive fluoroimmunoassays have been developed to detect high and low molecular weight toxins, respectively, in complex samples. Recognition molecules (usually antibodies) were first immobilized in specific locations on the waveguide and the resultant patterned array was used to interrogate up to 12 different samples for the presence of multiple different analytes. Upon binding of a fluorescent analyte or fluorescent immunocomplex, the pattern of fluorescent spots was detected using a CCD camera. Automated image analysis was used to determine a mean fluorescence value for each assay spot and to subtract the local background signal. The location of the spot and its mean fluorescence value were used to determine the toxin identity and concentration. Toxins were measured in clinical fluids, environmental samples and foods, with minimal sample preparation. Results are shown for rapid analyses of staphylococcal enterotoxin B, ricin, cholera toxin, botulinum toxoids, trinitrotoluene, and the mycotoxin fumonisin. Toxins were detected at levels as low as 0.5 ng mL(-1).

Biosensing Techniques↗

PubMed2002

Demonstration of four immunoassay formats using the array biosensor.

The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.

Antibodies↗