Biomedical subjects

Michael E Lassman

Publications and source records attributed to Michael E Lassman.

10 recordsLinked to original sources

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↗

PubMed2005

Antimicrobial peptides for detection of bacteria in biosensor assays.

Bacteria, plants, and higher and lower animals have evolved an innate immune system as a first line of defense against microbial invasion. Some of these organisms produce antimicrobial peptides (AMPs) as a part of this chemical immune system. AMPs exert their antimicrobial activity by binding to components of the microbe's surface and disrupting the membrane. The overall goal of this study was to apply the AMP magainin I as a recognition element for Escherichia coli O157:H7 and Salmonella typhimurium detection on an array-based biosensor. We immobilized magainin I on silanized glass slides using biotin-avidin chemistry, as well as through direct covalent attachment. Cy5-labeled, heat-killed cells were used to demonstrate that the immobilized magainin I can bind Salmonella with detection limits similar to analogous antibody-based assays. Detection limits for E. coli were higher than in analogous antibody-based assays, but it is expected that other AMPs may possess higher affinities for this target. The results showed that both specific and nonspecific binding strongly depend on the method used for peptide immobilization. Direct attachment of magainin to the substrate surface not only decreased nonspecific cell binding but also resulted in improved detection limits for both Salmonella and E. coli.

Anti-Infective Agents↗

PubMed2005

Application of a homogenous assay for the detection of 2,4,6-trinitrotoluene to environmental water samples.

A homogeneous assay was used to detect 2,4,6-trinitrotoluene (TNT) spiked into environmental water samples. This assay is based on changes in fluorescence emission intensity when TNT competitively displaces a fluorescently labeled, TNT analog bound to an anti-TNT antibody. The effectiveness of the assay was highly dependent on the source of the sample being tested. As no correlation between pH and assay performance was observed, ionic strength was assumed to be the reason for variation in assay results. Addition of 10 phosphate-buffered saline to samples to increase their ionic strength to that of our standard laboratory buffer (about 0.17 M) significantly improved the range over which the assay functioned in several river water samples.

Buffers↗

PubMed2005

A hybrid quantum dot-antibody fragment fluorescence resonance energy transfer-based TNT sensor.

We demonstrate the use of luminescent QDs conjugated to antibody fragments to develop solution-phase nanoscale sensing assemblies, based on fluorescence resonance energy transfer (FRET) for the specific detection of the explosive 2,4,6-trinitrotoluene (TNT) in aqueous environments. The hybrid sensor consists of anti-TNT specific antibody fragments attached to a hydrophilic QD via metal-affinity coordination. A dye-labeled TNT analogue prebound in the antibody binding site quenches the QD photoluminescence via proximity-induced FRET. Analysis of the data collected at increasing dye-labeled analogue to QD ratios provided an insight into understanding how the antibody fragments self-assemble on the QD. Addition of soluble TNT displaces the dye-labeled analogue, eliminating FRET and resulting in a concentration-dependent recovery of QD photoluminescence. Sensor performance and specificity were evaluated.

Cadmium Compounds↗

PubMed2005

Self-assembled TNT biosensor based on modular multifunctional surface-tethered components.

We demonstrate a self-assembled reagentless biosensor based on a modular design strategy that functions in the detection of TNT and related explosive compounds. The sensor consists of a dye-labeled anti-TNT antibody fragment that interacts with a cofunctional surface-tethered DNA arm. The arm consists of a flexible biotinylated DNA oligonucleotide base specifically modified with a dye and terminating in a TNB recognition element, which is an analogue of TNT. Both of these elements are tethered to a Neutravidin surface with the TNB recognition element bound in the antibody fragment binding site, bringing the two dyes into proximity and establishing a baseline level of fluorescence resonance energy transfer (FRET). Addition of TNT, or related explosive compounds, to the sensor environment alters FRET in a concentration-dependent manner. The sensor can be regenerated repeatedly through washing away of analyte and specific reformation of the sensor assembly, allowing for subsequent detection events. Sensor dynamic range can be usefully altered through the addition of a DNA oligonucleotide that hybridizes to a portion of the cofunctional arm. The modular design of the sensor demonstrates that it can be easily adapted to detect a variety of different analytes.

Biosensing Techniques↗

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↗

PubMed2004

General strategy for biosensor design and construction employing multifunctional surface-tethered components.

Biosensors function by reversibly linking bioreceptor-target analyte binding with closely integrated signal generation and can either continuously monitor analyte concentrations or be returned to baseline readout values by removal of analyte. We present an approach for producing fully reversible, reagentless, self-assembling biosensors on surfaces. In the prototype biosensor, quencher-dye-labeled biotin-linked E. coli maltose binding protein (MBP) bound in a specific orientation to a NeutrAvidin-coated surface is employed as a bioreceptor. To complete sensor formation, a modular tether arm consisting of a flexible biotinylated DNA oligonucleotide, a fluorescence resonance energy-transfer (FRET) donor dye, and a distal beta-cyclodextrin (beta-CD) analyte analogue is bound in an equimolar amount to the same surface by means of DNA-directed immobilization. After self-assembly, a baseline level of FRET quenching is observed due to specific interaction between the beta-CD of the flexible tether arm and the sugar binding site of MBP, which brings the two dyes into proximity. Addition of the target analyte, the nutrient maltose, displaces the linked beta-CD-dye of the DNA-based tether arm, and a concentration-dependent change in FRET results. Biosensor sensitivity and dynamic range can be controlled by either using MBP variants having different binding constants or by binding of modulator DNA oligonucleotides that are complementary to the flexible DNA tether. The sensor can be regenerated and returned to baseline quenching levels by washing away analyte. A complex set of interactions apparently exists on the sensing surface that may contribute to sensor behavior and range. This approach may represent a general way to assemble a wide range of useful biosensors.

Base Sequence↗

PubMed2004

Fragmentation of biotinylated cyclic peptides.

Electrospray ionization coupled with tandem mass spectrometry (MS/MS) was used to determine the preferred binding site(s) of biotin NHS ester with a series of cyclic peptides with antibiotic properties. The peptides investigated are polymyxins, cyclic peptides produced by Bacillus polymyxa. In spite of the 1:1 stoichiometry used in the labeling reaction, multiple biotin molecules were incorporated into intact polymyxin peptides. Given the amine specificity of the activated biotin and the large number of amino acids with primary amines in the polymyxins, it was not clear by inspection which binding sites were more reactive than others. MS/MS was used to characterize the structure of the biotinylated peptides. MS/MS spectra of cyclic peptides often lead to ambiguous structure determinations due to the potential for multiple ring openings which result in the generation of multiple ion series. The MS/MS spectra of polymyxin peptides are especially difficult to characterize due to the lack of variety in their amino acids; however, the added complexity of the biotin aided the elucidation of the fragmentation pathways. MS/MS spectra of the species with biotin additions were used to rationalize the preferential binding sites of these molecules.

Anti-Bacterial Agents↗

PubMedDate not supplied

A fluorescence resonance energy transfer sensor based on maltose binding protein.

A fluorescence resonance energy-transfer (FRET) sensing system for maltose based on E. coli maltose binding protein (MBP) is demonstrated. The FRET donor portion of the sensing system consists of MBP modified with long wavelength-excitable cyanine dyes (Cy3 or Cy3.5). The novel acceptor portion of the sensor consists of beta-cyclodextrin (beta-CD) modified with either the cyanine dye Cy5 or the dark quencher QSY9. Binding of the modified beta-CD to dye-conjugated MBP results in assembly of the FRET complex. Added maltose displaces the beta-CD-dye adduct and disrupts the FRET complex, resulting in a direct change in fluorescence of the donor moiety. In the use of these FRET pairs, MBP dissociation values for maltose were estimated (0.14-2.90 microM). Maltose limits of detection were in the 50-100 nm range.

Biosensing Techniques↗