Biomedical subjects

Frances S Ligler

Publications and source records attributed to Frances S Ligler.

34 records · Page 2Linked to original sources

PubMed2004

Colored thin films for specific metal ion detection.

This paper describes the investigation of chitosan and poly(allylamine) (PAH) for the creation of a multi-film, color-based dipstick for the detection of metal ions in solution. Thin, colored films of chitosan and PAH cross-linked with hexamethylene 1,6-di(aminocarboxysulfonate) (HDACS) are created where color is due to film thickness and optical interference effects. The films are investigated for their ability to selectively detect aqueous metal ions via changes in thickness and/or color. Chitosan-HDACS films were selective for Cr(VI) over all other metal ions tested including Cr(acac)3 and Cr(NO3)3 x 9H2O, and PAH-HDACS films were selective for Cu(II) and Cu(I) salts over all other metal ions tested. The irreversible, selective changes due to metal ion solutions were not caused by varying the pH. Potomac River water was also tested using the two films, with results indicating the presence of Cu(II) in the aqueous sample.

Allylamine↗

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↗

PubMed2004

Detection of Salmonella enterica serovar typhimurium by using a rapid, array-based immunosensor.

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 x 10(4) 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 x 10(3) CFU/g.

Animals↗

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↗

PubMed2003

Method for printing functional protein microarrays.

Piezoelectric dispensing of proteins from borosilicate glass capillaries is a popular method of protein biochip fabrication that offers the advantages of sample recovery and noncontact with the printing substrate. However, little regard has been given to the quantitative aspects of dispensing minute volumes (1 nL or less) at the low protein concentrations (20 micrograms/mL or less) typically used in microprinting. Specifically, loss of protein sample due to nonspecific adsorption to the glass surface of the dispensing capillaries can limit the amount of protein delivered to the substrate. We demonstrate the benefits of a low ionic strength buffer containing the carrier protein BSA that effectively minimizes the ionic strength-dependent phenomenon of nonspecific protein adsorption to borosilicate glass. Over the concentration range of 20-2.5 micrograms/mL, the dispensing of a reference IgG in 10 mM PBS including 0.1% BSA resulted in the deposition of 3.6- to 44-fold more IgG compared to the deposition of IgG in standard 150 mM PBS in the absence of BSA. Furthermore, when the IgG was dispensed with carrier protein, the resulting spots exhibited a more uniform morphology. In a direct immunoassay for cholera toxin, capture antibody spots dispensed in 10 mM PBS containing 0.1% BSA produced fluorescent signals that were 2.8- to 4.3-fold more intense than antibody spots that were dispensed in 150 mM PBS without BSA. Interestingly, no differences were observed in the specific activities of the capture antibodies as a result of printing in the different buffers. The implications of these results on the future development of protein biochips are discussed.

Adsorption↗

PubMed2003

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.

Biosensing Techniques↗

PubMed2002

Nine-analyte detection using an array-based biosensor.

A fluorescence-based multianalyte immunosensor has been developed for simultaneous analysis of multiple samples. While the standard 6 x 6 format of the array sensor has been used to analyze six samples for six different analytes, this same format has the potential to allow a single sample to be tested for 36 different agents. The method described herein demonstrates proof of principle that the number of analytes detectable using a single array can be increased simply by using complementary mixtures of capture and tracer antibodies. Mixtures were optimized to allow detection of closely related analytes without significant cross-reactivity. Following this facile modification of patterning and assay procedures, the following nine targets could be detected in a single 3 x 3 array: Staphylococcal enterotoxin B, ricin, cholera toxin, Bacillus anthracis Sterne, Bacillus globigii, Francisella tularensis LVS, Yersiniapestis F1 antigen, MS2 coliphage, and Salmonella typhimurium. This work maximizes the efficiency and utility of the described array technology, increasing only reagent usage and cost; production and fabrication costs are not affected.

Antigens↗

PubMed2002

A microarray immunoassay for simultaneous detection of proteins and bacteria.

We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).

Animals↗

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↗

PubMed2002

Integrating waveguide biosensor.

A capillary biosensor is demonstrated which uses the waveguiding properties of the capillary to integrate the signal over an increased surface area without simultaneously increasing the background noise from the detector. This biosensor achieves limits of detection of 30-50 pg/mL in immunoassays using a diode laser for excitation and a PMT for detection. This is approximately 2 orders of magnitude greater sensitivity than was achieved using the same immunoassay reagents in a fiber optic biosensor or a planar array biosensor. Two different approaches to using the capillaries as immunosensors are described, either of which could be adapted for multianalyte sensing.

Animals↗

PubMed2002

Attachment of plastic fluidic components to glass sensing surfaces.

Two types of flow manifolds have been developed in order to provide a reliable method for attaching plastic microfluidic systems to glass sensing surfaces. A permanently mounted flow manifold has grooves in the inter-channel barriers to confine the epoxy used for attachment. The reusable flow manifold has inter-channel barriers composed of both plexiglass for rigidity and polydimethysiloxane for leak-free attachment.

Biosensing Techniques↗

PubMed2002

Fabrication of a capillary immunosensor in polymethyl methacrylate.

A method for fabricating capillary-based immunosensors in a coupon milled from an inexpensive, commodity plastic (PMMA, plexiglass) is demonstrated. The key feature of the technique is the use of sol-gel technology to deposit a glass-like (Si [bond] OH) film on surfaces of the plastic capillary channels to facilitate antibody immobilization. The utility of this method was demonstrated in the context of continuous flow displacement immunosensors for the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). These sensors exhibited sensitivity to low microg/l RDX concentrations and peak-to-peak signal variations that were generally less than 10% for multiple injections at a single RDX concentration. The useful lifetime of the coupons in these experiments was greater than 10 h even after multiple exposures to high (1000 microg/l) RDX levels. This sensor platform has the physical characteristics needed for a portable field instrument: small, light-weight, and rugged.

Biosensing Techniques↗

PubMed2002

Voltage-induced inhibition of antigen-antibody binding at conducting optical waveguides.

Optical waveguides coated with electrically conducting indium-tin oxide (ITO) are demonstrated here as a new class of substrate for fluorescent immunosensors. These waveguides combine electrochemical control with evanescent excitation and image-based detection. Presented here are preliminary results utilizing these waveguides that demonstrate influence of waveguide voltage on antigen binding. Specifically, waveguide surfaces were bisected into electrically addressable halves, anti-ovalbumin immobilized in patterns on their surfaces, and a 1.3 V bias applied between waveguide halves in the presence of Cy5-labeled ovalbumin in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween-20. Fluorescence imaging indicated that binding of the antigen to positively biased waveguide halves was inhibited nearly 10-fold compared with negatively biased waveguide halves and unbiased controls. Furthermore, it is shown that ovalbumin binding to positively biased waveguide regions is regenerated after removal of applied voltage. These results suggest that electrochemical control of immunosensor substrates can be used as a possible strategy toward minimizing cross-reactive binding and/or nonspecific adsorption, immunosensor regeneration, and controlled binding.

Animals↗

PubMed2002

A comparison of imaging methods for use in an array biosensor.

An array biosensor has been developed which uses an actively-cooled, charge-coupled device (CCD) imager. In an effort to save money and space, a complementary metal-oxide semiconductor (CMOS) camera and photodiode were tested as replacements for the cooled CCD imager. Different concentrations of CY5 fluorescent dye in glycerol were imaged using the three different detection systems with the same imaging optics. Signal discrimination above noise was compared for each of the three systems.

Biosensing Techniques↗

PubMedDate not supplied

Applications of array biosensor for detection of food allergens.

Although food is a necessity, compounds within food products can be dangerous and life-threatening for people with food allergies. These allergy-causing compounds, such as proteins from eggs and milk, must be identified on the labels of commercial products. Unintentional contamination of food with these compounds occurs as a result of storage, manufacturing procedures, or cleaning procedures. A sensitive, specific, and rapid method to identify foods containing allergens is required by the food industry. The array biosensor, a rapid detection system, may provide a solution to this need. The array biosensor performs fluorescent immunoassays on the surface of a planar waveguide by first running samples, then fluorescently labeled antibodies, over a surface patterned with capture antibodies. An optical image is captured by a charged-coupled device camera and converted into fluorescence values. Signal intensity and spot location provide information on the compound and its concentration. The array biosensor has been successfully demonstrated for toxin, bacteria, and virus detection at low levels in under 20 min in food, clinical samples, and environmental matrixes. An assay for detection of ovalbumin as an indicator of egg contamination has been developed with limits of detection of 25 pg/mL in buffer and 1.3 ng/mL (13 ng/g) in non-egg pasta extract (buffer:pasta 10:1, v/w).

Allergens↗