Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
Anal Chim Acta ; 1077: 266-272, 2019 Oct 24.
Article in English | MEDLINE | ID: mdl-31307718

ABSTRACT

Fluorescence quenching based immunoassay format for the detection of a trace amount of some nitro-explosives with a high degree of selectivity is reported in this study. The immunoassay comprises anti-explosive antibodies functionalized microtitre strips specific to the targeted explosives, pentaerythritol tetranitrate (PETN), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and 2,4,6-trinitrotoluene (TNT). UV induced photolysis of nitro-explosive bound to targeted antibodies generates primarily nitrite ions which after the quick reaction with the detector molecule, 2,3-diaminonaphthalene (DAN), a fluorophore, quenches its fluorescence intensity, however, proportionately undergo cyclization to produce a highly fluorescent product, 2,3-naphthotriazole (NAT). The synthesized product, NAT, was verified using various chromatographic and spectrophotometric techniques. This newly developed antibody-based detection method, utilizing DAN dye, demonstrated a high selectivity towards PETN, RDX, and TNT. This method can be used as an economical testing kit for direct quantification of explosives, implying the great potential for quick, low-cost trace detection of explosives.


Subject(s)
Explosive Agents/analysis , Immunoassay/methods , Pentaerythritol Tetranitrate/analysis , Spectrometry, Fluorescence/methods , Triazines/analysis , Trinitrotoluene/analysis , 1-Naphthylamine/analogs & derivatives , Antibodies, Immobilized/immunology , Explosive Agents/immunology , Explosive Agents/radiation effects , Fluorescent Dyes/chemistry , Pentaerythritol Tetranitrate/immunology , Pentaerythritol Tetranitrate/radiation effects , Photolysis , Triazines/immunology , Triazines/radiation effects , Triazoles/chemistry , Trinitrotoluene/immunology , Trinitrotoluene/radiation effects , Ultraviolet Rays
2.
J Mol Recognit ; 29(2): 88-94, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26463875

ABSTRACT

An improved antibody against the explosive pentaerythritol tetranitrate (PETN) was developed. The immunogen was designed by the concept of bioisosteric replacement, which led to an excellent polyclonal antibody with extreme selectivity and immunoassays of very good sensitivity. Compounds such as nitroglycerine, 2,4,6-trinitrotoluene, 1,3,5-trinitrobenzene, hexogen (RDX), 2,4,6-trinitroaniline, 1,3-dinitrobenzene, octogen (HMX), triacetone triperoxide, ammonium nitrate, 2,4,6-trinitrophenol and nitrobenzene were tested for potential cross-reactivity. The detection limit of a competitive enzyme-linked immunosorbent assay was determined to be around 0.5 µg/l. The dynamic range of the assay was found to be between 1 and 1000 µg/l, covering a concentration range of three decades. This work shows the successful application of the bioisosteric concept in immunochemistry by exchange of a nitroester to a carbonate diester. The antiserum might be used for the development of quick tests, biosensors, microtitration plate immunoassays, microarrays and other analytical methods for the highly sensitive detection of PETN, an explosive frequently used by terrorists, exploiting the extreme difficulty of its detection.


Subject(s)
Antibodies/metabolism , Explosive Agents/immunology , Pentaerythritol Tetranitrate/immunology , Antibody Affinity , Antibody Specificity , Biosensing Techniques , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Limit of Detection
3.
Talanta ; 144: 439-44, 2015 Nov 01.
Article in English | MEDLINE | ID: mdl-26452845

ABSTRACT

The development of explosives detection technologies has increased significantly over the years as environmental and national security agencies implement tighter pollution control measures and methods for improving homeland security. 2, 4, 6-Trinitrotoluene (TNT), known primarily as a component in munitions, has been targeted for both its toxicity and carcinogenic properties that if present at high concentrations can be a detriment to both humans, marine and plant ecosystems. Enabling end users with environmental detection and monitoring systems capable of providing real-time, qualitative and quantitative chemical analysis of these toxic compounds would be extremely beneficial. Reported herein is the development of a multi-channeled microfluidic device immobilized with single chain fragment variable (scFv) recombinant proteins specific for the explosive, TNT. Fluorescence displacement immunoassays performed under constant flow demonstrated trace level sensitivity and specificity for TNT. The utility of three multi-channeled devices immobilized with either (1) scFv recombinant protein, (2) biotinylated-scFv (bt-scFv) and (3) monoclonal anti-TNT (whole IgG molecule) were investigated and compared. Fluorescence dose response curves, crossreactivity measurements and limits of detection (LOD) for TNT were determined. Fluorescence displacement immunoassays for TNT in natural seawater demonstrated detection limits at sub-parts-per-billion levels (0.5 ppb) utilizing the microfluidic device with immobilized bt-scFv.


Subject(s)
Antibodies, Immobilized/immunology , Explosive Agents/analysis , Lab-On-A-Chip Devices , Single-Chain Antibodies/immunology , Trinitrotoluene/analysis , Water Pollutants, Chemical/analysis , Antibodies, Immobilized/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Avidin/chemistry , Biotinylation , Explosive Agents/immunology , Fluorescence , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Limit of Detection , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Seawater/chemistry , Single-Chain Antibodies/chemistry , Trinitrotoluene/immunology , Water Pollutants, Chemical/immunology
4.
J Mol Recognit ; 25(2): 89-97, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22290770

ABSTRACT

Antibodies are a promising tool for the fast and selective trace detection of explosives. Unfortunately, the production of high-quality antibodies is not trivial and often expensive. Therefore, excellent antibodies are a rare and limiting resource in fields such as biosensing, environmental analysis, diagnostics, cancer therapy, and proteomics. Here, we report the synthesis, bioconjugation, and application of the structurally optimized hapten 6-(2,4,6-trinitro)-phenylhexanoic acid to improve the selectivity and sensitivity of antibodies for the detection of one of the most important explosives, trinitrotoluene. With a conjugate of bovine serum albumin and a highly purified N-hydroxy-succinimide (NHS)-activated hapten, two rabbits were immunized to obtain polyclonal antibodies. The immunization process was monitored by enzyme-linked immunosorbent assay to gain information about the progress of antibody titer and affinity. Finally, the polyclonal antibodies reached an affinity constant of (5.1 ± 0.6) × 10(9) l/mol (rabbit R1) and (2.3 ± 0.2) × 10(9) l/mol (rabbit R2). The respective assays show a minimum test midpoint (IC(50) value) of 0.1 ± 0.01 µg/l (R1) and 0.2 ± 0.02 µg/l (R2) and a working range of 0.005 to 150 µg/l (R1) and 0.007 to 200 µg/l (R2), which corresponds to more than four orders of magnitude for both. This is quite remarkable for a competitive immunoassay, which is often believed to have a narrow dynamic range. The limit of detection was calculated to 0.6 ng/l (R1) and 1.5 ng/l (R2), which is up to 100 times improvement in relation to the assay of Zeck et al. (1999) on the basis of a monoclonal antibody. The excellent selectivity of the polyclonal antibodies was comprehensively examined by determining the cross-reactivity to common explosives and other nitroaromatics including nitro musk components. The widely held belief that polyclonal antibodies generally display higher cross-reactivities than monoclonals could be disproved.


Subject(s)
Antibodies/immunology , Antibody Specificity/immunology , Drug Design , Explosive Agents/immunology , Haptens/chemistry , Haptens/immunology , Trinitrotoluene/immunology , Animals , Antibody Affinity/immunology , Calibration , Cattle , Cross Reactions/immunology , Explosive Agents/chemical synthesis , Explosive Agents/chemistry , Immunization , Nitrosation , Rabbits , Serum Albumin, Bovine/immunology , Titrimetry , Trinitrotoluene/chemical synthesis , Trinitrotoluene/chemistry
5.
Biosens Bioelectron ; 26(4): 1297-301, 2010 Dec 15.
Article in English | MEDLINE | ID: mdl-20688506

ABSTRACT

In recent years, there has been a growing focus on use of one-dimensional (1-D) nanostructures, such as carbon nanotubes and nanowires, as transducer elements for label-free chemiresistive/field-effect transistor biosensors as they provide label-free and high sensitivity detection. While research to-date has elucidated the power of carbon nanotubes- and other 1-D nanostructure-based field effect transistors immunosensors for large charged macromolecules such as proteins and viruses, their application to small uncharged or charged molecules has not been demonstrated. In this paper we report a single-walled carbon nanotubes (SWNTs)-based chemiresistive immunosensor for label-free, rapid, sensitive and selective detection of 2,4,6-trinitrotoluene (TNT), a small molecule. The newly developed immunosensor employed a displacement mode/format in which SWNTs network forming conduction channel of the sensor was first modified with trinitrophenyl (TNP), an analog of TNT, and then ligated with the anti-TNP single chain antibody. Upon exposure to TNT or its derivatives the bound antibodies were displaced producing a large change, several folds higher than the noise, in the resistance/conductance of SWNTs giving excellent limit of detection, sensitivity and selectivity. The sensor detected between 0.5 ppb and 5000 ppb TNT with good selectivity to other nitroaromatic explosives and demonstrated good accuracy for monitoring TNT in untreated environmental water matrix. We believe this new displacement format can be easily generalized to other one-dimensional nanostructure-based chemiresistive immuno/affinity-sensors for detecting small and/or uncharged molecules of interest in environmental monitoring and health care.


Subject(s)
Biosensing Techniques/methods , Explosive Agents/analysis , Nanotubes, Carbon , Trinitrotoluene/analysis , Biosensing Techniques/instrumentation , Biosensing Techniques/statistics & numerical data , Equipment Design , Explosive Agents/immunology , Nanotechnology , Sensitivity and Specificity , Single-Chain Antibodies , Trinitrotoluene/immunology
6.
Talanta ; 79(4): 1142-8, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19615523

ABSTRACT

A surface plasmon resonance (SPR) immunosensor for detection of 2,4-dinitrotoluene (2,4-DNT), which is a signature compound of 2,4,6-trinitrotoluene-related explosives, was developed by using a novel oligo (ethylene glycol) (OEG)-based sensor surface. A rabbit polyclonal antibody against 2,4-DNT (anti-DNPh-KLH-400 antibody) was prepared, and the avidity for 2,4-DNT and recognition capability were investigated by indirect competitive ELISA. The sensor surface was fabricated by immobilizing a 2,4-DNT analog onto an OEG-based self-assembled monolayer formed on a gold surface via an OEG linker. The fabricated surface was characterized by Fourier-transform infrared-refractive absorption spectrometry (FTIR-RAS). The immunosensing of 2,4-DNT is based on the indirect competitive principle, in which the immunoreaction between the anti-DNPh-KLH-400 antibody and 2,4-DNT on the sensor surface was inhibited in the presence of free 2,4-DNT in solution. The limit of detection for the immunosensor, calculated as three times the standard deviation of a blank value, was 20 pg mL(-1), and the linear dynamic range was found to be between 1 and 100 ng mL(-1). Additionally, the fabricated OEG-based surface effectively prevented non-specific adsorption of proteins, and the specific response to anti-DNPh-KLH-400 antibody was maintained for more than 30 measurement cycles.


Subject(s)
Dinitrobenzenes/analysis , Explosive Agents/analysis , Immunoassay/methods , Polyethylene Glycols/chemistry , Surface Plasmon Resonance/methods , Absorption , Animals , Antibodies/immunology , Binding, Competitive , Cattle , Dinitrobenzenes/chemistry , Dinitrobenzenes/immunology , Explosive Agents/chemistry , Explosive Agents/immunology , Female , Haptens/immunology , Molecular Weight , Rabbits , Spectroscopy, Fourier Transform Infrared , Surface Properties
7.
J Immunol Methods ; 339(1): 47-54, 2008 Nov 30.
Article in English | MEDLINE | ID: mdl-18755196

ABSTRACT

Llamas possess unique subclasses of antibodies that lack light chains, and thus are made by the pairing of two heavy chains. IgG was purified from two llamas which had been immunized with trinitrobenzene-keyhole limpet hemocyanin. Conventional IgG1 and heavy chain IgG2 and IgG3 subclasses were fractionated using affinity chromatography. The effectiveness of heavy chain antibodies for the detection of trinitrotoluene (TNT) using a competitive fluid array immunoassay was evaluated and compared to both the llama IgG1 as well as a murine monoclonal anti-TNT antibody. It was found that heavy chain antibody bound TNT with selectivity similar to conventional antibodies, yet the heavy chain antibodies possessed greater thermal stability. The titer of the heavy chain antibodies however was found to be 10-fold lower than the IgG1; thus analytical assays were best demonstrated using the llama IgG1 conventional antibody. The TNT competitive immunoassay on the Luminex fluid analyzer had a dynamic range from approximately 100 ng/mL to 10 microg/mL. Utilizing the same two-step competitive assay format the dynamic range of the monoclonal antibody was found to have a broad range (1 ng/mL to 1 microg/mL). This method was demonstrated on TNT contaminated soil extracts using both the llama IgG1 and the mouse monoclonal validating the utility of method for analysis of field samples.


Subject(s)
Camelids, New World , Explosive Agents/analysis , Immunoglobulin G/chemistry , Immunoglobulin Heavy Chains/chemistry , Trinitrotoluene/analysis , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Camelids, New World/immunology , Explosive Agents/immunology , Immunoassay/methods , Immunoglobulin G/immunology , Immunoglobulin Heavy Chains/immunology , Mice , Sensitivity and Specificity , Trinitrotoluene/immunology
SELECTION OF CITATIONS
SEARCH DETAIL
...