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One of the important analytical challenges is the fast and reliable trace detection of explosives in the context of security issues, ammunition disposal, and environmental pollution. Antibodies (Ab) are a promising tool for this purpose and the combination with a
surface acoustic wave (SAW) sensor opens the opportunity of highly selective and fast, label-free detection. A robust and sensitive method for the detection of the explosive trinitrotoluene (TNT) was developed. The detection limit was determined to be around 0.5 μg/L. The fast signal response of less than 1 minute shows that this approach is suitable for security and other time-critical applications. In addition, the very low crossreactivity highly reduces the number of false-positives in relation to competing techniques, including sniffer dogs. Due to the multianalyte ability of the SAW system, several explosives might be detected in parallel. Terminal amino groups were functionalized with trinitrophenyl (TNP) groups by reaction with trinitrobenzene sulfonic acid (TNBS). In less than 1 minute, a good signal response was obtained. 50 μL of sample was used. No non-specific interaction with the SAM surface was observed. A complete measuring cycle needed 19 minutes including a surface regeneration step with 50 μL of acetonitrile/water/propionic acid (50:50:1) and 150 μL of SDS solution (0.1 %, pH 1 with 100 mM glycine and 100 mM NaCl). A good long-term stability could be shown for at least 6 hours. Two polyclonal antibodies (R1, R2, affinity purified with Protein A) and a monoclonal antibody (A1.1.1) were tested successfully. A commercially available SAW sensor (sam5 blue, SAW Instruments) was used for liquid handling and detection. Self-assembled monolayers (SAM) of alkanethiol derivatives were
prepared on gold surfaces leading to non-fouling and hydrophilic properties, due to attached polyethylene glycol (PEG) residues. A continuous flow of buffer (phosphate-buffered saline, PBS plus Tween 20) of 100 μL/min was applied to the sensor system. TNT antibodies were pre-incubated with the samples containing traces of explosives. Polyclonal and monoclonal antibodies were tested.
The limit of detection (LOD) was determined to 0.5 μg/L for all three antibodies (3s from 12 replicates).
In 2019 over 30 000 people were killed or injured by explosions caused by explosives like TNT, PETN, HMX and RDX. Therefore, highly sensitive assays for the detection of TNT are needed. In this study we compared two commercially available TNT antibodies: A1.1.1 and EW75C with a highly optimized indirect competitive ELISA based on a BSA-TNA conjugate. As a result, a precision profile for both antibodies was determined with a LOD of 170 pmol L-1 for the clone A1.1.1 and a LOD of 3,2 nmol L-1 for the clone EW75C. The measurements showed that the clone A1.1.1 is a highly sensitive antibody for the detection of TNT while the clone EW75C does show medium performance at most.
In the cross-reactivity characterization of both antibodies many substances, closely related to the structure of TNT were tested. Both antibodies showed strong cross reactivity with trinitroaniline and trinitrobenzene. For the clone A1.1.1, which is known to originate from immunization of mice with an TNP-glycine-KLH conjugate, this has to be expected. Interestingly the clone EW75C, which was not characterized yet, showed similar behavior. This suggests a TNA-conjugate as immunogen for the EW75C antibody as well. None of both antibodies showed cross-reactions to the high explosives PETN, HMX and RDX. Also, the cross-reactions of nitro musks with the antibodies were investigated. Despite their prohibition, nitro musks are still used in Asia especially and are particularly popular in India. The overall superior clone A1.1.1 showed a significant cross-reactivity to musk ambrette. For practical reasons the influence of musk ambrette on this assay when used in natural environment should be investigated.
In further experiments, the highly sensitive TNT antibody A1.1.1 was digested with papain to obtain monovalent Fab-fragments. Due to its high stability against the digestion, a custom protocol for the IgG1 subclass of mice, to which the clone A1.1.1 belongs, was developed, resulting in a quantitative digestion of the intact antibody to Fab fragments. The success of the digestion was determined with MALDI-TOF-MS and SDS-PAGE. It was shown that this protocol worked for many different antibodies of IgG1 subclass as well.
The globe's seas were used as dumping ground after the world wars and those millions of ammunition, most of all containing 2,4,6-trinitrotoluene (TNT), represent a pressing danger for fishermen, dredging operations, submarine cable installations and tourism. We developed an extremely selective indication method for TNT based on a specific reaction that produces a highly fluorescent compound. The indication system was integrated into a microfluidic PDMS chip for the solid-liquid extraction of TNT from water samples, offering environmental monitoring possibilities. Combining the advantages of a light-up indicator, microfluidics and a smartphone as detector, the embedded sensor allows for the remote and rapid detection of TNT down to ng in surface and sea waters.
The mechanism of this system is based on kinetic competition. This biosensor consists of a monolithic glass column with a vast excess of immobilized hapten, which traps the fluorescently labeled antibody as long as no explosive is present. If the explosive 2,4,6-trinitrotoluene (TNT) is introduced some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein. The fluorescence is detected by highly sensitive laser-induced fluorescence with a conventional CMOS camera. The system achieved limits of detection of approx.1 pM (1 ppt) of the fluorescent label and around 100 pM (20 ppt) of TNT. The total assay time is less than 8 minutes. A cross-reactivity test with 5000 pM solutions of pentaerythritol tetranitrate (PETN), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) showed no cross reactivity.
A robust and sensitive method for the detection of the explosive trinitrotoluene (TNT) was developed. The detection limit was determined to be around 0.5 µg/L. The fast signal response of less than 1 minute shows that this approach is suitable for security and other time-critcal applications. In addition, the very low cross-reactivity highly reduces the number of false-positives in relation to competing techniques, including sniffer dogs. Due to the multianalyte ability of the SAW system, several explosives might be detected in parallel.
Autoclave methods are commonly used when pressure progress under thermal stress is of interest. One of them is the mini-autoclave method by Kühner AG. To test the robustness of the equipment under powerful exposure, two known high energetic materials, DNT and TNT, were investigated. The sample mass was increased stepwise in the range from 0.25 g to 1.0 g. For one test (1 g TNT), gas burners instead of the usually employed 2-zone heating block, were used to intensify the test conditions. Results showed the expected slower pressure generation of DNT in comparison to TNT. The mechanical robustness of the apparatus could be approved in all runs. Pressure data were used to calculate the energy release of decomposition. The values were in good agreement with the heat of decomposition, estimated by DSC.