Analytische Chemie
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In particular, the rapid development of lateral flow assays as indispensable tools for everyone to contain the SARS-CoV-2 pandemic has fuelled the global demand for analytical tests that can be used outside dedicated laboratories. In addition to their use in medical diagnostics, rapid tests and assays have become increasingly important in various fields such as food safety, security, forensics, and environmental management. The advantage is obvious: taking the assay directly to the sample minimizes the time between suspicion and decision-making, allowing faster action. Especially today, when mobile communication devices with powerful computing capabilities and built-in cameras are ubiquitous, more people than ever before around the world have the basic skills to operate a powerful detector at their fingertips. This sets the stage for a much wider use of analytical measurements in terms of prognosis and prevention, enabling professional laypersons in particular.
However, current strip-based systems are primarily focused on single parameter analysis, whether it is SARS-CoV-2 biomarkers, blood glucose levels, or lead concentrations in water samples. Industrial applications of such methods also often still rely on single-parameter assays, requiring multiple runs even for a limited number of key parameters. Overcoming these limitations depends on developing low-number multiplexing strategies that ensure robustness, reliability, speed, ease of use, and sensitivity.
This lecture will give an overview of several generic approaches developed in recent years to address these challenges. It will highlight how the synergy of supramolecular (bio)chemistry, luminescence detection, hybrid (nano)materials and device miniaturization can result in powerful (bio)analytical assays that can be used at a point-of-need.1-5 Selected examples will introduce key aspects of such systems that include tailored signaling mechanisms and recognition elements, materials functionalization and device integration, including hybrid nanomaterials, gated indicator release systems, strip modification, and smartphone-based analysis.
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.
Rapid testing methods for the use directly at a point-of-need are expected to unfold their true potential especially when offering adequate capabilities for the simultaneous measurement of multiple analytes of interest. However, the implementation of multiplexing features while retaining simplicity, performance and portability is one of the prominent challenges in the field. Keeping in mind these challenges, we decided to combine the use of stimuli-responsive materials for small-molecule sensing relying on chemical signal amplification and their incorporation on paper strips for lateral-flow assays in a straightforward manner. Considering the modularity, high sensitivity and selectivity of antibody-gated indicator delivery systems, a multiplexed assay for three small-molecule explosives TATP, TNT and PETN was developed, allowing to detect the analytes simultaneously with a single test strip at lower ppb concentrations in liquid phase in <5 min, using a fluorescence reader or a smartphone for readout.
Because of the versatility of the hybrid material and the modularity of the assay architecture, it is obvious that this generic approach should be easily transferable to food or environmental analysis, point-of-care diagnostics and other areas of application in which the rapid screening for multiple parameters from liquid samples without clean-up in a dedicated laboratory is in demand.
The mixture of ammonium nitrate (AN) prills and fuel oil (FO), usually referred to as ANFO, is extensively used in the mining industry as a bulk explosive. One of the major performance predictors of ANFO mixtures is the fuel oil retention, which is itself governed by the complex pore structure of the AN prills. In this study, we present how X-ray computed tomography (XCT), and the associated advanced data processing workflow, can be used to fully characterise the structure and morphology of AN prills. We show that structural parameters such as volume fraction of the different phases and morphological parameters such as specific surface area and shape factor can be reliably extracted from the XCT data, and that there is a good agreement with the measured oil retention values. Importantly, oil retention measurements (qualifying the efficiency of ANFO as explosives) correlate well with the specific surface area determined by XCT. XCT can therefore be employed non-destructively; it can accurately evaluate and characterise porosity in ammonium nitrate prills, and even predict their efficiency.
Shock tube systems are non-electric explosive fuses employed in blasting and demolition applications to trigger the detonation of explosive charges. Their working principle is based on the explosive reaction of a fine explosive powder on the tubing's inner surface, generating a shock wave traveling at a velocity of 2,100 m/s along the length of the tube, without destroying it. One of
the key aspects of the manufacturing process of these shock tubes is the size and morphology of the explosive powder grains and their distribution on the inner wall of the tube, in order to propagate the shockwave efficiently and reliably. For the first time, synchrotron X-ray computed tomography has been used to characterize non-destructively the explosive powder grains, typically Al/HMX between 10 and 20 μm in size, in terms of morphology and 3D distribution but also to characterise the presence and location of defects
within the shock tube walls.
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.