Antimicrobial drug residues in food are strictly controlled and monitored by national laws in most territories. Tetracyclines are a major broad-spectrum antibiotic class, active against a wide range of Gram-positive and Gram-negative bacteria, and they are the leading choice for the treatment of many conditions in veterinary medicine in recent years. In dairy farms, milk from cows being treated with antibiotic drugs, such as tetracyclines, is considered unfit for human consumption. Contamination of the farm bulk tank with milk containing these residues presents a threat to confidence of supply and results in financial losses to farmers and dairy. Real-time monitoring of milk production for antimicrobial residues could reduce this risk and help to minimise the release of residues into the environment where they can cause reservoirs of antimicrobial resistance. In this article, we review the existing literature for the detection of tetracyclines in cow’s milk. Firstly, the complex nature of the milk matrix is described, and the test strategies in commercial use are outlined. Following this, emerging biosensors in the low-cost biosensors field are contrasted against each other, focusing upon electrochemical biosensors. Existing commercial tests that identify antimicrobial residues within milk are largely limited to beta-lactam detection, or non-specific detection of microbial inhibition, with tests specific to tetracycline residues less prevalent. Herein, we review a number of emerging electrochemical biosensor detection strategies for tetracyclines, which have the potential to close this gap and address the industry challenges associated with existing tests.

A rapid, low cost sensor for Covid-19 was developed using a perfluorodecanthiol layer and ACE2.

A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing antibodies and identifies a set of mutations in the gp120 C terminus that exposes the membrane-proximal external region of gp41, with potential utility in HIV vaccine design. Our understanding of which regions of the HIV-1 envelope are targets for broadly neutralizing antibodies (likely required for an HIV-1 vaccine) has expanded greatly in recent years, offering insights for vaccine design. For instance, much of solvent-exposed gp41 is now known to be targeted by antibodies at the gp120-gp41 interface. In this study, we isolated the neutralizing monoclonal antibody CAP248-2B, and used structural biology to characterize its epitope which spanned both the gp120-gp41 and gp41-gp41 interfaces in a manner distinct from other antibodies. CAP248-2B extends the interface target to include the gp120 C terminus, effectively encircling the base of native pre-fusion trimers. While CAP248-2B recapitulated the donor’s plasma breadth, it had poor potency against some isolates as a consequence of low neutralization plateaus. Unlike many broadly neutralizing antibodies, these plateaus did not appear to be a consequence of glycan heterogeneity, suggesting additional mechanisms that contribute towards incomplete neutralization. Finally, we characterized viral escape pathways from CAP248-2B, and identified a cluster of unusual mutations in the gp160 cleavage sites that made HIV-1 viruses more sensitive to antibodies targeting highly conserved membrane-proximal epitopes. These mutations might improve the immunogenicity of gp41, and thereby inform HIV-1 immunogen design.

Tetracycline antibiotics are used extensively in veterinary medicine, but the majority of the administrated dose is eliminated unmodified from the animal through various excretion routes including urine, faeces and milk. In dairy animals, limits on residues secreted in milk are strictly controlled by legislation. Tetracyclines (TCs) have metal chelation properties and form strong complexes with iron ions under acidic conditions. In this study, we exploit this property as a strategy for low cost, rapid electrochemical detection of TC residues. TC-Fe(III) complexes in a ratio of 2:1 were created in acidic conditions (pH 2.0) and electrochemically measured on plasma-treated gold electrodes modified with electrodeposited gold nanostructures. DPV measurements showed a reduction peak for the TC-Fe(III) complex that was observed at 50 mV (vs. Ag/AgCl QRE). The limit of detection in buffer media was calculated to be 345 nM and was responsive to increasing TC concentrations up to 2 mM, added to 1 mM FeCl₃. Whole milk samples were processed to remove proteins and then spiked with tetracycline and Fe(III) to explore the specificity and sensitivity in a complex matrix with minimal sample preparation, under these conditions the LoD was 931 nM. These results demonstrate a route towards an easy-to-use sensor system for identification of TC in milk samples taking advantage of the metal chelating properties of this antibiotic class.

Testing outside the laboratory environment, such as point of care testing, is a rapidly evolving area with advances in the integration of sample handling, measurement and sensing elements widely reported. Low cost, simple to use systems are important in this context because they provide a route to devices that can be used outside the laboratory and could be implemented in low resource settings where advanced diagnostic testing is often unavailable. Here, we present an open source highly simplified electrochemical platform, called SimpleStat, that has been programmed to perform differential pulse voltammetry and can be used to detect the presence of OXA-1 DNA sequences for oxacillin resistance. This DNA sensor can be used to specifically detect the presence of the OXA-1 gene, contrasted to the tetA gene which encodes for tetracycline resistance. These measurements were performed with both polycrystalline gold electrodes as a benchmark and electrodes integrated into the SimpleStat printed circuit.