Dithiocarbamate fungicides (DTFs) are widely used to control various fungal diseases in crops and ornamental plants. Maximum residual limits in the order of ppb-ppm are currently imposed by legislation to prevent toxicity problems associated with excessive use of DTFs. The specific analytical determination of DTFs is complicated by their low solubility in water and organic solvents. This review summarizes the current analytical procedures used for the analysis of DTF, including chromatography, spectroscopy, and sensor-based methods and discusses the challenges related to selectivity, sensitivity, and sample preparation. Biosensors based on enzymatic inhibition demonstrated potential as analytical tools for DTFs and warrant further research, considering novel enzymes from extremophilic sources. Meanwhile, Raman spectroscopy and various sensors appear very promising, provided the selectivity issues are solved.

Mpox—formerly monkeypox—is a re-emerging zoonotic virus disease, with large numbers of human cases reported during multi-country outbreaks in 2022. The close similarities in clinical symptoms that Mpox shares with many orthopoxvirus (OPXV) diseases make its diagnosis challenging, requiring laboratory testing for confirmation. This review focuses on the diagnostic methods used for Mpox detection in naturally infected humans and animal reservoirs, disease prevalence and transmission, clinical symptoms and signs, and currently known host ranges. Using specific search terms, up to 2 September 2022, we identified 104 relevant original research articles and case reports from NCBI-PubMed and Google Scholar databases for inclusion in the study. Our analyses observed that molecular identification techniques are overwhelmingly being used in current diagnoses, especially real-time PCR (3982/7059 cases; n = 41 studies) and conventional PCR (430/1830 cases; n = 30 studies) approaches being most-frequently-used to diagnose Mpox cases in humans. Additionally, detection of Mpox genomes, using qPCR and/or conventional PCR coupled to genome sequencing methods, offered both reliable detection and epidemiological analyses of evolving Mpox strains; identified the emergence and transmission of a novel clade ‘hMPXV-1A’ lineage B.1 during 2022 outbreaks globally. While a few current serologic assays, such as ELISA, reported on the detection of OPXV- and Mpox-specific IgG (891/2801 cases; n = 17 studies) and IgM antibodies (241/2688 cases; n = 11 studies), hemagglutination inhibition (HI) detected Mpox antibodies in human samples (88/430 cases; n = 6 studies), most other serologic and immunographic assays used were OPXV-specific. Interestingly, virus isolation (228/1259 cases; n = 24 studies), electron microscopy (216/1226 cases; n = 18 studies), and immunohistochemistry (28/40; n = 7 studies) remain useful methods of Mpox detection in humans in select instances using clinical and tissue samples. In animals, OPXV- and Mpox-DNA and antibodies were detected in various species of nonhuman primates, rodents, shrews, opossums, a dog, and a pig. With evolving transmission dynamics of Mpox, information on reliable and rapid detection methods and clinical symptoms of disease is critical for disease management.

A mini-review of the reported biosensor research occurring in South Africa evidences a strong emphasis on electrochemical sensor research, guided by the opportunities this transduction platform holds for low-cost and robust sensing of numerous targets. Many of the reported publications centre on fundamental research into the signal transduction method, using model biorecognition elements, in line with international trends. Other research in this field is spread across several areas including: the application of nanotechnology; the identification and validation of biomarkers; development and testing of biorecognition agents (antibodies and aptamers) and design of electro-catalysts, most notably metallophthalocyanine. Biosensor targets commonly featured were pesticides and metals. Areas of regional import to sub-Saharan Africa, such as HIV/AIDs and tuberculosis diagnosis, are also apparent in a review of the available literature. Irrespective of the targets, the challenge to the effective deployment of such sensors remains shaped by social and economic realities such that the requirements thereof are for low-cost and universally easy to operate devices for field settings. While it is difficult to disentangle the intertwined roles of national policy, grant funding availability and, certainly, of global trends in shaping areas of emphasis in research, most notable is the strong role that nanotechnology, and to a certain extent biotechnology, plays in research regarding biosensor construction. Stronger emphasis on collaboration between scientists in theoretical modelling, nanomaterials application and or relevant stakeholders in the specific field (e.g., food or health monitoring) and researchers in biosensor design may help evolve focused research efforts towards development and deployment of low-cost biosensors.

To decrease the burden of laborious and reagent-intensive screening of modified aptamers, their binding function requires assessment in assay formats compatible with the end diagnostic application. Here, we report on the use of an alternative and cost-effective approach: a rapid lateral flow assay (LFA) utilising streptavidin-conjugated gold nanoparticles (AuNP) as reporter molecules to screen novel ssDNA aptamers for their ability to detect CD4. Crossover-SELEX was employed to identify CD4-targeting aptamers from a ssDNA library enriched against a recombinant human CD4 protein (hCD4) conjugated to magnetic-beads and to endogenous CD4 expressed by U937 cells. Counter-selection with IgG-conjugated beads and CD4-negative Ramos RA-1 cells was employed. Following SELEX, four sequences (U4, U14, U20 and U26) were selected for candidate screening. Fluorescence confocal microscopy showed comparable localization of the Cy5-labeled aptamer U26, compared to antibodies binding CD4's cytoplasmic domain. Aptamer-hCD4 binding kinetics were evaluated by a qPCR-based magnetic-bead binding assay to unmodified aptamers. U26 exhibited the highest binding affinity (K_d = 2.93 ± 1.03 nM) to hCD4-conjugated beads. Citrate-stabilized gold nanoparticles (mean particle diameter, 10.59 ± 1.81 nm) were functionalized with streptavidin to allow immobilization of biotin-labeled aptamers. Except for U4, the aptamer–gold nanoparticle conjugates (Apt–AuNP) remained stable under physiological conditions with their size (approx. 15 nm) appropriate for use in the LFAs. Lateral-flow based screening was used to evaluate the suitability of the Apt–AuNPs as CD4-detecting reporter molecules by immobilizing hCD4 and flowing the nanoparticle conjugates across the LFA. Using this approach, two novel sequences were identified as being suitable for the detection of hCD4: visual detection at 9 min was obtained using U20 or U26. After 20 min, equivalent colorimetric hCD4 responses were observed between anti-CD4 monoclonal antibody (ΔI = 94.19 ± 3.71), an existing CD4 aptamer F1-62 (ΔI = 90.31 ± 19.31) and U26 (ΔI = 100.14 ± 14.61) LFA's, each demonstrating high specificity to hCD4 compared to IgG. From the above, Crossover-SELEX allowed for the successful identification of ssDNA aptamers able to detect hCD4. Streptavidin-conjugated AuNPs, when bound to candidate aptamers, show potential application here as screening tools for the rapid evaluation of aptamer performance in low-cost lateral flow diagnostics.

Despite the widespread knowledge of the influence that electrode pretreatments have on electroanalysis, pretreatments of gold electrodes for impedimetric aptasensors remain study-specific. This may be a neglected reason behind the limited consensus in reports focusing on creation and testing of aptasensors. To investigate this, several commonly reported pretreatments were applied to gold electrodes that were subsequently used to fabricate thrombin-specific impedimetric aptasensors, which are widely reported in the literature. Layer-by-layer electrochemical impedance spectroscopy analyses were conducted to determine the effect the pretreatment selection has on impedimetric responses during fabrication and testing of the aptasensor. Pretreatments were evaluated using factors crucial for biosensor deployment: the repeatability of interelectrode measurements and whether biosensors produced statistically significant responses when exposed to 2 μM thrombin. Individual pretreatments were found to impart unique physicochemical properties to electrode surfaces, indicated by variations in measured capacitances (impedimetry) and electrode surface topographies (scanning electron microscopy). Impedimetrically measured resistivity (R_total) of electrodes increased during layering of the biosensor across all the investigated pretreatments: from 852 ± 830 Ω (bare electrodes) to 3117 ± 1199 Ω (fabricated aptasensors exposed to thrombin), consistent with prior literature. Crucially, the magnitude and reproducibility of R_total values measured during fabrication and testing of the assembled aptasensor were strongly contingent on the pretreatment. Depending on the pretreatment, interelectrode sensor responses exhibited relative standard deviations between 38 and 150%. These findings propose that the choice of gold-surface pretreatment protocols is an overlooked—yet crucial—factor to consider when developing analytically valid impedimetric aptasensors.