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The use of lateral flow assays exploiting antibodies is well established in different fields due to their advantages, which include low cost, ease of production and rapid response, with the only required end-user intervention being sample addition. In recent years, aptamer-based lateral flow assays are garnering increasing interest offering a highly cost-effective and more flexible alternative to antibodies. In this review, an overview of the aptamer-based lateral flow assays developed to date is provided, highlighting the advantages of using aptamers and their ability to be incorporated into formats not possible with antibodies. [ABSTRACT FROM AUTHOR]

Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications. Image 1 • Ultrasensitive molecular detection of Yersinia pestis using biotinylated dNTPs and tailed primer is demonstrated. • Optimisation of ratio of biotinylated dNTPs: natural dNTPs is carried out using PCR and ELONA. • Optimised parameters were exploited using RPA. • Lateral flow assays for detection of single tailed amplicon and biotinylated dNTPs with SA-AuNPs was developed. • Real samples were tested. [ABSTRACT FROM AUTHOR]

Highlights • Duplex PCR amplification of pork in beef and chicken products. • Tailed primers allow the generation of duplex flanked by single stranded tails. • Enzyme-linked oligonucleotide assay for direct detection via hybridisation of PCR products. • Sensitivity of 71–188 pg of genomic DNA from each species was achieved. • The presence of 0.5–1% pork in binary DNA add-mixtures and in raw meat mixtures could be detected. Abstract In this work, a duplex PCR–Enzyme Linked Oligonucleotide Assay (ELONA) is reported for the sensitive and reliable detection of pork adulteration in beef and chicken products, two of the most widely consumed meat types in the world. The strategy relies on the use of species-specific tailed primers for duplex amplification and simple dilution of the PCR reactions for direct colorimetric detection via hybridization, eliminating the need for any other post-amplification steps. A high sensitivity was achieved, with as low as 71–188 pg of genomic DNA able to be detected using mixtures of control DNA from each species. The strategy was validated using DNA add-mixtures as well as DNA extracted from raw meat mixtures and 0.5–1% w/w pork could be easily detected when mixed with beef or chicken. The proposed approach is simple, sensitive and cost-effective compared to equivalent commercial kits suitable for detecting adulterant pork levels in meat products. [ABSTRACT FROM AUTHOR]

The rapid and sensitive detection of small molecules is garnering increasing importance, and aptamers show great promise in replacing expensive, elaborate detection platforms exploiting chromatographic separation or antibody-based assays. The characterization of aptamer interaction with small molecule targets is not facile, and there is a mature need for a rapid, high-throughput technique for the analysis of aptamer-small molecule kinetics and affinity. In this work we present methodologies for the evaluation of aptamer-small molecule interactions, using the aptamers reported against the steroid 17β-estradiol as a model system. Microscale thermophoresis, apta-PCR affinity assay and surface plasmon resonance were explored to evaluate the reported aptamers’ binding properties in terms of affinity and specificity, and were demonstrated to be successfully applied to the analysis of aptamer-small molecule interactions. [ABSTRACT FROM AUTHOR]