The ingestion of histamine-rich foods or deficiencies in histamine-degrading enzymes can lead to histamine accumulation and produce intolerance, mimicking an allergic reaction with symptoms ranging from mild to even severe and potentially fatal anaphylaxis. Standard laboratory techniques like liquid chromatography are complex and lengthy, while antibody-based tests are costly and suffer from specificity issues due to the small size of the analyte. In this work, we sought to develop easy-to-use and cost-effective lateral flow assays for the rapid and accurate aptamer-based detection of histamine in fish and human whole blood. To this end, we optimised our previously selected histamine aptamer by truncation and combined it with a short partially complementary DNA probe for assay development. A microplate assay was initially designed for the detection of histamine in fish after a simple extraction procedure and the performance of the assay was comparable to standard liquid chromatographic methods. Aptamer-based lateral flow assays were then developed for the detection of histamine in fish and fingerprick blood. Canned tuna and sardines with known levels of histamine were successfully analysed, and the presence of histamine in spiked fingerprick blood samples was readily assessed by visual inspection. The limits of detection achieved were 23 nM (< 0.13 ppm) in fish and 8.4 nM (< 1 ng/mL) in blood, in combination with short duration (less than 10 minutes), in line with assay requirements for food safety control as well as rapid intervention and management of serious allergic reactions.

Retinol-binding protein 4 (RBP4) has been implicated in insulin resistance in rodents and humans with obesity and T2DM, making it a potential biomarker for the early diagnosis of T2DM. However, diagnostic tools for low-level detection of RBP4 are still lagging behind. Therefore, there is an urgent need for the development of T2DM diagnostics that are rapid, cost-effective and that can be used at the point-of-care (POC). Recently, nano-enabled biosensors integrating highly selective optical detection techniques and specificity of aptamers have been widely developed for the rapid detection of various targets. This study reports on the development of a rapid gold nanoparticles (AuNPs)-based aptasensor for the detection of RBP4. The retinol-binding protein aptamer (RBP-A) is adsorbed on the surface of the AuNPs through van der Waals and hydrophobic interactions, stabilizing the AuNPs against sodium chloride (NaCl)-induced aggregation. Upon the addition of RBP4, the RBP-A binds to RBP4 and detaches from the surface of the AuNPs, leaving the AuNPs unprotected. Addition of NaCl causes aggregation of AuNPs, leading to a visible colour change of the AuNPs solution from ruby red to purple/blue. The test result was available within 5 min and the assay had a limit of detection of 90.76 ± 2.81 nM. This study demonstrates the successful development of a simple yet effective, specific, and colorimetric rapid assay for RBP4 detection.

In previous work, a 93-mer aptamer was selected against the anaphylactic allergen, β-conglutin and truncated to an 11-mer, improving the affinity by two orders of magnitude, whilst maintaining the specificity. This 11-mer was observed to fold in a G-quadruplex, and preliminary results indicated the existence of a combination of monomeric and higher-order structures. Building on this previous work, in the current study, we aimed to elucidate a deeper understanding of the structural forms of this 11-mer and the effect of the structure on its binding ability. A battery of techniques including polyacrylamide gel electrophoresis, high-performance liquid chromatography in combination with electrospray ionization time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight, thermal binding analysis, circular dichroism and nuclear magnetic resonance were used to probe the structure of both the 11-mer and the 11-mer flanked with TT- at either the 5′ or 3′ end or at both ends. The TT-tail at the 5′ end hinders stacking effects and effectively enforces the 11-mer to maintain a monomeric form. The 11-mer and the TT- derivatives of the 11-mer were also evaluated for their ability to bind its cognate target using microscale thermophoresis and surface plasmon resonance, and biolayer interferometry confirmed the nanomolar affinity of the 11-mer. All the techniques utilized confirmed that the 11-mer was found to exist in a combination of monomeric and higher-order structures, and that independent of the structural form present, nanomolar affinity was observed.

The analysis of circulating cell-free (cf) DNA from plasma, serum or urine, has the potential to