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• Heterogeneous amplicons created by LAMP were analyzed using AF4 • Amplicons with two tags (biotin, fluorescein) were obtained due to 12 combination of primers • AF4 showed differences for amplicons with the tags at different positions • AF4 showed differences for complexes of amplicons with receptors depending on the tags position • AF4 showed that the fraction of small amplicons provides intense coloring in test strip Loop-mediated isothermal amplification (LAMP) is a rapid and efficient method for DNA amplification, producing concatemers of varying lengths (amplicons). This study explores the characterization of LAMP amplicons using asymmetric flow field-flow fractionation (AF4) and their realization in LAMP – lateral flow assay (LFA) for point-of-care diagnostics. We examined LAMP products from the invA gene of Salmonella enterica using two specific primer sets and three methods: fluorescent staining with SYBR Green, electrophoretic detection, and AF4. LAMP products labeled with biotin and fluorescein (introduced through primers, 12 combinations) were detected by LFA due to binding the labels. The efficiency of amplicon detection by test strips varied depending on the primer combination: the color intensities for the best and worst combinations differed by more than 7 times. AF4 enabled precise detection of components from primers to amplicons up to 500 bp and revealed differences influenced by labeling and primer combinations. An important advantage of AF4, which was demonstrated for the first time, was the characterization of native complexes of amplicons with receptors. The analysis of amplicons in complexes with streptavidin, anti-fluorescein antibodies, or both receptors together using AF4 revealed significant differences in the fractograms. The shape and maxima of the absorption peaks at 260 nm depended on retention time in the range from 20 to 35 min, indicating the formation of complexes of different compositions. AF4 and LFA showed that smaller amplicons (200-300 bp) primarily contribute to the binding of detected labels, while longer amplicons (1000-1500 bp) do not enhance the signal due to structural complexities. Our findings demonstrate the potential of AF4 as a tool for deep comprehension of LAMP processes and the composition of the formed amplicon-receptor complexes, offering valuable insights for enhancing LAMP-LFA systems. This work provides a foundation for the advanced application of AF4 for nucleic acid amplification technologies. [Display omitted] [ABSTRACT FROM AUTHOR]

An ultrasensitive electrochemical biosensor is described for the determination of microRNAs. It is based on the use of DNA-templated copper nanoparticles (Cu NPs) as signalling probe. MicroRNA-222 was selected as the model analyte. The probe was obtained from two different oligonucleotides (containing complementary bases) via hybridization chain reaction to form long DNA concatemers as template. The Cu NPs were formed by reaction of ascorbate with copper sulfate. The biosensor was fabricated as follows: (a) Capture probe (cDNA) with a thiolated group was immobilized on reduced graphene oxide modified with gold nanoparticles (rGO/Au NPs), (b) materials was placed on a glassy carbon electrode (GCE); (c) the modified electrode (cDNA/rGO/Au NPs/GCE) was sequentially hybridized with microRNA-222 and signal probe; this results in the formation of a sandwich structure of cDNA-microRNA-signal probe on surface of the modified electrode. Differential pulse voltammetry was employed to record the electrochemical response of biosensor in pH 6.0 solution. As a result, a sensitive oxidation current with a peak potential at 0.10 V (vs. SCE) was obtained corresponding to Cu NPs. The experimental conditions were optimized. Under optimal conditions, the biosensor exhibited wide linear response range (0.5 fM to 70 nM) and low limit of detection (0.03 fM; at S/N = 3). The assay possesses high selectivity and can discriminate analyte microRNA from single-base mismatched microRNA. [ABSTRACT FROM AUTHOR]

Enzyme-linked immunospot (ELISPOT) is one of the most important methods to measure the number of specific cells by detecting protein secretion at a single-cell level. However, traditional ELISPOT based on enzyme-substrate color development can only detect one target. Therefore, scientists developed multiple-target ELISPOT based on enzyme-substrate coloring. Besides, FluoroSPOT that can detect 2-4 fluorescent signals are developed. Nevertheless, the maximum detection targets of multiple-target ELISPOT and FluoroSPOT are around 4, and the signal amplification system can be further optimized. Fluorescence-based oligo-linked immunospot (FOLISPOT), which utilized DNA-barcoded antibodies to provide a highly multiplexed method with signal amplification, was developed to detect multiple targets simultaneously. In this method, multiple targets can be detected in one round and multiple rounds of detection can be conducted, and thus a large number of targets can be detected. Besides, signal amplification is achieved by DNA complementary pairing and modular orthogonal DNA concatemers, and thus cells secreting limited amounts of proteins can be detected. According to the studies, FOLISPOT can detect more spots than ELISPOT and can detect targets that are undetectable by ELISPOT. Furthermore, FOLISPOT can be utilized to detect more than 6 targets, by allowing sequential detection of multiple targets in one round and sequential detection in multiple rounds., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

An enzyme-free signal amplification strategy for colorimetric detection of RNA molecules was developed. The detecting process was started by the hybridization of the target RNA via two helper oligonucleotides to bi-functionalized (initiator and linker oligonucleotides-modified) gold nanoparticles. Afterwards, in presence of two auxiliary oligonucleotides, the nanoparticle-confined initiators triggered the formation of DNA concatemers containing hemin-binding aptamers through a modified hybridization chain reaction [HCR] strategy. In addition to the mediatory role, the helper oligonucleotides, with a toehold-mediated strand displacement [TMSD] ability, helped unwind the secondary structures of the RNA molecule and provided a binding site for a biotinylated capture probe. Upon binding, the complex was harvested on the streptavidin-coated microwell, and subsequently the formation of HRP-mimicking DNAzymes was stimulated by adding hemin molecules. The assay was successfully employed for detecting target nucleic acids, which bears secondary structures, in isothermal conditions (room temperature) without heat denaturation. The assay detected DNA molecule with LOD value of 1 pM with the ability to differentiate a spurious target containing a single-nucleotide substitution. On real RNA samples, the assay truly discriminated Escherichia coli O157:H7's 16s rRNA from closely related bacteria with a detection limit of ≥5×10 5 CFU. Compared to enzyme-based signal amplifications, the assay can detect the target RNA with a sensitivity similar to the one already found for RT-PCR, NASBA, and RT-LAMP assays. Overall, the analytical platform eliminates the need for enzymatic reactions, heat denaturation, and complicated instruments during the detection process. [ABSTRACT FROM AUTHOR]

Glass capillary-based nanopore is exploited for single-molecule conformational sensing of multiarm DNA concatemers during translocation. Both translocation frequency and orientation preference were found related with the number of arms of the DNA concatemers.

Summary In the ciliated protozoan Paramecium tetraurelia , Piwi-associated small RNAs are generated upon the elimination of tens of thousands of short transposon-derived DNA segments as part of development. These RNAs then target complementary DNA for elimination in a positive feedback process, contributing to germline defense and genome stability. In this work, we investigate the formation of these RNAs, which we show to be transcribed directly from the short (length mode 27 bp) excised DNA segments. Our data support a mechanism whereby the concatenation and circularization of excised DNA segments provides a template for RNA production. This process allows the generation of a double-stranded RNA for Dicer-like protein cleavage to give rise to a population of small regulatory RNAs that precisely match the excised DNA sequences. Video Abstract [ABSTRACT FROM AUTHOR]