SplintR ligation-triggered in-situ rolling circle amplification on magnetic bead for accurate detection of circulating microRNAsYang, Yuan, Luo
et alPeerJ (2025) 13, e19082
Abstract: The circulating microRNAs (miRNAs), endogenous noncoding RNAs, post-transcriptionally participate in multiple processes during cell growth and development. Moreover, dysregulation of miRNAs expression is intricately associated with cancer. Currently, challenges of high homology, sequence similarity, and low abundance encountered in the detection of target miRNAs in complex samples need to be addressed. Biosensors established for miRNAs detection suffer from limitations in terms of sensitivity, specificity and high cost. Herein, a miRNA detection method based on in-situ RCA on magnetic bead catalyzed by SplintR ligase was proposed to achieve high sensitivity and high specificity. The following steps are included: (1) formation of P1-P2-miRNA double-stranded complex under catalyzation of SplintR ligase, and the release of P1-P2 single strand under denaturation; (2) enrichment of P1-P2 single chain by streptavidin-modified magnetic beads (SM-MB); (3) in situ RCA on surface of magnetic beads; (4) fluorescence detection. After optimization of experimental conditions, miRNA-155 detection with improved sensitivity and specificity was achieved. The detection limit was low to 36.39 fM, and one-base mismatch discrimination was demonstrated. Also, the clinical practicability for circulating miRNA-155 detection was preliminarily validated in human serum samples.©2025 Yang et al.
An electrochemical DNA biosensor based on denatured vesicle-mediated chain exchange amplification combined with electric field-assistance for nucleic acid detectionCui, Sun, Liao
et alAnal Methods (2025)
Abstract: Electrochemical DNA biosensors have been extensively used in food safety, clinical medicine and environmental monitoring due to their high specificity and sensitivity. However, electrochemical DNA biosensors based on nucleic acid hybridization still face challenges in achieving rapid and sensitive detection. In this study, a sensitive and rapid electrochemical DNA biosensor was developed using Strand Exchange Amplification (SEA) technology, with its performance evaluated against the bovine genome as the target. Additionally, gold nanoparticles (AuNPs) were employed to modify the electrode surface, a strategy to enhance both the density of probe modification and the amplification efficiency. Furthermore, the biosensor's sensitivity has been shown to be augmented by the exceptional conductivity of AuNPs. Despite the biosensor's simplicity and sensitivity, the detection time remains a limiting factor. To address this, the incorporation of an electric field within the biosensor framework has been proposed as a strategy to enhance the coupling rate of the nucleic acid amplification and streptavidin-biotin systems. This modification is anticipated to reduce the overall detection time, enabling rapid and precise real-time nucleic acid analysis. The biosensor demonstrated the capability to detect genome DNA as low as 1 fg μL-1 within 65 min, underscoring its significant potential for applications, such as detecting meat adulteration.
High-fidelity telomerase activity assay based on light-triggered nucleic acid separation system for the diagnosis of bladder cancerCheng, Zhang, Han
et alBiosens Bioelectron (2025) 278, 117355
Abstract: In conventional methods for telomerase activity assay, the obtained telomerase sample contains a large amount of impurity, which seriously affect the accuracy of the assay. Herein, we propose a light-triggered nucleic acid separation strategy to realize high fidelity Telomerase activity assay (Lit-Telo). In the light-triggered nucleic acid separation system, a 5' terminal biotinylated and photo-cleavable (PC) linker-functionalized telomerase substrate probe (Bio-PCTS) is designed. Telomerase extends telomeric repeat DNA (TTAGGG) to the 3' terminal of Bio-PCTS probe to produce telomerase extension product, which can be captured by streptavidin coated 96-well plate. Thus, the impurity can be removed from the reaction to realize the purification of telomerase extension product. A few seconds of UV light irradiation can disrupt the PC-linker in Bio-PCTS probe, allowing the easy and quick release of the telomerase extension product DNA fragment from the bottom of 96-well plate into the reaction solution for subsequent detection. Asymmetric-PCR-based TRAP and LbaCas12a/crRNA system were elucidated and optimized to realize the enhanced detection of telomerase activity. The proposed Lit-Telo platform achieved a limit-of-detection of telomerase activity equivalent to 8 HeLa cells. 26 bladder specimens were collected for telomerase activity assay using both fluorescence detection based Lit-Telo (Fluo Lit-Telo) visual detection based Lit-Telo (Visual Lit-Telo). ROC (receiver operating characteristic curve) analysis of the data indicated the good detection accuracy of Fluo Lit-Telo and Visual Lit-Telo methods with the AUC value of 93.94% and 92.12%, respectively. These results demonstrated the potential of the Lit-Telo platform in the in vitro diagnosis of bladder cancer.Copyright © 2025 Elsevier B.V. All rights reserved.
Optimization of IgG1 immune complexes to stimulate cytokine production in innate cellsDeb, Lott, Miceli
et alJ Immunol Methods (2025) 539, 113851
Abstract: Fcγ receptors are key immunoreceptors, that when bound by IgG immune complexes, trigger activation of downstream signaling pathways. However, there are limited in vitro assays that stimulate innate cells via Fcγ receptors that allow for evaluation of drugs or chemicals on antibody-triggered signaling. Our study investigated the activation of Fcγ receptors in innate cells using immune complexes. Our findings revealed that immobilized IgG antibodies did not elicit a significant immune response, so we designed two IgG1 immune complexes: trinitrophenyl-bovine serum albumin (TNP-BSA)/TNP-IgG1 and streptavidin-biotinylated IgG1 (Strept-Biotin IgG1). Strept-Biotin IgG1 immune complex was particularly effective, significantly enhancing IL-6, TNFα, and C3a levels, whereas TNP-BSA/TNP-IgG1 immune complex showed a modest IL-6 increase. Both TNP-BSA/TNP-IgG1 and Strept-Biotin IgG1 stimulated CD86 marker expression on F4/80+ macrophages. We also confirmed the binding of Strept-Biotin IgG1 to innate cells with fluorochrome-conjugated streptavidin. To further understand the Fcγ receptor-mediated activation of innate cells, we blocked the downstream phosphatidylinositol 3-kinase (PI3K) pathway. We found out that the PI3K inhibitor successfully suppressed IL-6 cytokine release and C3a production. However, specific Fcγ receptor-blocking antibodies failed to block IL-6 cytokine production and only modestly suppressed TNFα cytokine release, suggesting either that the antibodies were not effective blockers or that these immune complexes use other receptors. Regardless, the use of the Strept-Biotin IgG1 immune complex to stimulate cytokine production and other immune signaling was consistent with Fcγ receptor activation on innate cells which might be useful in assessing the effects of drugs or chemicals in innate cells.Copyright © 2025 Elsevier B.V. All rights reserved.
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Star Staining
