Search Results (652)

Ochratoxin A (OTA) is a highly toxic mycotoxin commonly detected in food and agricultural products, requiring sensitive analytical methods for reliable monitoring. Herein, we report an ultrasensitive turn-on electrochemical aptasensor for OTA detection based on a target-induced displacement of an aptamer–complementary DNA (cDNA) duplex assembled on an electrochemically reduced graphene oxide (ERGO)-modified glassy carbon electrode (GCE). In the absence of OTA, a methylene blue (MB)-labeled aptamer hybridized with cDNA is immobilized on the ERGO surface via π–π stacking interactions, forming a rigid duplex that suppresses electron transfer and yields a low electrochemical signal. Upon OTA binding, the aptamer undergoes a conformational transition into a G-quadruplex structure, leading to dissociation of the cDNA strand. This target-induced folding brings the MB redox tag into close proximity to the ERGO surface, markedly accelerating electron transfer and enhancing the cathodic reduction current of MB, thereby producing a pronounced signal-on response in square-wave voltammetry (SWV). The ERGO-modified electrode provides a conductive and stable interface without chemical linkers. Under optimized conditions, the aptasensor shows a linear response to OTA from 10 fM to 100 pM with an ultralow LOD of 0.67 fM, together with high selectivity, good reproducibility, and satisfactory stability. This work demonstrates a simple and effective turn-on aptasensing strategy for sensitive electrochemical detection of OTA. Full article

Aptamers, short single-stranded DNA or RNA oligonucleotides, are emerging as transformative tools in cardiology for the diagnosis, treatment, and theranostics of cardiovascular diseases (CVDs). This review highlights their dual utility. In diagnostics, aptamers enable the construction of highly sensitive biosensors for key cardiac biomarkers (e.g., troponins, myoglobin, C-reactive protein, natriuretic peptides), outperforming conventional assays and enabling early detection and point-of-care testing. Therapeutically, aptamers offer targeted, controllable, and reversible anticoagulation, as demonstrated by clinical-stage candidates like BT200 (anti-vWF) and NU172 (anti-thrombin), whose action can be rapidly reversed with antidote oligonucleotides. Furthermore, aptamers serve as precision delivery vehicles (e.g., Gint4.T, RNA-Apt30) for transporting therapeutic peptides or nucleic acids specifically to cardiomyocytes. Recent integration with nanomaterials (quantum dots, graphene, liposomes, DNA origami) has led to advanced biosensing and drug delivery platforms. Despite challenges like stability and the polyethylene glycol (PEG) immunogenicity, ongoing clinical trials underscore the significant potential of aptamer technology to bridge precise diagnostics and targeted therapy, paving the way for innovative, personalized CVD interventions.) Full article

Background/Objectives: Leishmaniasis is a disease affecting millions of people caused by parasites of the genus Leishmania. The GP63 protein of Leishmania infantum (LiGP63) is one of its major surface antigens and a main virulence factor, playing a role in the adhesion of extracellular promastigote stages to macrophages and in the survival of intracellular amastigotes. Methods: Here, DNA aptamers have been developed against LiGP63 through the systematic evolution of ligands by exponential enrichment. Results: Twenty individual aptamer sequences were characterized using confocal fluorescence microscopy and flow cytometry analysis, and 14 of them had targeting to more than 70% of L. infantum promastigotes with different subcellular localization patterns. Subsequent dot blot analyses narrowed down the selection to five candidates for further characterization through an aptamer-linked immobilized sorbent assay where it was possible to detect endogenous LiGP63 in L. infantum promastigote lysates. The five selected aptamers recognized the recombinant LiGP63 protein with binding affinities ranging from 0.3 to 2.1 µM. Promastigotes preincubated with LiGP63Apt-4, -27 and -28 exhibited a significantly reduced adhesion to and infection of RAW 264.7 macrophages. Moreover, when LiGP63Apt-4 and -28 were conjugated to liposomes, these two aptamers significantly enhanced the targeting to L. infantum promastigotes compared to plain liposomes. Conclusions: Given their improved stability and cost-effectiveness over antibodies, the aptamers evolved here represent promising candidates for new therapeutic and diagnostic approaches and for future nanoparticle-based drug delivery strategies in leishmaniasis. Full article

Background: This study aimed to develop a superior aptamer-based therapeutic for targeted glioblastoma intervention by conducting a comparative analysis of two DNA aptamers: the original U2 sequence, selected against glioblastoma cells exhibiting high EGFRvIII expression, and its modified, shortened, and stabilized variant, Gol1. Methods: The effects of the investigated aptamers on primary human glioblastoma cells with graded receptor expression levels and on a rat 101/8 glioblastoma tissue model were rigorously studied. Results: The results demonstrated the significant superiority of the stabilized Gol1 aptamer, which exhibited exceptional binding affinity for the EGFRvIII receptor. Pronounced antiproliferative and antimigratory effects against EGFRvIII-positive human tumor cells, ultimately inducing complete cell death. Transcriptomic analysis revealed a sophisticated dual mechanism of action for Gol1: the specific activation of neuronal differentiation genes concurrent with the suppression of key alternative splicing factors. Crucially, in vivo confirmation showed highly selective accumulation of the FAM-labeled Gol1 aptamer exclusively within tumor tissue, with a maximum concentration gradient observed in the invasive border zone and a complete absence of accumulation in intact brain parenchyma. Conclusions: These comprehensive findings confirm that the Gol1 aptamer constitutes a highly promising and versatile platform for developing novel targeted theranostic strategies against glioblastoma, offering a precise approach for both diagnostic imaging and therapeutic intervention. Full article

Breast cancer continues to be a major challenge in global health, in part due to significant inequalities in access to costly diagnostic and therapeutic technologies based on antibodies. Their manufacturing requires complex and expensive bioproduction systems, resulting in limited availability of these tools—essential for early detection and targeted treatment—in many regions, particularly in Latin America. This gap has highlighted the need for cost-effective and scalable theranostic alternatives, increasing interest in aptamers. Obtained through SELEX technology, aptamers are synthetic DNA or RNA oligomers that fold into functional structures. Among their advantages are high affinity for their target, low immunogenicity, and chemical synthesis, which assures reproducible production. Aptamers have expanded the landscape of diagnostic platforms through the development of sensitive aptasensors, liquid biopsy strategies, and imaging systems based on nanomedicines. They also contribute to targeted therapy by recognizing cancer biomarkers selectively and enabling controlled drug delivery. This review presents a critical summary of advances in aptamer-based theranostics for breast cancer, addressing molecular mechanisms, structural folding, selective ligand binding, and nanomaterial interfacing. We also discuss applications in extracellular vesicle capture, cancer stem cell detection, and therapeutic conjugates, emphasizing their advantages and limitations relative to approaches based on antibodies. Overall, current advances show aptamers as emerging tools capable of democratizing precision oncology, particularly in regions where access to advanced technologies remains limited. Full article

Aptamers are powerful tools for detecting and analyzing biomolecules that consist of proteins or nucleic acids. However, their application to aptamers against viroids—highly structured self-replicating RNAs—has not yet been explored. In this study, a magnetic bead- and high-throughput sequencing-based SELEX (MB-HTS-SELEX) protocol for selecting potential aptamers against potato spindle tuber viroid (PSTVd) is presented. Full-length biotinylated-PSTVd RNA was transcribed in vitro, immobilized on streptavidin-coated magnetic beads, and incubated with a library of ~3.32 × 10¹⁴ molecules of random single-stranded oligo-DNAs (oligo-ssDNAs) of 20, 30, or 40 nucleotides (L20, L30, or L40, respectively) flanked by primer binding sites for downstream PCR amplification. Simultaneous biotin labeling of the anti-aptamer strand of the resulting double-stranded DNA (dsDNA) amplicons facilitated strand separation using streptavidin-coated magnetic beads. After 10 selection rounds, high-throughput sequencing, followed by bioinformatics analysis of the generated sequences, allowed for the detection of several enriched sequences, representing putative PSTVd-binding aptamers. Subsequent pull-down assays showed that the most abundant oligo-ssDNA in L30 was docked on PSTVd molecules. This combination method may ameliorate the selection of high-affinity aptamers against PSTVd, reduce the number of selection cycles, time, and other costs of aptamer production, thereby promoting future massive and cost-effective viroid detection and characterization. Full article

In on-site rapid detection, the electrochemical method boasts high sensitivity and rapid response capabilities, while the colorimetric method can provide intuitive visual readings suitable for on-site screening. Therefore, this study developed an innovative dual-mode electrochemical/colorimetric aptasensor for the accurate detection of malathion (MAL) in vegetables. The sensor combines magnetic Fe₃O₄@ZIF-8-DNA composites and CuZr-MOF-cDNA probes, enabling simultaneous detection of the target through electrochemical reactions and colorimetric changes. The introduction of CuZr-MOF not only enhances the sensor’s conductivity but also significantly amplifies the electrochemical signal through its catalytic properties. The magnetic Fe₃O₄@ZIF-8-DNA composite facilitates solid–liquid separation under an external magnetic field. When the target MAL is present, the aptamer binds to the target, causing the CuZr-MOF-cDNA probes to release from the composite, altering the number of free probes in the supernatant and generating varying intensities of colorimetric signals. Meanwhile, the MAL captured in the precipitate by the aptamer is quantitatively detected through electrochemical methods. Experimental results demonstrate that as the target concentration increases, the colorimetric signal intensifies while the electrochemical signal weakens, showing a good linear relationship between the two. The aptasensor’s limit of detection (LOD) for colorimetric and electrochemical modes was 1.57 × 10⁻¹¹ M and 4.76 × 10⁻¹¹ M, respectively, with recoveries ranging from 87.71% to 107.68% and relative standard deviations between 3.23% and 10.75%. This method exhibits high sensitivity, excellent selectivity, and strong reliability, providing a novel technique for the accurate quantification of MAL in vegetables, particularly suited for on-site rapid detection. Full article

The increasing impact of infectious, cardiovascular and neurodegenerative diseases has intensified the demand for early and decentralized diagnostics. Label-free electrochemical biosensors are promising candidates, offering high sensitivity, low reagent consumption and miniaturizable, low-cost architectures for point-of-care (PoC) testing. This review summarizes advances in immobilization strategies, recognition elements such as DNA, antibodies, aptamers, and molecularly imprinted polymers, as well as electrode platforms including glassy carbon, screen-printed, and 3D-printed systems, with an emphasis on DNA biosensors, multiplexed configurations, and applications to disease biomarkers. Beyond analytical performance, we critically examine the barriers that keep most devices at the proof-of-concept stage, including bioreceptor stability and immobilization, limited validation in real samples, reliance on conventional materials, challenges in scalable manufacturing, transport, and storage, and the absence of fully integrated PoC systems. Finally, we discuss significant advances in sensitivity, reproducibility, and application to real samples, but note that translation to real-world use and commercialization remains limited. Full article

Background/Objectives: Interleukin-17A (IL-17A) is a key pathogenic cytokine in autoimmune arthropathies. Current monoclonal antibody inhibitors targeting the IL-17/IL-17RA axis demonstrate clinical efficacy but face significant limitations, including immunogenicity, the loss of therapeutic response, and cold-chain storage. Our study evaluated oligonucleotide aptamers targeting IL-17A and its receptor as an alternative to monoclonal antibodies to suppress an IL-17A-induced inflammatory response in cell models relevant to immunoinflammatory rheumatic diseases. Methods: We examined three aptamers: 2′-F-RNA aptamers Apt21-2 and Apt3-4 specific to IL-17A and DNA aptamer RA10-6 targeting the receptor of IL-17A. Their ability to suppress IL-17A functional activity was assessed in peripheral blood mononuclear cells (PBMCs) from healthy donors and personalized fibroblast-like synoviocytes (FLSs) from patients with axial spondyloarthritis (axSpA) and rheumatoid arthritis (RA). Inhibition was measured by quantifying IL-6 and MMP-13 secretion using ELISA and flow cytometry, using secukinumab as a reference control. Results: In PBMC, all aptamers suppressed IL-17A-stimulated IL-6 secretion and cell proliferation in a concentration-dependent manner (17–200 nM), with a 65–85% efficacy, comparable to that of secukinumab. In axSpA-derived FLS, we observed time-dependent efficacy: At 4 h, all three aptamers suppressed IL-6 to the same extent as secukinumab; at 24 h, RA10-6 maintained high efficacy while Apt21-2 and Apt3-4 showed reduced activity. A combination of receptor-targeting RA10-6 with anti-IL-17A aptamers resulted in synergistic IL-6 suppression. All aptamers reduced MMP-13 to basal levels. RA-derived FLS showed diminished responses to all inhibitors. Conclusions: Aptamers demonstrate high specificity and sustained efficacy in suppressing IL-17A signaling for an in vitro model of spondyloarthritis, with superior performance over antibodies. Disease-dependent differential efficacy in RA FLS reflects heterogeneity consistent with limited clinical anti-IL-17 efficacy in RA. These findings show the strong potential of the studied aptamers as an alternative to monoclonal antibodies for IL-17-associated inflammatory arthropathies, particularly spondyloarthritis. Full article

Split G-quadruplex DNAzymes offer unique opportunities for building gated biosensors with a wide range of applications. Splitting G4 DNAzymes involves separating guanine tracts in the G-quadruplex DNA sequence into two non-functional sequences that reconstitute into a functional G-quadruplex with peroxidase activity upon hybridisation of the aptamer probe region within the split system with the target molecule. Several studies have demonstrated the reassembly of split G4 DNAzymes and their applications in the detection of various analytes. This approach offers unique opportunities for modular biosensor construction, target-dependent activation, lack of requirement for labelling, amplification-free high sensitivity, and specificity over traditional G4 sensing. In this review, we explore the strategies of splitting G-quadruplex and their applications in biomedical diagnosis, environmental sensing, food safety monitoring, cell detection, and the integration of the technology with nanomaterials for enhanced stability and sensitivity. We considered the classical intermolecular split strategies that utilise binary probes and intramolecular split systems, which integrate the spacer DNA that allow for single probes as the model G4 sequence. Finally, we explore the current challenges required to develop split G-quadruplex DNAzymes into tools for routine practical applications. Full article

This paper presents the optimization of surface modification for aptameric graphene nanosensors for the measurement of biomarkers in undiluted physiological media. In these sensors, graphene transduces the binding between an aptamer and the intended target biomarker into a measurable signal while being coated with a polyethylene glycol (PEG) nanolayer to minimize nonspecific adsorption of matrix molecules. We perform a systematic study of the aptamer and PEG attachment schemes and parameters, including the impact of the serial or parallel PEG–aptamer attachment scheme, PEG molecular weight and surface density, and aptamer surface density on the sensor behavior, such as the responsivity to biomarker concentration changes, and importantly, they are used for operation in physiological media and have the ability to reject nonspecific binding to interfering molecules. We then use the understanding from this parametric study to identify graphene nanosensor designs that are optimally functionalized with PEG and aptamers to be strongly responsive to target biomarkers and effectively reduce nonspecific adsorption of interferents, thereby enabling sensitive and specific biomarker measurements in undiluted physiological media. The experimental results show that nanosensors that were optimized via serial modification with 5000 Da PEG at 15 mM and a 94 nt DNA aptamer at 500 nM allowed specific measurement of C-reactive protein (CRP) in undiluted human serum with a limit of detection (LOD) down to 27 pM, representing an up to 1000-fold improvement compared to previously reported CRP measurements. Full article

The blood–brain barrier (BBB) restricts therapeutic delivery to the central nervous system (CNS), hindering the treatment of neurological disorders, such as Alzheimer’s disease, Parkinson’s disease, brain cancers, and stroke. Aptamers, short single-stranded DNA or RNA oligonucleotides that can fold into unique 3D shapes and bind to specific target molecules, offer high affinity and specificity, low immunogenicity, and promising BBB penetration via receptor-mediated transcytosis targeting receptors such as the transferrin receptor (TfR) and low-density lipoprotein receptor-related protein 1 (LRP1). This review examines aptamer design through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and its variants, mechanisms of BBB crossing, and applications in CNS disorders. Recent advances, including in silico optimization, in vivo SELEX, BBB chip-based MPS-SELEX, and nanoparticle–aptamer hybrids, have identified brain-penetrating aptamers and enhanced the brain delivery efficiency. This review highlights the potential of aptamers to transform CNS-targeted therapies. Full article

Food allergies represent a growing public health concern, requiring analytical methods capable of detecting trace levels of allergenic ingredients in increasingly complex and processed food matrices. In recent years, nucleic acid-based electrochemical sensors have emerged as a powerful alternative to protein-targeting assays, offering improved stability and sequence specificity, as well as compatibility with portable, low-cost sensing platforms. This review provides a comprehensive overview of nucleic acid-based sensing strategies developed for detecting either allergen proteins or nucleic acids related to allergenic species. Particular attention is given to the methodological approaches implemented, which for DNA detection include sandwich-type designs and DNA switches, while for protein detection rely on aptamer-based assays in a labelled or label-free setup. The review also discusses the impact of pre-analytical steps, such as nucleic acid extraction and PCR-based amplification, on assay reproducibility, cost and applicability at the point of need. Although significant improvements in analytical performance have been achieved, challenges remain in terms of simplifying workflows, standardizing methods, validating them on a large scale, and developing continuous monitoring schemes for timely intervention. The review highlights emerging opportunities, including multiplexed detection platforms, robust extraction protocols, and the harmonization of allergen thresholds, which are key to supporting the practical implementation of nucleic acid-based sensors. Full article

There is an increasing demand to design user-friendly specific assays for the detection of analytes of interest for healthcare, environment, and agrifood. Modern biotechnology has approached this problem by using proteins, enzymes, or RNA/DNA fragments (aptamers) as biological recognition elements for biosensors/assays. The idea is to exploit the extremely wide range of selective affinities sculpted into the various proteins or aptamers by biological evolution. The number of compounds specifically recognized by different proteins and aptamers is very large and ranges from small molecules to macromolecules. The advantages of using proteins and aptamers as molecular recognition elements (MRE) for assays/biosensors are many, and involve relatively low costs in design and synthesis, water solubility, and finally high specificity. Many of the analytes of interest in the food control industry are relatively small. In this case, aptamers and antibodies are widely used as specific MREs in designing advanced biosensors. Aflatoxin B1 (AFB1) is the most frequently found aflatoxin in contaminated food samples, and is one of the most potent natural compounds in terms of genotoxicity and carcinogenicity. Aflatoxin M1 (AFM1) is the hydroxylated metabolite of AFB1 and is usually found in milk and milk products as a carry-over of AFB1 in animals that have ingested contaminated feed. AFM1 is also found in human milk, and has been shown to be hepatotoxic and carcinogenic. Here, we present recent advances in assays and biosensors based on the use of antibodies and aptamers as MREs that have been developed for monitoring the presence of AFM1 in milk and dairy products. The limitations and advantages of aptamer- and antibody-based assays/biosensors are discussed, as well as future research perspectives. Full article

Excessive use of oxytetracycline (OTC) in veterinary medicine has increased the presence of antibiotics in food, which accelerates the development of antimicrobial resistance. We report the development of a highly sensitive electrochemical aptasensor for OTC detection, based on a glassy carbon electrode (GCE) modified with reduced graphene oxide (rGO) and multiwalled carbon nanotubes (MWCNTs) nanocomposite. DNA aptamers specific to OTC were covalently attached to the nanocomposite surface via carbodiimide chemistry. Differential pulse voltammetry (DPV) showed a decrease in peak current due to the binding of OTC to the aptamers. The sensor exhibited a limit of detection (LOD) of 1.72 ng/mL, which is below the maximum residue limit (MRL) for OTC (100 ng/mL) established by European Union. The sensor has been tested on a spiked milk sample. Full article