Search Results (52)

Tetrodotoxin (TTX) is a potent marine neurotoxin, necessitating sensitive and user-friendly on-site assays. To address long workflows of traditional immunoassays and limited signal amplification in colorimetric microfluidics, we developed a nanozyme-catalyzed colorimetric magnetic microfluidic immunosensor (Nano-CMI). This platform combines an aptamer–antibody sandwich capture format with catalytic amplification via AuNR@Pt@m-SiO₂ (APMS) nanozymes on a magnetically actuated microfluidic chip. Magnetic actuation simplifies sample handling and washing, while APMS catalysis enhances sensitivity and visual readout. The Nano-CMI has been used for the detection of TTX samples ranging from 0.2 to 20 ng/mL with a detection limit of 0.2 ng/mL in 10 min, following the linear equation: y = −31.14ln x + 110.15, and the entire “capture-reaction-detection” workflow can be completed within 1 h. With rapid response, minimal hands-on time, and robust performance, this platform offers a practical, high-sensitivity solution for on-site TTX screening in food safety and customs inspection. Full article

Fluorescence Anisotropy (FA) is a sensitive and efficient technique for quantifying biomolecular interactions, offering advantages such as minimal sample requirements and elimination of separation of bound from unbound species. Thus, it is well suited for aptamer–protein binding affinity studies. However, accurately determining equilibrium dissociation constants (K_D) in FA requires low concentrations of fluorescently labeled aptamers to prevent ligand depletion. A significant challenge arises at low aptamer concentrations due to an unexpected and physically nonmeaningful increase in apparent anisotropy, which impairs accurate data fitting. This anomalous increase in apparent anisotropy may arise from non-specific adsorption of aptamers to surfaces. In this study, we investigated the use of non-ionic surfactants to mitigate these effects and stabilize the anisotropy signal at low aptamer concentrations using the thrombin aptamer as a model system. We evaluated the impact of varying concentrations of two surfactants (Tween 20 and Triton X-100) on plots of anisotropy as a function of aptamer concentration and determined aptamer–protein binding affinities. Addition of 0.1% Tween 20 corrects the anomalous increase in anisotropy at low aptamer concentrations, enabling the use of aptamer concentrations as low as 5 nM in binding assays. Triton X-100 was less effective. By incorporating optimized concentrations of Tween 20, we demonstrated improved assay reproducibility and accuracy in K_D determination, expanding the dynamic range of usable aptamer concentrations in FA-based binding affinity studies. Similar benefits were observed with the clinically relevant aptamer s10yh2 and human serum albumin. These findings provide a practical strategy for enhancing the robustness of FA measurements and may be applicable to other aptamer–target systems and high-throughput assay formats. Full article

Human norovirus (HuNoV), particularly the GII.4 genotype, is a leading cause of acute gastroenteritis worldwide, posing a significant public health and economic burden due to its low infectious dose. To address the need for rapid and sensitive detection, we developed a colorimetric biosensor utilizing a structure-optimized aptamer and gold nanoparticles (AuNPs). Biotin-modified aptamers could protect AuNPs from aggregation in salt solution. Upon specific binding to GII.4 HuNoV virus-like particles (VLPs), this protective effect is disrupted, leading to AuNP aggregation and a measurable color shift quantified by the A620/A520 absorbance ratio. Under optimized conditions, the assay demonstrated a linear response (y = 0.004597x + 0.3277, R² = 0.9922) to GII.4 HuNoV VLP concentrations ranging from 0.1 to 3.0 μg/mL, with the recovery rates between 91.74% and 106.43%. The biosensor exhibited high specificity for GII.4 HuNoV, showing minimal cross-reactivity with other common diarrheal pathogens, and achieved an exceptional detection limit of 27.2 copies/mL in a fecal matrix. Molecular docking and point mutation confirmed the critical roles of specific nucleotide bases (T20, C22, G31, and G44) in the aptamer and the Asn55 residue in the viral capsid for binding. This work establishes a sensitive, rapid, and cost-effective aptamer-based colorimetric platform suitable for the large-scale monitoring of GII.4 HuNoV. Full article

In large-scale radiological events, there is a need to triage affected individuals based on their biological absorbed dose. Biodosimetry measures biological responses in relation to the received dose. Radiation-responsive protein biomarkers in peripheral blood lymphocytes, especially intracellular proteins, have been validated for biodosimetry with immunochemical-based measurement methods. However, these antibody-based assays can suffer from stability and batch-to-batch variations. Aptamers are single-stranded oligonucleotide alternatives to antibodies that are stable and much smaller in size, making them ideal probes for intracellular targets. However, few aptamers have been developed against intracellular targets, and these efforts are especially hampered due to the time-consuming nature of the conventional aptamer selection method. An efficient method for isolating aptamers against intracellular radiation-responsive proteins is not available yet. Herein, we used a microfluidic aptamer isolation method to develop an aptamer against the intracellular radiation biomarker BAX in blood lymphocytes. The isolated aptamer has a dissociation constant of 6.95 nM against human BAX protein and a bright detail similarity score of 1.9 when colocalizing with anti-BAX aptamer intracellularly. The in situ labeling of the intracellular BAX protein also shows the aptamer can be used to differentiate 2.5 Gy or 3 Gy of radiation in ex vivo human and in vivo mouse peripheral blood samples exposed to X-rays. In conclusion, this proof-of-concept study indicates that the microfluidic-enabled aptamer isolation method could be used for the development of a panel of targeted intracellular proteins for radiation biodosimetry applications. Full article

Background: Hepatocellular carcinoma is associated with high mortality and increasing incidence. Sorafenib, a cornerstone of therapy for advanced hepatocellular carcinoma, presents certain disadvantages, including low bioavailability and poor water solubility. This work describes a new strategy for sorafenib-targeted delivery aimed at improving treatment efficiency and reducing side effects. Methods: Magnetic nanoparticles coated with azelaic acid were modified with aptamer molecules that specifically recognize human liver cancer cell line HepG2, ensuring specificity for the tumor tissue. The nanoparticles were further loaded with sorafenib. The obtained drug delivery system was extensively characterized using UV-Vis spectrophotometry, transmission electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, and electrochemical impedance spectroscopy. Results: The drug delivery system demonstrated a higher release of sorafenib at acidic pH compared to pH 7.4. The cell internalization of the bare and aptamer-modified magnetic nanoparticles was assessed in HepG2 and human normal foreskin fibroblasts BJ cell lines, demonstrating that the aptamer significantly enhances internalization in tumor cells, while having no impact on healthy cells. Conclusions: The sorafenib-modified nanoparticles exhibited excellent cytocompatibility with BJ cells across all tested concentrations, while showing cytotoxicity towards HepG2 cells at higher concentrations, confirming the selectivity of the system. Full article

While databases are emerging across various domains, from small molecules to genomics and proteins, aptamer databases remain scarce, if not entirely absent. Such databases could serve as a comprehensive resource for advancing research, innovation, and the applications of aptamer technology across multiple fields. This advancement would likely lead to improvements in healthcare, environmental monitoring, and biotechnology. Furthermore, the establishment of aptamer databases would facilitate molecular modelling and machine learning, opening doors to further advancements in understanding and utilizing aptamers. Against this backdrop, in this study, we present and benchmark the Base Randomization Algorithm (BRA) as a potential solution to the scarcity of aptamer databases. Through statistical analysis, we examine key factors such as minimum free energy (MFE), base compositions, and base arrangements. Notably, sequences generated using the BRA exhibit a Gaussian distribution pattern. We also examine the details of how each base within a sequence is chosen using mathematical principles, ensuring that the sequences are valid and optimized statistically. Additionally, we explore how the length of the randomized generated sequences can affect the folding of their structures at both the secondary and tertiary levels. Based on composition analysis, we propose that the base mean of the dataset can be approximated as ${\overline{x}}_B\approx P\left(x\right) \times N$, for dataset of sequences with the same length and ${\overline{x}}_B\approx P\left(x\right) \times M,$ where $M$ is the median and $N$ the mean, for a dataset with randomized length that follows a Gaussian distribution. Full article

The WHO considers the Epidermal Growth Factor Receptor (EGFR) one of the key biomarkers of glioblastoma (GB). EGFR can be identified and targeted using molecular recognition elements (MoREs), like aptamers and aptamer–drug conjugates (ApDCs). Understanding the kinetics of anti-EGFR ApDC interactions with EGFR as well as the kinetics of their internalization into the cells is a crucial step for the further development of anti-EGFR ApDCs. For the first time, a novel approach was implemented to study real-time kinetics by measuring the cellular index (CI) using impedance (xCELLigence). Doxorubicin (DOX) was used as an indicator drug. Because DOX intercalates into the DNA double helix, aptamer–DOX non-covalent complexes were obtained. For the anti-EGFR DNA aptamer GR20, an additional duplex was constructed by synthesizing the extra region (GR20h) and via hybridization with the complementary oligonucleotide (h’) to form a duplex (hh’), thus creating the aptamer construct with complementary oligonucleotide (ACCO) GR20hh’. The original HPLC method quantified the assembly efficiency of an ACCO. The ACCO GR20hh’ retained affinity for the recombinant extracellular domain of EGFR, as measured using Biolayer Interferometry (BLI). According to cytofluorimetry, the ACCO GR20hh’ interacts with cells of continuous culture from GB patient (CCGBP) surgical samples. The DOX–ACCO GR20hh’ complexes are more efficiently internalized by EGFR+ cells lines A-431 and CCGBP 107 than DOX alone. Full article

In photoelectrochemical (PEC) biosensing, efficient electron-hole separation is crucial to obtain preferred photocurrent response and analytical performance; thus, constructing developed heterointerfaces with high carrier transfer efficiency is an effective method for sensitive evaluation of analytes. Herein, a 1D ZnIn₂S₄ nanosheet-decorated 2D In₂O₃ tube was developed to integrate with a prostate antigen (PSA)-sensitive aptamer for sensitive PSA antigen detection. 1D In₂O₃ tubes were first prepared by two-step hydrothermal and annealing methods, followed by the in-situ growth of ZnIn₂S₄ nanosheets. Morphology, optical properties, structure, and PEC performance of prepared In₂O₃-ZnIn₂S₄ were characterized by scanning electron microscopy, transmission electron microscopy, ultraviolet–visible spectrophotometry, X-ray diffraction, X-ray photoelectron spectroscopy, and an electrochemical workstation. Benefiting from the photoelectric effect and specific 1D/2D hierarchical structure, In₂O₃-ZnIn₂S₄ displayed enhanced optical absorption and photocarrier separation, thus a superior photoelectrochemical response. Proposed bioassay protocol possessed a linear range from 0.001 to 50 ng/mL and a detection limit at 0.00037 ng/mL. In addition, this biosensor exhibited satisfactory anti-interface ability and stability, which also could be extended to other quantitative platforms for detecting other proteins. Full article

We developed a sensor consisting of V₂O₅ nanorods and a reduced graphene oxide (rGO) nanocomposite (V₂O₅/rGO) with immobilized DNA aptamers (Apt-NH@V₂O₅/rGO) for the sensitive electrochemical detection of Hg (II). The V₂O₅ nanorods anchored on rGO nanosheets were synthesized using a hydrothermal method. The nanocomposite was analyzed by various powerful physical methods that include X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), field emission scanning electron microscopy (FE-SEM), Raman spectroscopy, the Brunauer–Emmett–Teller (BET) method, and Fourier transform infrared spectroscopy (FTIR). The FE-SEM of V₂O₅ disclosed the nanorod-like structure and uniform anchoring of V₂O₅ on the rGO nanosheet. Moreover, the BET results showed that the V₂O₅/rGO nanocomposite possesses excellent porosity. Furthermore, a glassy carbon electrode (GCE) was modified with Apt-NH@V₂O₅/rGO and used for the electrochemical detection of Hg(II) by differential pulse voltammetry (DPV). The aptasensor exhibited excellent sensitivity and selectivity toward Hg(II) detection, with a limit of detection (LOD) of 5.57 nM, which is below the maximum permissible limit established by WHO for rivers (30 nM). The sensor also exhibited significant stability and good repeatability. Full article

The binding interactions between okadaic acid (OA) aptamers and OA molecules are crucial for developing effective detection methods. This study aims to identify the recognition site and establish a reliable detection protocol through computational simulations and experimental validations. After determining the target sequence (OA-2), molecular docking simulations using Sybyl-X and H-dock were conducted to predict the binding affinity and interaction sites of OA aptamers with their targets. These predictions were subsequently validated through experiments based on the Förster resonance energy transfer (FRET) principle. The combined approach not only confirmed the computational predictions, identifying the “major region” as the recognition basis of OA-2, but also provided deeper insights into the binding mechanisms. Subsequently, a classical AuNPs-aptamer colorimetric detection method was established based on the OA-2 sequence and applied to the detection of real shellfish samples, achieving a limit of quantification (LOQ) of 5.0 μg kg⁻¹. The recoveries of OA in spiked samples ranged from 79.0% to 122.9%, with a relative standard deviation (RSD) of less than 14.7%. The results of this study contribute to the development of robust detection methods for OA aptamer–target interactions, enhancing the potential for practical applications in toxin detection and monitoring. Full article

CD44, a pivotal cell surface molecule, plays a crucial role in many cellular functions, including cell-cell interactions, adhesion, and migration. It serves as a receptor for hyaluronic acid and is involved in lymphocyte activation, recirculation, homing, and hematopoiesis. Moreover, CD44 is a commonly used cancer stem cell marker associated with tumor progression and metastasis. The development of CD44 aptamers that specifically target CD44 can be utilized to target CD44-positive cells, including cancer stem cells, and for drug delivery. Building on the primary sequences of our previously selected thioaptamers (TAs) and observed variations, we developed a bead-based X-aptamer (XA) library by conjugating drug-like ligands (X) to the 5-positions of certain uridines on a complete monothioate backbone. From this, we selected an XA with high affinity to the CD44 hyaluronic acid binding domain (HABD) from a large combinatorial X-aptamer library modified with N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (ADDA). This XA demonstrated an enhanced binding affinity for the CD44 protein up to 23-fold. The selected CD44 X-aptamers (both amine form and ADDA form) also showed enhanced binding affinity to CD44-overexpressing human ovarian cancer IGROV cells. Secondary structure predictions of CD44 using MFold identified several binding motifs and smaller constructs of various stem-loop regions. Among our identified binding motifs, X-aptamer motif 3 and motif 5 showed enhanced binding affinity to CD44-overexpressing human ovarian cancer IGROV cells with ADDA form, compared to the binding affinities with amine form and scrambled sequence. The effect of ADDA as a binding affinity enhancer was not uniform within the aptamer, highlighting the importance of optimal ligand positioning. The incorporation of ADDA not only broadened the XA’s chemical diversity but also increased the binding surface area, offering enhanced specificity. Therefore, the strategic use of site-directed modifications allows for fine-tuning aptamer properties and offers a flexible, generalizable framework for developing high-performance aptamers that target a wide range of molecules. Full article

Tandem SAM-II/SAM-V riboswitch belongs to a class of riboswitches found in the marine bacterium ‘Candidatus Pelagibacter ubique’. Previous studies have demonstrated that these riboswitches have the potential for digital modulation of gene expression at both the transcriptional and translational levels. In this study, we investigate the conformational changes in the tandem SAM-II/SAM-V riboswitch binding to S-adenosylmethionine (SAM) using selective 2′-hydroxyl acylation analyzed by the primer extension (SHAPE) assay, small-angle X-ray scattering (SAXS), and oligos depressing probing. Our findings reveal that the linker between SAM-II/SAM-V aptamers blocks the SAM response of the SAM-II domain. This result proposes a new mechanism for gene expression regulation, where the ligand-binding functions of tandem riboswitches can be selectively masked or released through a linker. Full article

In this paper, one of the great challenges faced by silicon-based biosensors is resolved using a biomaterial multilayer. Tiny biomolecules are deposited on silicon substrates, producing devices that have the ability to act as iridescent color sensors. The color is formed by a coating of uniform microstructures through the interference of light. The system exploits a flat, RNA-aptamer-coated silicon-based surface to which captured microbes are covalently attached. Silicon surfaces are encompassed with the layer-by-layer deposition of biomolecules, as characterized by atomic force microscopy and X-ray photoelectron spectroscopy. Furthermore, the results demonstrate an application of an RNA aptamer chip for sensing a specific bacterium. Interestingly, the detection limit for the microbe was observed to be 2 × 10⁶ CFUmL⁻¹ by visually observed color changes, which were confirmed further using UV-Vis reflectance spectrophotometry. In this report, a flexible method has been developed for the detection of the pathogen Sphingobium yanoikuyae, which is found in non-beverage alcohols. The optimized system is capable of detecting the specific target microbe. The simple concept of these iridescent color changes is mainly derived from the increase in thickness of the nano-ordered layers. Full article

The high affinity of the biotin–streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin–streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer–protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag–streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin–streptavidin interaction while increasing throughput and improving assay efficiency. Full article

Ketamine is one of the most commonly abused drugs globally, posing a severe risk to social stability and human health, not only it is being used for recreational purposes, but this tasteless, odourless, and colourless drug also facilitates sexual assaults when it is mixed with drinks. Ketamine abuse is a threat for safety, and this misuse is one of the main uses of the drug. The crucial role of ketamine detection is evident in its contributions to forensic investigations, law enforcement, drug control, workplace integrity, and public health. Electrochemical sensors have gained considerable interest among researchers due to their various advantages, such as low cost and specificity, and particularly screen-printed paper-based electrode (SPBE) biosensors have gained attention. Here, we reported an ePAD (electrochemical paper-based analytical device) for detecting the recreational drug ketamine. The advantages of using a paper-based electrode are that it reduces the electrode’s production costs and is disposable and environmentally friendly. At the same time, nanographite sheets (NGSs) assisted in amplifying the signals generated in the cyclic voltammetry system when ketamine was present. This ePAD was developed by immobilizing a ketamine aptamer on NGS electrodes. The characterization of proper synthesized NGSs was performed by Scanning Electron Microscopy (SEM), XRD (X-ray Diffraction), Fourier-transform infrared spectroscopy (FTIR), and UV-Vis spectroscopy. Electrochemical techniques, including cyclic voltammetry (CV) and linear sweep voltammetry (LSV), were employed to validate the results and confirm each attachment. Furthermore, the versatility of the proposed sensor was explored in both alcoholic and non-alcoholic beverages. The developed sensor showed a low LOD of about 0.01 μg/mL, and the linear range was between 0.01 and 5 μg/mL. This approach offers a valid diagnostic technique for onsite service with minimal resources. This cost effective and portable platform offers desirable characteristics like sensitivity and selectivity and can also be used for POC (point of care) testing to help in the quick identification of suspicious samples and for testing at trafficking sites, amusement parks, and by the side of the road. Full article