Graphene-based materials are actively being investigated as sensing elements for the detection of different analytes. Both graphene grown by chemical vapor deposition (CVD) and graphene oxide (GO) produced by the modified Hummers\' method are actively used in the development of biosensors. The production costs of CVD graphene- and GO-based sensors are similar; however, the question remains regarding the most efficient graphene-based material for the construction of point-of-care diagnostic devices. To this end, in this work, we compare CVD graphene aptasensors with the aptasensors based on reduced GO (rGO) for their capabilities in the detection of NT-proBNP, which serves as the gold standard biomarker for heart failure. Both types of aptasensors were developed using commercial gold interdigitated electrodes (IDEs) with either CVD graphene or GO formed on top as a channel of liquid-gated field-effect transistor (FET), yielding GFET and rGO-FET sensors, respectively. The functional properties of the two types of aptasensors were compared. Both demonstrate good dynamic range from 10 fg/mL to 100 pg/mL. The limit of detection for NT-proBNP in artificial saliva was 100 fg/mL and 1 pg/mL for rGO-FET- and GFET-based aptasensors, respectively. While CVD GFET demonstrates less variations in parameters, higher sensitivity was demonstrated by the rGO-FET due to its higher roughness and larger bandgap. The demonstrated low cost and scalability of technology for both types of graphene-based aptasensors may be applicable for the development of different graphene-based biosensors for rapid, stable, on-site, and highly sensitive detection of diverse biochemical markers.

Target specificity, one of aptamer characteristics that determine recognition efficiency of biosensors, is generally considered to be an intrinsic property of aptamer. However, a high-affinity aptamer may have additional target binding specificity, little is known about the specificity of aptamer binding to multiple targets, which may result in false-positive results that hinder the accuracy of detection. Herein, an aptamer OBA3 with dual target ochratoxin A (OTA) and norfloxacin (NOR) was used as an example to explore the binding specificity mechanism and developed rapid fluorescent aptasensing methods. The nucleotide 15th T of aptamer OBA3 was demonstrated to be critical for specificity and affinity binding of target OTA via site-saturation mutagenesis. Substituting the 15th T base for C base could directly improve recognition specificity of aptamer for NOR and remove the binding affinity for OTA. The combination of π-π stacking interactions, salt bridges and hydrogen bonds between loop pocket of aptamer and quinolone skeleton, piperazinyl group may contributes to the fluoroquinolone antibiotics (NOR and difloxacin)-aptamer recognition interaction. Based on this understanding, a dual-aptamer fluorescent biosensor was fabricated for simultaneous detection of OTA and NOR, which has a linear detection range of 50-6000 nM with a detection limit of 31 nM for OTA and NOR. Combined with T15C biosensor for eliminating interference of OTA, the assay was applied to milk samples with satisfactory recovery (94.06-100.93%), which can achieve detection of OTA and NOR individually within 40 min.

As a potent computational methodology, molecular dynamics (MD) simulation provides advantageous knowledge about biological compounds from the molecular viewpoint. In particular, MD simulation gives exact information about aptamer strands, such as the short synthetic oligomers, their orientation, binding sites, folding-unfolding state, and conformational re-arrangement. Also, the effect of the different chemicals and biochemicals as the components of aptamer-based sensors (aptasensors) on the aptamer-target interaction can be investigated by MD simulation. Liquid crystals (LCs) as soft substances with characteristics of both solid anisotropy and liquid fluidity are new candidates for designing label-free aptasensors. To now, diverse aptasensors have been developed experimentally based on the optical anisotropy, fluidity, and long-range orientational order of LCs. Here, we represent a computational model of an LC-based aptasensor through a detailed MD simulation study. The different parameters are defined and studied to achieve a comprehensive understanding of the computational design of the LC-based aptasensor, including the density of LCs, their orientation angle, and lognormal distribution in the absence and presence of aptamer strands, both aptamer and target molecules with various concentrations, and interfering substance. As a case study, the tobramycin antibiotic is considered the target molecule for the computational model of the LC-based aptasensor.Communicated by Ramaswamy H. Sarma.

Neutrophils play a pivotal role in innate immunity by releasing ROS through respiratory bursts to neutralize various pathogenic factors. However, excessive ROS release can cause tissue damage. Adenosine is an endogenous anti-inflammatory molecule inhibiting respiratory burst to protect the host. Adenosine aptamers with antibody-like properties and good stability are expected to act as adenosine antagonists with functional modulation capability. This study compares the effects of adenosine and its aptamer on the respiratory bursts of salivary polymorphonuclear leukocytes and circulating polymorphonuclear leukocytes using a programmable stopped-flow injection approach, ensuring rapid and efficient analysis while maintaining the neutrophils\' viability. The results show that primed salivary polymorphonuclear leukocytes exhibit specificities that differ from circulating polymorphonuclear leukocytes. Adenosine aptamer can function as an inhibitory antagonist that distinguishes between physiologically controlled and excessive priming of neutrophils, showing potential application prospects in immunotherapy.

BACKGROUND: Scaffold (SCA) functionalization with aptamers (APT) provides adsorption of specific bioactive molecules on biomaterial surfaces. The aim of this study was to observe if SCA enriched with anti-fibronectin APT can favor coagulum (PhC) and osteoblasts (OSB) differentiation.
METHODS: 20 μg of APT was functionalized on SCA by simple adsorption. For PhC formation, SCAs were inserted into rat calvaria defects for 17 h. Following proper transportation (buffer solution PB), OSBs (UMR-106 lineage) were seeded over PhC + SCAs with and without APT. Cells and PhC morphology, PhC cell population, protein labeling and gene expression were observed in different time points.
RESULTS: The APT induced higher alkaline phosphatase and bone sialoprotein immunolabeling in OSB. Mesenchymal stem cells, leukocytes and lymphocytes cells were detected more in the APT group than when scaffolds were not functionalized. Additionally, an enriched and dense fibrin network and different cell types were observed, with more OSB and white blood cells in PhC formed on SCA with APT. The gene expression showed higher transforming growth factor beta 1 (TGF-b1) detection in SCA with APT.
CONCLUSIONS: The SCA functionalization with fibronectin aptamers may alter key morphological and functional features of blood clot formation, and provides a selective expression of proteins related to osteo differentiation. Additionally, aptamers increase TGF-b1 gene expression, which is highly associated with improvements in regenerative therapies.

The development of highly sensitive diagnostic systems for the early revelation of diseases in humans is one of the most important tasks of modern biomedical research, and the detection of the core antigen of the hepatitis C virus (HCVcoreAg)-a protein marker of the hepatitis C virus-is just the case. Our study is aimed at testing the performance of the nanoribbon biosensor in the case of the use of two different types of molecular probes: the antibodies and the aptamers against HCVcoreAg. The nanoribbon sensor chips employed are based on \"silicon-on-insulator structures\" (SOI-NR). Two different HCVcoreAg preparations are tested: recombinant β-galactosidase-conjugated HCVcoreAg (\"Virogen\", Watertown, MA, USA) and recombinant HCVcoreAg (\"Vector-Best\", Novosibirsk, Russia). Upon the detection of either type of antigen preparation, the lowest concentration of the antigen detectable in buffer with pH 5.1 was found to be approximately equal, amounting to ~10-15 M. This value was similar upon the use of either type of molecular probes.

Molecular interactions play a vital role in regulating various physiological and biochemical processes in vivo. Kinetic capillary electrophoresis (KCE) is an analytical platform that offers significant advantages in studying the thermodynamic and kinetic parameters of molecular interactions. It enables the simultaneous analysis of these parameters within an interaction pattern and facilitates the screening of binding ligands with predetermined kinetic parameters. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was the first proposed KCE method, and it has found widespread use in studying molecular interactions involving proteins/aptamers, proteins/small molecules, and peptides/small molecules. The successful applications of NECEEM have demonstrated its promising potential for further development and broader application. However, there has been a dearth of recent reviews on NECEEM. To address this gap, our study provides a comprehensive description of NECEEM, encompassing its origins, development, and applications from 2015 to 2022. The primary focus of the applications section is on aptamer selection and screening of small-molecule ligands. Furthermore, we discuss important considerations in NECEEM experimental design, such as buffer suitability, detector selection, and protein adsorption. By offering this thorough review, we aim to contribute to the understanding, advancement, and wider utilization of NECEEM as a valuable tool for studying molecular interactions and facilitating the identification of potential ligands and targets.

ssDNA aptamers have been increasingly used to detect heavy metal ions as recognition elements due to their high affinity and specificity. However, the specific recognition and binding mechanisms between aptamers and most heavy metals were still unclear, which limits the development of aptamer-based detection methods. In this work, the interaction mechanisms of CD-2-1 aptamers with Cd2+ in aqueous solutions were investigated using molecular dynamic simulations. The most stable complex was found where Cd2+ binding at aptamer\'s stem-loop junction and preferred at the phosphate backbone or bases. Noteworthily, two binding modes of Cd2+ combining aptamer in aqueous solution were discovered: direct and indirect. In the former mode, Cd2+ directly coordinated O atoms of bases. For the latter, Cd2+ connected to bases with coordinated water molecules as bridges. Electrostatic interaction was found to be the main driving force, and differences of residues role between two binding modes were elucidated. Buffer molecules in aqueous solutions can stabilize aptamer-Cd2+ complex by hydrogen bonds. This study revealed the specific interaction mechanisms of aptamer with Cd2+ at an atomic level, which provided theoretical references for aptamer-based Cd2+ detection methods establishment as well as an efficient technical route of screening potential aptamers for heavy metal ions.

In this study, a novel electrochemical aptamer sensor for detecting ochratoxin A (OTA) was constructed. First, a gold-copper alloy film was prepared via electrochemical deposition, and copper was selectively dissolved in constant potential mode for obtaining the nano-porous gold modified screen-printed carbon electrodes (NPG/SPCE). Then, 2-mercaptoethylamine was dropped on the NPG/SPCE surface and Au-S covalent bonds were formed for immobilizing the metal. Glutaraldehyde as cross-linking agent was added, which resulted in immobilization and attachment of PAMAM to the 2-mercaptoethylamine through the dehydration condensation reaction. During the preparation process, the nano-porous gold and PAMAM-modified layers were characterized by SEM, XRD, and IR spectroscopy, respectively. The characterization results showed that the nano-porous gold and PAMAM composite films were successfully modified. Finally, the OTA aptamer was cross-linked with PAMAM by glutaraldehyde to complete construction of the Apt/PAMAM/NPG/SPCE sensor. The electrochemical performance of this sensor was tested in ochratoxin A solutions with the DPV method. The results showed that the sensor\'s reproducibility, stability, and specificity were good. The spiked recoveries in red wine ranged from 99.65%∼101.6%, with a linear range of 0.5 ng/mL∼20 ng/mL and a minimum detection limit of 0.141 ng/mL. Thus, the novel biosensor may provide a promising tool for the trace detection of OTA.

Molecular docking (MD) analysis is currently the most commonly used theoretical simulation method to investigate the interaction of aptamers (receptors) and small molecules (ligands) and understand the recognition mechanism between them at a molecular level. Using the specific aptamers of tetracycline antibiotics (tetracycline (TET), oxytetracycline (OTC), doxycycline (DOC)) as the docking models, three steady-state aptamers of tertiary structures (SATS) were established for each aptamer with the UNAFold and RNAComposer tools. The binding free energy (BFE), docking score (DS), and binding site (base) of the specific ligands (TET, OTC, and DOC) with their respective SATS were obtained by molecular docking. The results revealed one or more binding sites in the established SATS of the aptamers. The BFE and DS of different binding sites of one specific SATS varied significantly. The results also revealed that the site with the highest BFE represented the most dominant binding site, even if it was not the SATS with minimum energy. The BFE values could also be used to evaluate the affinity and specificity of the aptamer to its target. For the first time, this study proposes a method for MD analysis of the aptamer and its target based on different SATS, clarification of the binding mode, and prediction of the binding sites (bases). This study provides a theoretical basis for tailoring; structural optimization; and base modification of aptamers; identifying aptamers with high affinity and specificity.