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Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins and poses a high risk to human health. Highly sensitive and rapid detection is one of the most effective preventive measures to avoid potential hazards. Herein, an electrochemical aptasensor based on DNA nanotetrahedron and zeolitic imidazolate framework-67 loading gold nanoparticles, horseradish peroxidase, and aptamers was designed for the ultrasensitive detection of AFB1. The high specific surface area and large pore volume of zeolitic imidazolate framework-67 can increase the loading capacity and further improve the detection sensitivity of electrochemical aptasensors. DNA nanotetrahedron can enhance the capture ability of AFB1 with steady immobilization. The developed aptasensor showed good analytical performance for AFB1 detection, with a detection limit of 3.9 pg mL -1 and a wide linear range of 0.01-100 ng mL -1 . The aptasensor detected AFB1 in corn samples with recovery rates ranging from 94.19%-105.77% and has potential for use in food safety monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The nanozyme-linked aptamer-sorbent assay (NLASA) is a rapid way to screen and characterize aptamer binding to targets. In this paper, a MnO 2 @AuNPs@aptamer (Apt) based NLASA coupled with colorimetric-SERS dual-mode for Staphylococcus aureus (S. aureus) detection is presented. Cu,Fe-CDs were used as the reducing agent to synthesize MnO 2 and gold nanoparticles (AuNPs). Then, they were fabricated to obtain MnO 2 @AuNPs with oxidase (OXD)-like and SERS activities. The S. aureus aptamer was conjugated to MnO 2 @AuNPs and enhanced the OXD-like activity, which realized the specific capture of S. aureus in food matrices. In addition, S. aureus improves the oxidation of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) but inhibits 3,3',5,5'-tetramethylbenzidine (TMB) to generate Raman-active oxTMB with MnO 2 @AuNPs@Apt. This sensor was used for detections of S. aureus in a concentration ranged from 10 1 to 10 7  CFU/mL with a detection limit of 0.926 CFU/mL (colorimetric) and 1.561 CFU/mL (SERS), and the recovery is 85%-105% in real samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

To effectively monitor multi-residues of penicillin antibiotics (PENs) in milk, we developed a novel ratiometric electrochemical aptasensor enabling simultaneous detection of PENs. The aptasensor employed a broad-spectrum aptamer as a recognition element, niobium carbide functionalized with methylene blue (Nb 2 C-MB) as a reference signal generator, and a ferrocene-labeled aptamer (Fc-Apt) as an output signal. Electrodes were modified with Fe-N-C doped carbon nanotubes (Fe-N-C-CNTs) to amplify detection signals further. During detection, Fc-Apt binding to PENs decreased Fc current intensity (I Fc ) and increased MB current intensity (I MB ). The simultaneous detection of PENs was achieved using I MB /I Fc as a quantitative signal. Under optimal conditions, a good linear relationship between I MB /I Fc and antibiotic concentration was observed, indicating the aptasensor had a robustness. The limits of detection of aptasensor for four penicillin antibiotics and their mixed targets were 0.093-0.191 nM. This work provides a new approach to multi-residue detection of the same class of antibiotics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The misuse of antibiotics may contaminate the environment and cause harm to human health. Therefore, rapid and accurate detection of antibiotics is essential. In this study, a novel electrochemiluminescence resonance energy transfer (ECL-RET) pair was designed using a new ECL emitter (CPM, Ce-TBAPy) as the donor and Co-MOF@AuPt as the acceptor. Moreover, a highly sensitive and specific "on-off-on" ECL aptasensor was constructed for detecting sulfadiazine (SDZ). The aptasensor exhibited a broad linear range (from 10.0 fg mL -1 to 100 ng mL -1 ) for the SDZ concentration, with limit of detection and limit of quantification values of 1.14 fg mL -1 and 3.75 fg mL -1 , respectively. The aptasensor achieved good results in spiking experiments with milk and egg samples, and successfully quantified SDZ in fish meal quality control sample. The prepared aptasensor presents great potential for food and environmental safety by detecting antibiotics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Sensitive and selective detection of trace aflatoxin B1 (AFB1) in foods is of great importance to guarantee food safety and quality but still challenging because of its trace amount and the interference from the complex food matrix. Here, we report the integration of aptamer (Apt) and an ordered 2D covalent organic framework (COF) to solid-state anodic aluminum oxide (AAO) nanochannels (Apt/COF/AAO) for selective and sensitive detection of trace AFB1. The high specificity of Apt for AFB1 led to a selective change in the surface charge of Apt/COF/AAO and in turn the current change of the nanochannel, permitting the selective and sensitive determination of trace AFB1 in complex food samples. The developed nanofluidic sensor gave a wide linear range (1-500 pg mL -1 ), low detection limit (0.11 pg mL -1 ), and good precision (relative standard deviation of 1.5% for 11 replicate determinations of 100 pg mL -1 ). In addition, the developed sensor was successfully used for the detection of AFB1 in food samples with the recovery of 86.9%-102.5%. The coupling of Apt-conjugated 2D COF with an AAO nanochannel provides a promising way for sensitive and selective determination of food contaminants in complex samples.

An aptamer is a single-stranded oligonucleotide that serves as a chemical antibody with a high specificity and binding affinity that can recognize a wide range of molecules. Effective modification and truncation of aptamers can enhance their binding affinities to particular targets while also broadening their application for uses, such as biosensors. However, a conventional trial-and-error methodology hinders this process. Herein, we demonstrate an in silico method to elucidate the binding site of single-stranded DNA aptamer specific to verrucarin A, a mycotoxin produced by molds in indoor buildings that causes adverse effects in living organisms. The novel ssDNA aptamer exhibited a binding affinity of 29.5 nM, demonstrating a relatively strong affinity compared to those of previously reported typical aptamers for small molecules. Furthermore, the selected aptamer was highly specific toward verrucarin A among structurally related mycotoxins (i.e., verrucarol and zearalenone). The specific binding site of the aptamer predicted via molecular dynamics and molecular docking simulations was highly consistent with the results observed via truncation, single base mutation, and circular dichroism experiments. The fluorescence assay revealed limits of detection and quantification of 4.1 and 12 nM for the aptamer, respectively. Comparing our developed aptasensor with LC-MS/MS methodology revealed that it could detect verrucarin A levels in phosphate-buffered saline and dust extracts with robust precision and consistency. Our findings provide insight for future studies exploring interaction mechanisms with intended targets and practical sensing applications, such as point-of-care detection of verrucarin A.

Monitoring biomarkers secreted by cardiomyocytes is critical to evaluate anticancer drug-induced myocardial injury (MI). Cardiac troponin I (cTnI) is considered the gold standard biomarker for MI. Herein, an electrochemical aptasensor is engineered for cTnI detection based on lanthanide europium metal-organic frameworks (Eu-MOFs) and a hybridization chain reaction-directed DNAzyme strategy. Three types of Eu-MOF morphologies were easily synthesized by changing the solvent, and the Eu-MOF modulated by mixing the solvent of dimethylformamide and H 2 O (D-Eu-MOF) exhibited the best performance compared to other morphologies of the Eu-MOFs. Multifunctional nanoprobes were constructed from D-Eu-MOF@Pt loaded with natural horseradish peroxidase and combined with an aptamer-initiated nuclear acid hybridization chain reaction to form G-quadruplex/hemin DNAzymes for signal amplification. A novel capture probe is constructed on the basis of DNA nanotetrahedrons modified on screen-printed gold electrodes to enhance the capture of the target and multifunctional nanoprobes for signal amplification. It exhibits a detection limit of 0.17 pg mL -1 and a linear range from 0.5 pg mL -1 to 15 ng mL -1 . The practicality of the platform is evaluated by measuring cTnI in real samples and secreted by cardiomyocytes after drug treatment, which provides great potential in drug-induced MI evaluation for clinical application.

One of the most harmful contaminants found in corn and its products is aflatoxin B1 (AFB1) and thus developing reliable detection methods is of great significance to consumers and the food industry. In this research, Au MBA @Ag nanoparticles (NPs) and AgNPs deposited on a silicon wafer (Si@AgNPs) were functionalized with an aptamer and its complementary strand, respectively, and self-assembled into a SERS aptasensor, which generated strong SERS signals. AFB1 bound to the aptamer prior to the complementary chain, causing Au MBA @Ag NPs to detach from Si@AgNPs. The complex dissociated, leading to a decrease in signal intensity from the solid-phase substrate. Under optimal conditions, the linear detection range was 0.05-20.0 ng mL -1 , and the detection limit was 0.039 ng mL -1 . Notably, the aptasensor demonstrated a recovery rate between 92.77% and 110.13% when utilized for the detection of AFB1 in corn flour and oil, indicating its good potential for detecting AFB1 in real sample matrices. In conclusion, a quantitative and reliable specific SERS detection system for AFB1 was developed in this study with significant applicability to food safety.

Bacterial infections have been seriously endangering public health and life, making it imperative to explore novel anti-infection strategies for their control. Herein, we constructed a DNA hydrogel encoded with aptamers (Apt-hydrogel) to inhibit Shiga toxin II (Stx2) toxicity, thereby alleviating Escherichia coli (EHEC) infection. The Apt-hydrogel was formed by two Y-shaped DNA scaffolds through rational design, where one end of Y was encoded with an aptamer sequence targeting the B subunit of Shiga toxin II (Stx2B). The Apt-hydrogel not only retained the high affinity of the aptamer but also provided protection for the aptamer, endowing it with better stability and biocompatibility. The results from in vitro and in vivo demonstrated good mediation effects of the Apt-hydrogel on Stx2 toxicity and confirmed its excellent inhibition activity. We hypothesized that the mechanism could be attributed to the high affinity of Apt-hydrogel for Stx2B, which effectively occupies the active site of Stx2B and its receptor Gb3. This interaction enhanced steric hindrance, thereby mediating their interaction and preventing Stx2 from entering the cell to exert toxicity. We anticipate that the novel Apt-hydrogel will expand the usage of aptamers and provide a new dimension for the Apt-hydrogel as a promising blocking assistant to inhibit Shiga toxin infections via a strong steric hindrance effect.

During a typical aptamer selection experiment, buffer molecules are used at the 10 to 50 mM range, whereas target molecules could be used at much lower concentrations even in low μM levels. Therefore, doubts existed regarding the potential enrichment of buffer binding aptamers, particularly for failed selections that cannot validate binding of enriched sequences. In this study, we used two common buffer molecules, Tris and HEPES, as target molecules. While we successfully isolated aptamers for Tris buffer, our attempts to generate aptamers for HEPES buffer failed. Thioflavin T (ThT) fluorescence spectroscopy showed the dissociation constant ( K d ) of the Tris buffer aptamer to be 2.9 mM, while isothermal titration calorimetry showed a K d of 43 μM. NMR spectroscopy also confirmed aptamer binding. Finally, we discussed the implications of this buffer selection work and recommended the use of certain buffers.

The incidence of primary hepatocellular carcinoma (HCC) has recently ranked fifth in the world, and the incidence rate is increasing year by year worldwide. Therefore, early diagnosis is the highest priority in the treatment of HCC. In this paper, four anti-HCC aptamers were obtained using magnetic bead SELEX technology. Among them, Apt-1 had the smallest K d value(5.9 nM) and the highest affinity. Flow cytometry results showed that the FITC-aptamers only specifically recognized HCC serum. Circular dichronism (CD) spectral characterization showed a positive peak near 275 nm and a negative peak near 250 nm for all aptamers, elucidating that the secondary structure formed by the candidate aptamers was a stem-loop B-DNA structure. In addition, molecular docking simulations showed that the binding of the HCC target to the candidate aptamer sequences was mainly dominated by hydrogen bonding. The results of the aptamer sensing performance analysis showed that under the optimized assay conditions, a linear relationship (ranging from 1 nM to 1 µM) was achieved, with a limit of detection (LOD) down to 0.75 nM and a LOQ of 2.32 nM. This was further validated in clinical samples, with a positive detection rate of more than 90%. Furthermore, aptamer-mediated in vivo delivery of luciferase mRNA showed that Apt-1-luciferase mRNA could be targeted to the liver and hepatic luciferase expression was significantly increased. These results demonstrate that the aptamer paves the way for clinical application, evidencing  significant potential to offer reference information for early diagnosis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Aptamers are valuable tools for applications such as cell imaging, drug delivery, and therapeutics. RNA aptamers, in particular, exhibit complex structural diversity and flexibility, affording higher affinity and specificity, broader target recognition, and easier chemical modification compared with DNA aptamers. However, traditional selection methods for RNA aptamers are time-consuming and involve numerous rounds of screening, thus limiting their widespread application. To overcome this challenge, we propose an efficient truncated selection approach termed ID-SELEX. This method incorporates a molecular identification marker whereby each template is labeled with a unique molecular identifier, or UMI. Such incorporation helps mitigate biases introduced by multiple polymerase chain reaction (PCR) amplification during high-throughput sequencing, ensuring accurate identification of aptamer candidates. Utilizing ID-SELEX, we successfully identified a panel of high-quality aptamers targeting the human colon cancer cell line HCT-8 in just 2 rounds of selection. Furthermore, we demonstrated the versatility of this strategy by selecting 6 RNA aptamers targeting mouse myoblast cell line C2C12 with only one round of selection. In summary, RNA aptamer selection based on ID-SELEX utilizes high-throughput sequencing and UMI labeling to enable the rapid screening of RNA aptamers across human and murine cell lines. As such, ID-SELEX has the potential to facilitate RNA aptamer discovery, providing a novel molecular tool for biomedical research, clinical applications, and precision medicine.

ATP and NAD + are small biomolecules that participate in a variety of physiological functions and are considered as potential biomarkers for disease diagnosis. In this study, we developed a ligation-dependent light-up aptamer transcriptional amplification assay for the sensitive and selective detection of ATP and NAD + . This assay relies on a specific DNA ligase that catalyzes the ligation of a nicked DNA template in the presence of a specific small molecule. We prepared a nicked template consisting of a duplex fragment with an overhang for the T7 promoter region and a single-stranded DNA with a complementary overhang sequence for the Broccoli aptamer. The nicked template was connected using a DNA ligase in the presence of a specific small molecule. The ligation product was subjected to in vitro transcription to amplify the light-up aptamer-mediated fluorescence signals. By integrating the target-dependent ligation and transcription amplification, significant signal amplification was achieved with 5.9 and 142 pM detection limits for ATP and NAD + , respectively. Moreover, good selectivity to discriminate between the target and its analogues was also realized. The application of this method to biological samples was evaluated using human serum and exhibited excellent recovery values.

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.

Effective targeting and delivery of large amounts of medications into the cancer cells enhance their therapeutic efficacy through saturation of cellular defensive mechanisms, which is the most privilege of nano drug delivery systems (NDDS) compared to traditional approaches. Herein, we designed dual-pH/redox responsive DTX-loaded poly (β-amino ester) (PBAS) micelles decorated with a chimeric peptide and TA1 aptamer. In vitro and in vivo results demonstrated that the designed nanoplatform possessed an undetectable nature in the blood circulation, but after exposure to the tumor microenvironment (TME) of 4T1 breast cancer, it suddenly changed into dual targeting nanoparticles (NPs) (containing two ligands, SRL-2 and TA1 aptamer). The dual targeting NPs destruction in the high GSH and low pH conditions of the cancer cells led to amplified DTX release (around 70% at 24 h). The IC50 value of DTX-loaded MMP-9 sensitive heptapeptide/TA1 aptamer-modified poly (β-amino ester) (MST@PBAS) micelles and free DTX after 48 h of exposure was determined to be 1.5 µg/ml and 7.5 µg/ml, respectively. The nano-formulated DTX exhibited cytotoxicity that was 5-fold stronger than free DTX (Pvalue˂0.001). Cell cycle assay test results showed that following exposure to MST@PBAS micelles, a considerable rise in the sub G1 population (48%) suggested that apoptosis by cell cycle arrest had occurred. DTX-loaded MST@PBAS micelles revealed significantly higher (Pvalue ˂ 0.001) levels of early apoptosis (59.8%) than free DTX (44.7%). Interestingly, in vitro uptake studies showed a significantly higher TME accumulation of dual targeted NPs (6-fold) compared to single targeted NPs (Pvalue < 0.001) which further confirmed by in vivo biodistribution and fluorescent TUNEL assay experiments. NPs treated groups demonstrated notable tumor growth inhibition in 4T1 tumor bearing Balb/c mice by only 1/10th of the DTX therapeutic dose (TD) as a drug model. In conclusion, cleverly designed nanostructures here demonstrated improved anticancer effects by enhancing tumor targeting, delivering chemotherapeutic agents more accurately, promoting drug release, reducing the therapeutic dosage, and lowering side effects of anticancer drugs., (© 2024. The Author(s).)

A sandwich-type electrochemical aptasensor for ultrasensitive detection of glypican-3 (GPC3) was constructed using GPC3 aptamer (GPC3 Apt ) labelled reduced graphene oxide-cerium oxide-gold nanoparticles (RGO-CeO 2 -Au NPs) as the signal probe and the same GPC3 Apt as the capture probe. The electrochemical redox properties of CeO 2 (Ce 3+ /Ce 4+ ) in the RGO-CeO 2 -Au NPs indicate the electrochemical signals. When the target GPC3 was present, an "aptamer-protein-aptamer" sandwich structure was formed on the sensing interface due to the specific binding between the protein and aptamers, resulting in an increased electrochemical redox signal detected by differential pulse voltammetry (DPV) technique. Under optimal conditions, the established aptasensor exhibited a logarithmic linear relationship between the current response and GPC3 concentration in the range 0.001-100.0 ng/mL, with a minimum detection limit of 0.74 pg/mL. Using the spike-recovery tests for measurement of the human serum samples, the recovery was from 99.26 to 114.01%, and the RSD range was 3.04 to 5.34%. Furthermore, the sandwich-type electrochemical aptasensor exhibited excellent performance characteristics such as good stability, high specificity, and high sensitivity, demonstrating effective detection of GPC3 in human serum samples and can be used as a clinical detection tool for GPC3., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The malachite green aptamer (MGapt) is known for its utility in RNA measurement in vivo and in lysate-based cell-free protein systems. However, MGapt fluorescence dynamics do not accurately reflect RNA concentration. Our study finds that MGapt fluorescence is unstable in commercial PURE systems. We discovered that the chemical composition of the cell-free reaction strongly influences MGapt fluorescence, which leads to inaccurate RNA calculations. Specific to the commercial system, we posit that MGapt fluorescence is significantly affected by the system's chemical properties, governed notably by the presence of dithiothreitol (DTT). We propose a model that, on average, accurately predicts MGapt measurement within a 10% margin, leveraging DTT concentration as a critical factor. This model sheds light on the complex dynamics of MGapt in cell-free systems and underscores the importance of considering environmental factors in RNA measurements using aptamers.

Small molecule-regulated RNA devices have the potential to modulate diverse aspects of cellular function, but the small molecules used to date have potential toxicities limiting their use in cells. Here we describe a method for creating drug-regulated RNA nanodevices (RNs) using acyclovir, a biologically compatible small molecule with minimal toxicity. Our modular approach involves a scaffold comprising a central F30 three-way junction, an integrated acyclovir aptamer on the input arm, and a variable effector-binding aptamer on the output arm. This design allows for the rapid engineering of acyclovir-regulated RNs, facilitating temporal, tunable, and reversible control of intracellular aptamers. We demonstrate the control of the Broccoli aptamer and the iron-responsive element (IRE) by acyclovir. Regulating the IRE with acyclovir enables precise control over iron-regulatory protein (IRP) sequestration, consequently promoting the inhibition of ferroptosis. Overall, the method described here provides a platform for transforming aptamers into acyclovir-controllable antagonists against physiologic target proteins., Competing Interests: Declaration of interests S.R.J. is the co-founder of Lucerna Technologies and has equity in this company. Lucerna has licensed technology related to Spinach, Broccoli, and other RNA-fluorophore complexes. T.H., J.L.L., and S.R.J. are authors of patents related to technology described in this manuscript. S.R.J. and J.L.L. are co-founders of Chimerna Therapeutics and have equity in this company., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Mushroom poisoning poses a significant global health concern, with high morbidity and mortality rates. The primary lethal toxins responsible for this condition are alpha-amanitin (ɑ-AMA) and beta-amanitin (β-AMA). As a promising bio-recognition molecules in biosensors, aptamers, have been broadly used in the field of food detection. However, the current SELEX-based methods for screening aptamers for structurally similar small molecules were limited by the labelling or salt ion induction. In this study, we aimed to develop a novel label-free SELEX strategy for the screening of aptamers with high affinity and constructed new aptasensors for the detection of ɑ-AMA and β-AMA., Results: A novel label-free SELEX strategy based on the positively charged gold nanoparticles (AuNPs) was proposed to simultaneous screening of aptamers for ɑ-AMA and β-AMA. Only 18 rounds of SELEX were required to obtain new aptamers. The candidate aptamers were analyzed by colloidal gold assay, and the sequences of ɑ-30 and β-37 displayed great affinity with Kd values of 22.26 nM and 23.32 nM, respectively, without interference from botanical toxins. Notably, the truncated aptamers ɑ-30-2 (50 bp) and β-37-2 (57 bp) exhibited higher affinity than their original counterpart (79 bp). Subsequently, the selected aptamers were utilized to construct recognition probes for electrochemical aptasensors based on hairpin cyclic cleavage of substrates by Cu 2+ dependent DNAzyme and Exo I-triggered recycling cascades. The detection platform showed excellent analytical performance with limits of detection as low as 4.57 pg/mL (ɑ-AMA) and 8.49 pg/mL (β-AMA). Moreover, the aptasensors exhibited superior performance in mushroom and urine samples., Significance: This work developed a simple and efficient label-free SELEX method for screening new aptamers for ɑ-AMA and β-AMA, which employed the positively charged AuNPs as the screening medium, without the need for chemical labelling of libraries or induction of salt ions. Furthermore, two novel electrochemical aptasensors were developed based on our newly obtained aptamers, which offer the new biosensing tool for ultrasensitive detection of the AMA poisoning, showing great potential in practical applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Biotoxins are pervasive in food and the environment, posing significant risk to human health. The most effective strategy to mitigate the risk arising from biotoxin exposure is through their specific and sensitive detection. Aptasensors have emerged as pivotal tools, leveraging aptamers as biorecognition elements to transduce the specificity of aptamer-target interactions into quantifiable signals for analytical applications, thereby facilitating the meticulous detection of biotoxins. When integrated with readily portable devices such as lateral flow assays (LFAs), personal glucose meters (PGMs), smartphones, and various meters measuring parameters like pH and pressure, aptasensors have significantly advanced the field of biotoxin monitoring. These commercially available devices enable precise, in situ, and real-time analysis, offering great potential for portable biotoxin detection in food and environmental matrices. This review highlights the recent progress in biotoxin monitoring using portable aptasensors, discussing both their potential applications and the challenges encountered. By addressing these impediments, we anticipate that a portable aptasensor-based detection system will open new avenues in biotoxin monitoring in the future.

The construction of far-red fluorescent molecular rotors (FMRs) is an imperative task for developing nucleic acid stains that have superior compatibility with cellular systems and complex matrices. A typical strategy relies on the methine extension of asymmetric cyanines, which unfortunately fails to produce sensitive rotor character. To break free from this paradigm, we have synthesized far-red hemicyanines using a dimethylamino thieno[3,2- b ]thiophene donor. The resultant probes, designated as ATh 2 Ind and ATh 2 Btz, possess excitation maxima (λ max ) of >600 nm and have been rigorously characterized by NMR, electrochemistry, and computational methods. The dyes possess alternating charge patterns like indodicarbocyanine (Cy5), but with twisted intramolecular charge transfer (TICT) rotational barriers at 60°, akin to the classical FMR thiazole orange (TO1). ATh 2 Btz also displays cyanine characteristics, enhancing its response upon binding to nucleic acids and allowing for efficient staining of cellular nuclei. When binding to the DNA aptamer for quinine (MN4), ATh 2 Btz exhibits a K d of 17 nM, a 660-fold light-up response, brightness (Φ fl x ε max ) of ∼37,000 M -1 cm -1 , and λ ex /λ em of 655/677 nm. The resulting far-red DNA-based MN4-ATh 2 Btz platform has been termed "pomegranate."

Treatment of lung cancer leptomeningeal carcinomatosis (LM) remains challenging partly due to the biological nature of the blood-brain barrier (BBB). Cisplatin has limited effects on LM, and it is notorious for neurotoxicity. Aptamers are small oligonucleotides considered as antibody surrogates. Here we report a DNA therapeutics, AptBCis1. AptBCis1 is a cisplatin-conjugated, BBB-penetrating, and cancer-targeting DNA aptamer. Its backbone, AptB1, was identified via in vivo SELEX using lung cancer LM orthotopic mouse models. The AptB1 binds to EAAT2, Nucleolin, and YB-1 proteins. Treatment with AptBCis1 1 mg/kg (equivalent to cisplatin 0.35 mg/kg) showed superior tumor suppressive effects compared to cisplatin 2 mg/kg in mice with lung cancer LM diseases. The cerebrospinal fluid platinum concentration in the AptBCis1 group was 10% of that in the cisplatin group. The data suggested the translational potential of AptBCis1 in lung cancer with LM and in cancers in which platinum-based chemotherapy remains as the standard of care.

This feature article delves into the realm of α-L-threose nucleic acid (TNA), an artificial nucleic acid analog characterized by a backbone comprising an unconventional four-carbon sugar, α-L-threose, with phosphodiester linkages connecting at the 2' and 3' vicinal positions of the sugar ring. Within this article, we encapsulate the potential, progress, current state of the art, and persisting challenges within TNA research. Kicking off with a historical overview of xeno nucleic acids (XNAs), the discussion transitions to the compelling attributes and structure-property relationships of TNAs as advanced tools when contrasted with natural nucleic acids. Noteworthy aspects such as their advantageous spatial arrangements of functional groups around the sugar ring, stable Watson-Crick base pairing, high binding affinity, biostability, biocompatibility, and in vivo bio-safety are highlighted. Moreover, the narrative unfolds the latest advancements in chemical and biological methodologies for TNA synthesis, spanning from monomer and oligomer synthesis to polymerization, alongside cutting-edge developments in enzyme engineering aimed at bolstering large-scale TNA synthesis for in vitro selection initiatives. The article sheds light on the evolution of TNA aptamers over time, expounding on the tools and selection techniques engineered to unearth superior binding aptamers and TNA catalysts. Furthermore, the article accentuates the recent applications of TNAs across diverse domains such as molecular detection, immunotherapy, gene therapy, synthetic biology, and molecular computing. In conclusion, we summarize the key aspects of recent TNA research, address persisting gaps and challenges, and provide crucial insights and future perspectives in the dynamic domain of TNA research.

This study reports the development of a photocatalytic electrochemical aptasensor for the purpose of detecting chloramphenicol (CAP) antibiotic residues in water by utilizing SYBR Green I (SG) and chemically exfoliated MoS 2 (ce-MoS 2 ) as synergistically signal-amplification platforms. The Au nanoparticles (AuNPs) were electrodeposited onto the surface of an indium tin oxide (ITO) electrode. After that, the thiolate-modified cDNA, also known as capture DNA, was combined with the aptamer. Subsequently, photosensitized SG molecules and ce-MoS 2 nanomaterial were inserted into the groove of the resultant double-stranded DNA (dsDNA). The activation of the photocatalytic process upon exposure to light resulted in the generation of singlet oxygen. The singlet oxygen effectively split the dsDNA, resulting in significant enhancement in the current of [Fe(CN) 6 ] 3-/4- . When the CAP was present, both SG molecules and ce-MoS 2 broke away from the dsDNA, which turned off the photosensitization response, leading to significant reduction in the current of [Fe(CN) 6 ] 3-/4- . Under the optimal conditions, the aptasensor exhibited a linear relationship between the current of [Fe(CN) 6 ] 3-/4- with logarithmic concentrations of CAP from 20 to 1000 nM, with a detection of limit (3σ) of 3.391 nM. The aptasensor also demonstrated good selectivity towards CAP in the presence of interfering antibiotics, such as tetracycline, streptomycin, levofloxacin, ciprofloxacin, and sulfadimethoxine. Additionally, the results obtained from the analysis of natural water samples using the proposed aptasensor were consistent with the findings acquired through the use of a liquid chromatograph-mass spectrometer. Therefore, with its simplicity and high selectivity, this aptasensor can potentially detect alternative antibiotics in environmental water samples by replacing the aptamers based on photosensitization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The development of dual-mode strategies with superior sensitivity and accuracy have garnered increasing attention for researchers in Aflatoxin B1 (AFB1) analysis. Herein, a colorimetric-electrochemiluminescence (ECL) dual-mode biosensor was constructed for onsite and ultrasensitive determination of AFB1. The multi-wall carbon nanotubes (MWCNTs) were integrated with the ZnO metal organic frameworks (MOFs) to accelerate the electron transfer and boost the ECL intensity of g-C 3 N 4 nanoemitters. Through the aptamer-based DNA sandwich assay, the CuO@CuPt nanocomposites were introduced onto the electrode and acted as the dual functional signal nanoprobes. Due to the good spectrum overlap between the CuO@CuPt nanoprobes and g-C 3 N 4 nanosheets, ECL signal could be efficiently quenched. Additionally, the CuO@CuPt nanoprobes show superior catalytic properties towards the TMB and H 2 O 2 colorimetric reactions, and an obvious color alteration from colorless to blue can be observed using the smartphone. Under optimized conditions, a sensitive and accurate dual-mode analysis of the AFB1 was accomplished with the colorimetric detection limit of 3.26 fg/mL and ECL detection limit of 0.971 fg/mL (S/N = 3). This study combines innovative nanomaterial properties of ZnO@MWCNTs, g-C 3 N 4 and CuO@CuPt for ultrasensitive dual-mode detection, which offers new opportunities for the innovative engineering of the dual-mode sensors and demonstrates significant potential in food safety analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A disposable dual-output biosensor to detect program death-ligand 1 (PD-L1) was developed for immunotherapy progress monitoring and early cancer detection in a single experimental setup. The aptamer probe was assembled on rGO composited with carboxylated terthiophene polymer (rGO-pTBA) to specifically capture PD-L1 protein labeled with a new redox mediator, ortho-amino phenol para sulphonic acid, for amperometric detection. Each sensing layer was characterized through electrochemical and surface analysis experiments, then confirmed the sensing performance. The calibration plots for the standard PD-L1 protein detection revealed two dynamic ranges of 0.5-100.0 pM and 100.0-500.0 pM, where the detection limit was 0.20 ± 0.001 pM (RSD ≤5.2%) by amperometry. The sensor reliability was evaluated by detecting A549 lung cancer cell-secreted PD-L1 and clinically relevant serum levels of soluble PD-L1 (sPD-L1) using both detection methods. In addition, therapeutic trials were studied through the quantification of sPD-L1 levels for a small cohort of lung cancer patients. A significantly higher level of sPD-L1 was observed for patients (221.6-240.4 pM) compared to healthy individuals (16.2-19.6 pM). After immunotherapy, the patients' PD-L1 level decreased to the range of 126.7-141.2 pM. The results indicated that therapy monitoring was successfully done using both the proposed methods. Additionally, based on a comparative study on immune checkpoint-related proteins, PD-L1 is a more effective biomarker than granzyme B and interferon-gamma., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To enhance the precision and reliability of early disease detection, especially in malignancies, an exhaustive investigation of multi-target biomarkers is essential. In this study, an advanced integrated electrochemical biosensor array that demonstrates exceptional performance was constructed. This biosensor was developed through a controllable porous-size mechanism and in-situ modification of carbon nanotubes (CNTs) to quantify multiplex biomarkers-specifically, C-reaction protein (CRP), carbohydrate antigen 125 (CA125), and carcinoembryonic antigen (CEA)-in human serum plasma. The fabrication process involved creating a highly ordered three-dimensional inverse-opal structure with the CNTs (pCNTs) modifier through microdroplet-based microfluidics, confined spatial self-assembly of nanoparticles, and chemical wet-etching. This innovative approach allowed for direct in-situ modification of nanomaterial onto the surface of electrode array, eliminating secondary transfer and providing exceptional control over structure and stability. The outstanding electrochemical performance was achieved through the synergistic effect of the pCNTs nanomaterial, aptamer, and horseradish peroxidase-labeled (HRP-) antibody. Additionally, the integrated biosensor array platform comprised multiple individually addressable electrode units (n = 11), enabling simultaneous multi-parallel/target testing, thereby ensuring accuracy and high throughput. Crucially, this integrated biosensor array accurately quantified multiplex biomarkers in human serum, yielding results comparable to commercial methods. This integrated technology holds promise for point-of-care testing (POCT) in early disease diagnosis and biological analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pancreatic cancer is one of the most malignant tumors with the highest mortality rates, and it currently lacks effective drugs. Aptamer-drug conjugates (ApDC), as a form of nucleic acid drug, show great potential in cancer therapy. However, the instability of nucleic acid-based drugs in vivo and the avascularity of pancreatic cancer with dense stroma have limited their application. Fortunately, VNP20009, a genetically modified strain of Salmonella typhimurium, which has a preference for anaerobic environments, but is toxic and lacks specificity, can potentially serve as a delivery vehicle for ApDC. Here, we propose a synergistic therapy approach that combines the penetrative capability of bacteria with the targeting and toxic effects of ApDC by conjugating ApDC to VNP20009 through straightforward, one-step click chemistry. With this strategy, bacteria specifically target pancreatic cancer through anaerobic chemotaxis and subsequently adhere to tumor cells driven by the aptamer's specific binding. Results indicate that this method prolongs the serum stability of ApDC up to 48 h and resulted in increased drug concentration at tumor sites compared to the free drugs group. Moreover, the aptamer's targeted binding to cancer cells tripled bacterial colonization at the tumor site, leading to increased death of tumor cells and T cell infiltration. Notably, by integrating chemotherapy and immunotherapy, the effectiveness of the treatment is significantly enhanced, showing consistent results across various animal models. Overall, this strategy takes advantage of bacteria and ApDC and thus presents an effective synergistic strategy for pancreatic cancer treatment., (© 2024. The Author(s).)

Studies have found that matrix metalloproteinase-9 (MMP-9) and interleukin-6 (IL-6) play an important role in tumorigenesis. In order to detect MMP-9 and IL-6 concentrations with high sensitivity and specificity, an efficient microfluidic-SERS sensing system was prepared based on surface-enhanced Raman scattering (SERS). The aptamer recognition-release mechanism and the dual signal amplification strategy were applied in the sensing system. The sensor system was developed using two kinds of nanomaterials with excellent SERS properties, namely gold-coated iron tetroxide particles (Fe 3 O 4 @AuNPs) and gold nanocages (AuNCs). In addition, Fe 3 O 4 @AuNPs also has magnetic adsorption properties. In the sensing system, single-stranded DNA1 (ssDNA1) and aptamer were modified on Fe 3 O 4 @AuNPs. Single-stranded DNA2 (ssDNA2) and Raman tags were modified on AuNCs. When the target was present, the aptamer bound to the target and detached from the Fe 3 O 4 @AuNPs, and ssDNA2 bound to the exposed ssDNA1. At this time, the Fe 3 O 4 @AuNPs@AuNCs@SERS tag complex was formed, and the SERS signal was enhanced for the first time. Under the action of an external magnet on the microfluidic chip, the complex was magnetized and enriched. The SERS signal was enhanced for the second time. Due to the high affinity between the aptamer and the target object, the sensing system has a strong specificity. The double amplification of the SERS signal gave the system excellent sensitivity. The limit of detection (LOD) relative to MMP-9 and IL-6 were as low as 0.178 pg/mL and 0.165 pg/mL, respectively. The microfluidic-SERS sensing system has a feasible prospect in the early screening of gastric cancer., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Riboswitches regulate downstream gene expression by binding to specific small molecules or ions with multiple mechanisms to transfer the binding information. In the case of the fluoride riboswitch, the transcription termination signal is conveyed through a transient excited state (ES). In this work, we performed conventional molecular dynamics (MD) simulations, totaling 180 μs, to obtain the ES structure and investigate the mechanism underlying information transmission in Mg 2+ /F - binding within the fluoride riboswitch aptamer. The Mg 2+ /F - binding pocket exhibits various conformations in its apo form. A series of ES structures were extracted from the MD trajectories of the apo form. The dynamics of the Mg 2+ /F - binding pocket influenced key pair A40-U48 in ES structures. The pathway connecting the binding pocket to the pair involves interactions between the phosphate groups of U7 and G8 and the nucleobases of G8-C47-U48. Our work presents a structural ensemble of the ES and elucidates a pathway for transferring Mg 2+ /F - binding information, thereby facilitating the understanding of how the holo-like apo state achieves transcriptional repression.

Nucleic acid aptamers possess unique advantages in specific recognition. However, the lack of in-depth investigation into their dynamic recognition mechanisms has restricted their rational design and potential applications in fields such as biosensing and targeted therapy. We herein utilized enhanced sampling molecular dynamics to address affinities of adenosine monophosphate (AMP) to the dual binding sites in the DNA aptamer, focusing on the dynamic recognition mechanism and pathways. The present results indicate that in addition to the widely known intermolecular interactions, inequivalence of chemical environments of the two binding sites leads to slightly higher stability of AMP binding to the site proximal to the aptamer terminus. In the presence of two AMPs captured by the two sites, each binding free energy is enhanced. In particular, an additional hydrogen bond of AMP to A10 is introduced in the dual-site binding complex, which increases the binding energy from -4.25 ± 0.47 to -9.48 ± 0.33 kcal mol -1 in the site close to the loop. For the dual-site recognition process, the free energy landscape and minimum free energy pathway calculations elucidate the crucial role of electrostatic interactions between the AMP phosphate groups and Na + ions in positively cooperative binding mechanisms.

The thrombin binding aptamer (TBA) is a prototypical platform used to understand the impact of chemically-modified nucleotides on aptamer stability and target affinity. To provide structural insight into the experimentally-observed effects of modification size, location, and number on aptamer performance, long time-scale molecular dynamics (MD) simulations were performed on multiple binding orientations of TBA-thrombin complexes that contain a large, flexible tryptophan thymine derivative (T-W) or a truncated analogue (T-K). Depending on modification position, T-W alters aptamer-target binding orientations, fine-tunes aptamer-target interactions, strengthens networks of nucleic acid-protein contacts, and/or induces target conformational changes to enhance binding. The proximity and 5'-to-3' directionality of nucleic acid structural motifs also play integral roles in the behavior of the modifications. Modification size can differentially influence target binding by promoting more than one aptamer-target binding pose. Multiple modifications can synergistically strengthen aptamer-target binding by generating novel nucleic acid-protein structural motifs that are unobtainable for single modifications. By studying a diverse set of modified aptamers, our work uncovers design principles that must be considered in the future development of aptamers containing chemically-modified nucleotides for applications in medicine and biotechnology, highlighting the value of computational studies in nucleic acids research., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In this research, we describe the first aptasensor for the detection of the Rift Valley Fever virus (RVFV). The process involved the selection of aptamers through the systematic evolution of ligands by the exponential enrichment (SELEX) technique. After 12 rounds of selection, 6 aptamers were selected and the corresponding binding affinities were assessed using fluorescence binding assays, revealing dissociation constants ranging from 15.45 to 40.98 nM. Notably, among the aptamers, RV2 and RV3 exhibited the highest binding affinities toward RVFV, with dissociation constants of 15.45 and 18.62 nM, respectively. Thiol-modified aptamers were subsequently immobilized onto screen-printed gold electrodes, facilitating the label-free detection of RVFV through square wave voltammetry. The voltammetric aptasensor demonstrated an excellent sensitivity, with a detection limit of 0.015 ng/mL. In addition, cross-reactivity assessments were conducted, where negligible response was obtained when the aptasensor was exposed to non-specific proteins., (© 2024. The Author(s).)

An alternative biomarker for assessing the cyanide levels in postmortem materials is crucial for investigating acute cyanide intoxication. Herein, an aptamer-ligand biorecognition system with high specificity was developed to detect acute cyanide poisoning via its secondary metabolite, 2-amino-2-thiazoline-4-carboxylic acid (ATCA). Potential aptamers were screened from a random library of 66-base single-stranded DNA using GO-SELEX, with individual aptamers being identified through single-stranded DNA sequencing. Molecular docking was employed to predict the affinity of these aptamers toward ATCA and selected counter-targets; these predictions were confirmed using thermodynamic analysis with an isothermal titration calorimeter. Owing to its label-free biomolecular binding interactions, Apt46 exhibited the highest affinity against ATCA and notable selectivity against structurally similar counter-targets. Thus, an amino-tagged Apt46 binding aptamer was attached to a carbon electrode modified with EDC-NHS-activated graphene oxide. The binding of Apt46 to ATCA was quantified by measuring current changes using differential pulse voltammetry. The aptasensor achieved a detection limit of 0.05 µg/mL and demonstrated suitability for detecting ATCA across various biological matrices, with the high recovery percentages ranging from 92.29 to 114.22%. Overall, the proposed ATCA aptasensor is promising for identifying ATCA metabolites in cases of acute cyanide exposure., (© 2024. The Author(s).)

By exploiting the programmability of DNA, dual-color DNA-templated silver nanoclusters have been synthesized to serve as a label-free fluorescent probe with a G5-linker at the 3' end. This advancement facilitates the modular design of universal FRET-based aptasensors using aptamers with a C5-linker at the 3' end for multiplexed detection, making them easily switch their applications.

Highly sensitive and specific imaging of ATP in living cells remains a challenge. Here, a novel aptasensor based endogenous enzyme-powered DNA walker for imaging ATP was proposed. The strategy leverages the highly expressed APE1 in tumor cells as the driving force of the DNA walker, achieving high sensitivity and superior imaging contrast. The method can detect ATP as low as 3.43 μM within 1 h. The approach can also effectively monitor intracellular ATP expression fluctuations and successfully differentiate between normal and cancer cells with high contrast.

Neuron-specific enolase (NSE), a tumor marker of small cell lung cancer (SCLC), has high application value in the early diagnosis of SCLC. In this study, a dual signal electrochemical aptasensor for NSE was constructed based on hemin/reduced graphene oxide/multi-walled carbon nanotube (H-rGO-MWCNT) nanocomposites. Hemin played a dual role, functioning not only as an in situ electrochemical probe but also exhibiting excellent peroxidase-like properties, effectively catalyzing the electroreduction of H 2 O 2 . Reduced graphene oxide and multi-walled carbon nanotubes exhibited excellent conductivity. Through their binding with hemin, the nanocomposites achieved a larger specific surface area, providing numerous active sites for capturing the NSE aptamer. In the presence of NSE, the specific adsorption between the antigen and the aptamer formed a stable antigen-aptamer structure, which inhibited the performance of hemin, resulting in the weakening of the electrochemical signals of hemin and H 2 O 2 . Leveraging these characteristics, the sensitive and cost-effective dual-signal electrochemical aptasensor has been fabricated for the detection of NSE. One signal corresponded to differential pulse voltammetry (DPV) of hemin, while the other signal was derived from chronoamperometry, capturing the catalytic reduction of H 2 O 2 . The linear ranges for NSE were 1 pg mL -1 to 1 μg mL -1 and 100 pg mL -1 to 100 ng mL -1 with the limit of detection (LOD) of 0.21 pg mL -1 and 11.22 pg mL -1 by DPV and chronoamperometry, respectively. In addition, this aptasensor exhibited good reproducibility, stability and specificity. The recovery of NSE in human blood serum samples was from 89% to 131%. It provided a promising strategy for the detection of NSE in clinical diagnostics.

Resonance energy transfer (RET) quenching is significantly important for developing electrochemiluminescence (ECL) sensors, but RET platforms face challenges like interference from other fluorescent substances and reliance on energy transfer efficiency. This study used Zn-PTC, formed by zinc ions coordinated with perylene-3,4,9,10-tetracarboxylate, as a dual-mechanism quencher to reduce the ECL intensity of carbon nitride nanosheets (Tg-CNNSs). Co 3 O 4 /NiCo 2 O 4 acts as a coreaction promoter, enhancing and stabilizing the luminescence of Tg-CNNSs. Zn-PTC absorbs energy from Tg-CNNSs, altering the fluorescence lifetime to confirm energy transfer, while energy-level matching demonstrates electron transfer. By leveraging both RET and electron transfer mechanisms, the designed ECL aptasensor significantly reduces signal fluctuations that may arise from a single mechanism, resulting in more stable and reliable detection outcomes. The ECL aptasensor designed for furanyl fentanyl (FUF) detection shows excellent performance with a detection limit of 5.7 × 10 -15 g/L, offering new pathways for detecting FUF and other small molecules.

High-throughput and label-free protein nanopore-based sensors are extensively used in DNA sequencing, single-protein analysis, molecular sensing and chemical catalysis with single channel recording. These technologies show great potential for identifying various harmful substances linked to public health by addressing the limitations of current portability and the speed of existing techniques. In this review, we provide an overview of the fundamental principles of nanopore sensing, with a focus on chemical modification and genetic engineering strategies aimed at enhancing the detection sensitivity and identification accuracy of protein nanopores. The engineered protein nanopores enable direct sensing, while the introduction of aptamers and substrates enables indirect sensing, translating the physical structure and chemical properties of analytes into readable signals. These scientific discoveries and engineering efforts have provided new prospects for detecting and monitoring trace hazardous substances.

Coagulation factor XIa (FXIa) is associated with a low risk of bleeding and has been identified as an effective and safe target for the development of novel anticoagulant drugs. In this study, we established an ultrasensitive competitive dual-enzyme cascade signal amplification method for the quantitative analysis and screening of FXIa inhibitors. Due to the specific recognition of FXIa's active site by the aptamer AptE40, the AptE40-QDs-EK recognition probe modified with enterokinase (EK) and the aptamer AptE40, was attached to the MNPs-FXIa capture probe. When FXIa inhibitor was present, it competed with AptE40 for binding to FXIa, resulting in the detachment of AptE40-QDs-EK from MNPs-FXIa. After magnetic separation, the enterokinase of AptE40-QDs-EK in the supernatant hydrolyzed N-terminal hexapeptide of trypsinogen, leading to the production of a large amount of trypsin as part of the first-stage signal cascade amplification. Next, trypsin could hydrolyze the hexameric arginine peptide (RRRRRR, R6), leading to the dissociation of RQDs from the R6-RQDs signal probe; this resulted in a dramatic increase in the fluorescence intensity of the supernatant as the second-stage signal cascade was amplified. The feasibility of the method was investigated using the FXIa inhibitor aptamer FELIAP as a positive model drug. Furthermore, the method was applied to screen the FXIa inhibitors in Eupolyphaga sinensis Walker. Two fractions with more active anticoagulated ingredients were successfully identified and validated via the conventional method, and the results were consistent. The established method provides a key technique for the sensitive detection, high-throughput analysis, and screening of the FXIa inhibitors.

Nowadays, continuous efforts have been devoted to designing stable and high-efficiency electrochemiluminescence (ECL) emitters as alternatives for tris(2,2'-bipyridine)-ruthenium(II) (Ru(bpy) 3 2+ ) in medical research. Herein, a novel ECL emitter was obtained by coordinating crystalline covalent triazinyl frameworks (cCTFs) with Ru 2+ (termed Ru-cCTFs), which exhibited strong ECL emission by the ligand to metal charge transfer (LMCT) route. After its integration with 4-mercaptopyridine (SH-Py), the resultant SH-Py-Ru-cCTFs achieved 2.3-fold enhancement in the ECL efficiency by employing Ru(bpy) 3 2+ as a standard, which involved a dynamic "intrarticular radical annihilation" ECL pathway. On such foundation, an automated ECL (A-ECL) aptasensor was constructed with an "on-off-on" model and magnetic separation upon linkage of the SH-Py-Ru-cCTFs with streptavidin (SA) magnetic beads (MBs). This automatic assay of miRNA-182 showed a wider linear range from 1.0 to 100.0 fM with a correlation coefficient ( R 2 ) of 0.994, a lower limit of detection (LOD) down to 0.28 fM, and faster operation within 41 min. Impressively, this bioassay facilely distinguished the stages of glioma disease from clinical blood samples with high accuracy. Hence, this research sheds light on how to develop advanced ECL luminophores and an automatic method, showing substantial insights into pathogenesis research of gliomas.

A novel sandwich-type electrochemical aptasensor based on supramolecularly immobilized affinity bioreceptor was prepared via host-guest interactions. This method utilizes an adamantane-modified, target-responsive hairpin DNA aptamer as a capture molecular receptor, along with a perthiolated β-cyclodextrin (CD) covalently attached to a gold-modified electrode surface as the transduction element. The proposed sensing strategy employed an enzyme-modified aptamer as the signalling element to develop a sandwich-type aptasensor for detecting prostate-specific antigen (PSA). To achieve this, screen-printed carbon electrodes (SPCEs) with electrodeposited reduced graphene oxide (RGO) and gold nanoferns (AuNFs) were modified with the CD derivative to subsequently anchor the adamantane-modified anti-PSA aptamer via supramolecular associations. The sensing mechanism involves the affinity recognition of PSA molecules on the aptamer-enriched electrode surface, followed by the binding of an anti-PSA aptamer-horseradish peroxidase complex as a labelling element. This sandwich-type arrangement produces an analytical signal upon the addition of H 2 O 2 and hydroquinone as enzyme substrates. The aptasensor successfully detected the biomarker within a concentration range of 0.5 ng/mL to 50 ng/mL, exhibiting high selectivity and a detection limit of 0.11 ng/mL in PBS.

Thermostable direct hemolysin (TDH) is a key virulence factor of Vibrio parahaemolyticus, capable of causing seafood-mediated outbreaks of gastroenteritis, posing a threat to the aquatic environment and global public health. In the present study, we explored a multivalent aptamer-mediated inhibition strategy to mitigate TDH toxicity. Based on the characteristic structure of TDH, a stable multivalent aptamer, Ap3-5, was rationally designed by truncation, key fragment evolution, and end fixation. Ap3-5 exhibited strong affinity (K d =39.24 nM), and thermal (T m =57.6 °C) and enzymatic stability. In silico studies also revealed that Ap3-5 occupied more active sites of TDH and covered its central pore, indicating its potential as a blocking agent for inhibiting TDH toxicity. In the hemolysis assay, Ap3-5 significantly suppressed the hemolytic effect of TDH. A cellular study revealed a substantial (∼80 %) reduction in TDH cytotoxicity. Supporting these findings, in vivo trials confirmed the inhibitory action of Ap3-5 on both the acute and intestinal toxicity of TDH. Overall, benefiting from the strong binding affinity, high stability, and multisite occupation of the multivalent aptamer with TDH, Ap3-5 displayed robust potential against TDH toxicity by inhibiting membrane pore formation, providing a new approach for alleviating bacterial infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Label-free nucleic acid fluorescent probes are gaining popularity due to their low cost and ease of application. However, the primary challenges associated with label-free fluorescent probes stem from their tendency to interact with other biomolecules, such as RNA, proteins, and enzymes, which results in low specificity. In this work, we have developed a simple detection platform that utilizes Fe 3 O 4 @PPy in combination with a label-free nucleic acid probe, 1,1,2,2-tetrakis[4-(2-bromo-ethoxy)phenyl]ethene (TTAPE) or Malachite Green (MG), for highly selective detection of metal ions, acetamiprid, and thrombin. Fe 3 O 4 @PPy not only adsorbs aptamers through electrostatic interactions, π-π bonding, and hydrogen bonding, but also quenches the fluorescence of the TTAPE/MG. Upon the addition of target compounds, the aptasensor separates from Fe 3 O 4 @PPy through magnetic separation. Moreover, by changing different aptamers, the aptasensor was applied to detect metal ions, acetamiprid, and thrombin, with the turned-on photoluminescence (PL) emission intensity recorded and showing linearity to the concentrations of targets. The robustness of method was demonstrated by applying it to real samples, which included vegetables (for detecting acetamiprid with LODs of 0.02 and 0.04 ng/L), serum samples (for detecting thrombin with LODs of 5.5 and 4.3 nM), and water samples (for detecting Pb 2+ with an LOD of 0.17 nM). Therefore, due to its impressive selectivity and sensitivity, the Fe 3 O 4 @PPy aptasensor could be utilized as a universal detection platform for various clinical and environmental applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Riboswitches are highly structured RNA regulators of gene expression. Although found in all three domains of life, they are particularly abundant and widespread in bacteria, including many human pathogens, thus making them an attractive target for antimicrobial development. Moreover, the functional versatility of riboswitches to recognize a myriad of ligands, including ions, amino acids, and diverse small-molecule metabolites, has enabled the generation of synthetic aptamers that have been used as molecular probes, sensors, and regulatory RNA devices. Generally speaking, a riboswitch consists of a ligand-sensing aptamer domain and an expression platform, whose genetic control is achieved through the formation of mutually exclusive secondary structures in a ligand-dependent manner. For most riboswitches, this involves formation of the aptamer's P1 helix and the regulation of its stability, whose competing structure turns gene expression ON/OFF at the level of transcription or translation. Structural knowledge of the conformational changes involving the P1 regulatory helix, therefore, is essential in understanding the structural basis for ligand-induced conformational switching. This review provides a summary of riboswitch cases for which ligand-free and ligand-bound structures have been determined. Comparative analyses of these structures illustrate the uniqueness of these riboswitches, not only in ligand sensing but also in the various structural mechanisms used to achieve the same end of regulating switch helix stability. In all cases, the ligand stabilizes the P1 helix primarily through coaxial stacking interactions that promote helical continuity.

Aptamer-based biosensors have been widely constructed and applied to detect diverse targets. Glutathione S-transferase (GST), a pivotal phase II metabolic enzyme, plays a critical role in biotransformation in vivo, and aberrant GST expression is associated with various health risks. Herein, aptamers targeting GST were systematically selected from a randomized single-stranded DNA (ssDNA) library of 79 nucleotides (nt) using a biotinylated GST-immobilized streptavidin agarose (SA) bead SELEX technology. Following rigorous screening across eight rounds, four aptamers with strikingly similar secondary structures emerged. Among these, Seq3 exhibited the highest affinity towards GST and was selected for further optimization. A semi-rational post-SELEX truncation strategy was then employed based on base composition analysis, secondary structure analysis and affinity assessment. This strategy enabled the systematic removal of redundant nucleotides in Seq3 without compromising its affinity, ultimately yielding a truncated aptamer, Seq3-3, which retains its specificity with a compact 39nt length. Building upon Seq3-3, a double-stranded fluorescent aptamer probe was ingeniously designed for the in vitro detection of GST. The detection mechanism hinges on the competitive displacement of the complementary chain from the probe, mediated by the target protein, leading to the separation of the antisense oligonucleotide from the double-stranded complex. This process triggers the restoration of the fluorescence signal, enabling sensitive detection, and the probe exhibits excellent response within a linear range of GST activity ranging from 0 to 1500 U/L. The results show that not only an efficient strategy for screening robust and practicable aptamers but also an ultrahighly sensitive detection platform for GST was established.

A colorimetric aptasensor based on the dual recognition of magnetic carbon dots (M-CDs) and copper-based metal-organic frameworks (Cu-MOFs) was constructed for the visual detection of Staphylococcus aureus ( S. aureus ). Aptamer-modified Cu-MOFs could specifically identify target bacteria and exhibited peroxidase-like activity by catalyzing the colorimetric reaction of the 3,3',5,5'-tetramethylbenzidine-H 2 O 2 (TMB-H 2 O 2 ) system. M-CDs electrostatically attracted bacteria because of their strong positive electrical properties and offered better capturing performance compared with magnetic beads owing to their smaller size. Visual detection of the target was achieved using the color change of the reaction catalyzed by the resuspension of the sandwich-structured complex. The bacteria were sensitively detected with a limit of detection (LOD) of 1 CFU mL -1 using the peroxidase catalytic activity of Cu-MOFs. Non-target bacteria produced negative results, demonstrating the excellent selectivity of the proposed aptasensor. The strategy showed good recovery in real sample detection, showing great potential for visual monitoring of pathogenic bacteria, and could provide a reference for further development of novel sensing platforms for bacterial detection.

Tumor-associated antigens (TAAs) are not exclusively expressed in cancer cells, inevitably causing the "on target, off tumor" effect of molecular recognition tools. To achieve precise recognition of cancer cells, by using protein tyrosine kinase 7 (PTK7) as a model TAA, a DNA molecular logic circuit Aisgc8 was rationally developed by arranging H + -binding i-motif, ATP-binding aptamer, and PTK7-targeting aptamer Sgc8c in a DNA sequence. Aisgc8 output the conformation of Sgc8c to recognize PTK7 on cells in a simulated tumor microenvironment characterized by weak acidity and abundant ATP, but not in a simulated physiological environment. Through in vitro and in vivo results, Aisgc8 demonstrated its ability to precisely recognize cancer cells and, as a result, displayed excellent performance in tumor imaging. Thus, our studies produced a simple and efficient strategy to construct DNA logic circuits, opening new possibilities to develop convenient and intelligent precision diagnostics by using DNA logic circuits.

Molecular recognition probes targeting cell surface proteins such as aptamers play crucial roles in precise diagnostics and therapy. However, the selection of aptamers against low-abundance proteins in situ on the cell surface, especially in scarce samples, remains an unmet challenge. In this study, we present a single-round, single-cell aptamer selection method by employing a digital DNA sequencing strategy, termed DiDS selection, to address this dilemma. This approach incorporates a molecular identification card for each DNA template, thereby mitigating biases introduced by multiple PCR amplifications and ensuring the accurate identification of aptamer candidates. Through DiDS selection, we successfully obtained a series of high-quality aptamers against cell lines, clinical specimens, and neurons. Subsequent analyses for target identification revealed that aptamers derived from DiDS selection exhibit recognition capabilities for proteins with varying abundance levels. In contrast, multiple rounds of selection resulted in the enrichment of only one aptamer targeting a high-abundance target. Moreover, the comprehensive profiling of cell surfaces at the single-cell level, utilizing an enriched aptamer pool, revealed unique molecular patterns for each cell line. This streamlined approach holds promise for the rapid generation of specific recognition molecules targeting cell surface proteins across a broad range of expression levels and expands its applications in cell profiling, specific probe identification, biomarker discovery, etc.

Background: Ultrasensitive detection is crucial for the early warning and intervention of risk factors, ultimately benefiting the environment and human health. Low levels of ochratoxin A (OTA) present a hidden yet significant threat, and rapid detection via high-performing biosensors is therefore essential., Results: A cascade isothermal amplification aptasensor (CIA-aptasensor) was designed for OTA detection. On the surface of a magnetic bead probe, the OTA level was converted into positively correlated trigger cDNA through its competitive binding with OTA-Apt. The released trigger cDNA activated catalytic hairpin assembly followed by coupling with a hybridization chain reaction to achieve CIA. After adding graphene oxide and SYBR Green I, the background interference was eliminated to specifically obtain OTA-related fluorescence. The ultrasensitive limit of detection was 0.22 pg mL -1 , an improvement of 1368-fold over conventional enzyme-linked aptamer sorbent assay by the same OTA-Apt, demonstrating satisfactory reliability and practicability. Thus, the CIA-aptasensor provides an enzyme- and label-free simplified homogeneous system with minimal background interference using isothermal conditions., Significance: This study provides a polymerase chain reaction-like approach for enhancing the sensitivity and performance of a biosensor, which could be extended for the application of CIA and label-free signaling strategy to other risk factors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)