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Here, a separation‐free and label‐free portable aptasensor is developed for rapid and sensitive analysis of tumor‐derived exosomes (TEXs). It integrated a parallel rolling circle amplification (RCA) reaction, selective binding of metal ions or small molecules to nucleic acid‐specific conformations, and a low‐cost, highly sensitive handheld fluorometer. Lung cancer, for example, is targeted with two typical biomarkers (mucin 1 and programmed cell death ligand 1 (PD‐L1)) on its exosomes. The affinity of aptamers to the targets modulated the amount of RCA products (T‐Hg2+‐T and cytosine (C)‐rich single‐stranded DNA), which in turn affected the fluorescence intensity of quantum dots (QDs) and methylene blue (MB). The results revealed that the limit of detection (LOD) of the handheld fluorometer for cell‐derived exosomes can be as low as 30 particles mL−1. Moreover, its specificity, sensitivity, and area under the curve (AUC) are 93% (14/15), 92% (23/25), and 0.956, as determined by the analysis of 40 clinical samples. Retesting 16 of these samples with the handheld fluorometer yielded strong concordance between the fluorometer results and those acquired from clinical computed tomography (CT) and pathology. [ABSTRACT FROM AUTHOR]

Overexpression of epidermal growth factor receptor (EGFR) and its mutations influence signal pathways leading to the proliferation, invasion, and increased survival of tumor cells. Despite the successful clinical application of antibodies against EGFR in patients with colorectal cancer and squamous cell carcinoma of the head and neck, these have been found to have low efficacy in glioblastoma. The treatment of gliomas therefore requires an EGFR-specific drug able to penetrate the tumor focus in the brain and which has low immunogenicity. We report here studies of aptamers (single-stranded DNA oligonucleotides) specific to EGFR, U2, and Gol1 and discuss their use as agents of this type. The work reported here includes preparation of a cell model of human glioma with overexpression of EGFR and EGFRvIII and its use to demonstrate the specificity of aptamers U2 and Gol1 to these receptors using classical methods and by apta-immunocytochemistry. Investigation of the effect of binding of aptamer Gol1 to the EGFRvIII receptor on subsequent steps in the signal pathway demonstrated changes in the levels of expression of genes associated with cell proliferation and survival [Jun, Fos, CCND1, PI3K and Akt3], while aptamer U2 did not produce any significant effect on cells in vitro. These results indicate that Gol1 aptamer has therapeutic potential against human glioblastoma tumor cells overexpressing the mutant EGFRvIII receptor. [ABSTRACT FROM AUTHOR]

The degradation of low-density lipoprotein receptor (LDLR) is induced by proprotein convertase subtilisin/kexin type 9 (PCSK9), resulting in elevated plasma concentrations of LDL cholesterol. Therefore, inhibiting the interactions between PCSK9 and LDLR is a desirable therapeutic goal for managing hypercholesterolemia. Aptamers, which are RNA or single-stranded DNA sequences, can recognize their targets based on their secondary structure. Aptamers exhibit high selectivity and affinity for binding to target molecules. The systematic evolution of ligands by exponential enrichment (SELEX), a combination of biological approaches, is used to screen most aptamers in vitro. Due to their unique advantages, aptamers have garnered significant interest since their discovery and have found extensive applications in various fields. Aptamers have been increasingly utilized in the development of biosensors for sensitive detection of pathogens, analytes, toxins, drug residues, and malignant cells. Furthermore, similar to monoclonal antibodies, aptamers can serve as therapeutic tools. Unlike certain protein therapeutics, aptamers do not elicit antibody responses, and their modified sugars at the 2'-positions generally prevent toll-like receptor-mediated innate immune responses. The focus of this review is on aptamer-based targeting of PCSK9 and the application of aptamers both as biosensors and therapeutic agents. [ABSTRACT FROM AUTHOR]

Nitrofuran (NF) contamination in food products is a global problem resulting in the banned utilization and importation of nitrofuran contaminated products. A novel chromogenic detection method using a specific DNA aptamer with high affinity and specificity to nitrofurans was developed. Single-stranded DNA aptamers specific to nitrofuran metabolites, including 3-amino-2-oxazolidinone (AOZ), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), were isolated using magnetic bead-SELEX. The colorimetric detection of nitrofurans using gold nanoparticles (AuNPs) exhibited an AOZ detection range of 0.01–0.06 ppb with a limit of detection (LOD) of 0.03 ppb. At the same time, this system could detect AMOZ and AHD at a range of 0.06 ppb and 10 ppb, respectively. The fast nitrofuran extraction method was optimized for food, such as fish tissues and honey, adjusted to be completed within 3–6 h. This novel apta-chromogenic detection method could detect NF metabolites with a sensitivity below the minimum required performance limit (MPRL). This analysis will be valuable for screening, with a shortened time of detection for aquaculture products such as shrimp and fish muscle tissues. [ABSTRACT FROM AUTHOR]

Blood is one of the most commonly found biological fluids at crime scenes, with the detection and identification of blood holding a high degree of evidential value. It can provide not only information about the nature of the crime but can also lead to identification via DNA profiling. Presumptive tests for blood are usually sensitive but not specific, so small amounts of the substrate can be detected, but false-positive results are often encountered, which can be misleading. Novel methods for the detection of red blood cells based on aptamer–target interactions may be able to overcome these issues. Aptamers are single-stranded DNA or RNA sequences capable of undergoing selective antigen association due to three-dimensional structure formation. The use of aptamers as a target-specific moiety poses several advantages and has the potential to replace antibodies within immunoassays. Aptamers are cheaper to produce, display no batch-to-batch variation and can allow for a wide range of chemical modifications. They can help limit cross-reactivity, which is a hindrance to current forensic testing methods. Within this study, a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process was used to generate aptamers against whole red blood cells. Obtained aptamer pools were analysed via massively parallel sequencing to identify viable sequences that demonstrate a high affinity for the target. Using bioinformatics platforms, aptamer candidates were identified via their enrichment profiles. Binding characterisation was also conducted on two selected aptamer candidates via fluorescent microscopy and qPCR to visualise and quantify aptamer binding. The potential for these aptamers is broad as they can be utilised within a range of bioassays for not only forensic applications but also other analytical science and medical applications. Potential future work includes the incorporation of developed aptamers into a biosensing platform that can be used at crime scenes for the real-time detection of human blood. [ABSTRACT FROM AUTHOR]

Leucomalachite green (LMG), a derivative antimicrobial agent, can accumulate in animal tissues for long periods and is categorized as a carcinogen affecting consumer health. Therefore, determining the contamination of aquatic products by LMG is necessary to prevent residual contamination. In the work described herein, the detection of LMG based on a colorimetric method using gold nanoparticles (AuNPs) was developed. The specificity of isolated single-stranded DNA aptamers to LMG was proven by using LMG and other antibiotics. The binding affinity of the LMG-aptamer showed a Kd value of 1.94 pM. Furthermore, a colorimetric detection module was successfully developed, showing a limit of detection (LOD) value of LMG at 250 nM using standard LMG substance. Taken together, this research demonstrates a rapid and sensitive method to detect chemical residues, reducing the possibility of contamination in fishery products. Considering these findings, the aptamer-based colorimetric approach might be feasible to utilize as a biosensing module, providing a rapid, inexpensive analysis method for detecting deposited chemical residues. [ABSTRACT FROM AUTHOR]

An electrochemical biosensor is reported for controlling CRISPR/Cas12a activity through the utilization of entropy-driven reactions, alongside the construction of a highly sensitive biosensor for B-type natriuretic peptide (BNP) detection. In the biosensor, entropy-driven reactions are employed to regulate the activity of CRISPR/Cas12a - a gene editing tool - capable of nonspecific cleavage of single-stranded DNA (ssDNA). The biosensor architecture encompasses an electrode that is modified with ssDNA probes designed to hybridize with target BNP aptamers. These aptamers, furnished with labeled ssDNA triggers, facilitate the activation of CRISPR/Cas12a through interaction with its guide RNA. Upon the presence of BNP, it associates with the aptamers, subsequently liberating the triggers that instigate the entropy-driven reactions. As a consequence of these reactions, more stable duplexes emerge between the triggers and guide RNA, thereby activating CRISPR/Cas12a. The activated CRISPR/Cas12a subsequently executes cleavage of ssDNA probes residing on the electrode surface, culminating in the generation of an electrochemical signal directly (the calibration plots of differential pulse voltammetric detection were acquired at a working potential of 0.2 V (vs. ref. electrode)) proportional to the BNP concentration. Validation of the biosensor's performance is undertaken, wherein BNP detection is demonstrated in both buffer and human serum samples. Evident in the findings is the biosensor's discernible sensitivity and specificity for BNP detection, exemplified by a detection limit of 13.53 fM and a lack of interference originating from other cardiac biomarkers, respectively. Furthermore, the biosensor's potential to discriminate between healthy individuals and those afflicted by heart failure, predicated on distinctive BNP levels, is illustrated. [ABSTRACT FROM AUTHOR]

To date, numerous aptamer-based biosensing platforms have been developed for sensitive and selective monitoring of target analytes, relying on analyte-induced conformational changes in the aptamer for the quantification of the analyte and the conversion of the binding event into a measurable signal. Despite the impact of these conformational rearrangements on sensor performance, the influence of the environment on the structural conformations of aptamers has rarely been investigated, so the link between parameters directly influencing aptamer folding and the ability of the aptamer to bind to the target analyte remains elusive. Herein, the effect a number of variables have on an aptamer's 3D structure was examined, including the pH of the buffering medium, as well as the anchoring of the aptamer on a solid support, with the use of two label-free techniques. Circular dichroism spectroscopy was utilized to study the conformation of an aptamer in solution along with any changes induced to it by the environment (analyte binding, pH, composition and ionic strength of the buffer solution), while quartz crystal microbalance with dissipation monitoring was employed to investigate the surface-bound aptamer's behavior and performance. Analysis was performed on an aptamer against oxytetracycline, serving as a model system, representative of aptamers selected against small molecule analytes. The obtained results highlight the influence of the environment on the folding and thus analyte-binding capacity of an aptamer and emphasize the need to deploy appropriate surface functionalization protocols in sensor development as a means to minimize the steric obstructions and undesirable interactions of an aptamer with a surface onto which it is tethered. [ABSTRACT FROM AUTHOR]

Cardiovascular disease (particularly atherosclerosis) is a leading cause of death around the world, and there still exists a need for improved diagnostic techniques and treatments to improve patient outcomes as well as minimize the disease's global burden. Aptamers are short, single-stranded DNA or RNA molecules that are accompanied by unique characteristics such as specificity, high binding affinity, ease of cellular internalization, and rapid tissue accumulation capabilities, offering great potential as theranostic agents in cardiovascular diseases with significantly improved sensitivity and accuracy. These theranostic agents provide a combination of therapy and diagnostics in which aptamers may diagnose and treat disease simultaneously. Therefore, this review article summarizes the role of aptamer-based probes for imaging and theranostics in cardiovascular disease. It also provides insight into current research and future treatment techniques that are very relevant for future clinical practice with the aim of improving the quality of life of cardiovascular disease patients. [ABSTRACT FROM AUTHOR]

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Gastric Cancer (GC) is one of the fifth most malignant tumors in the world and the third leading cause of cancer‐related deaths. At present, one of the methods to diagnose gastric cancer patients is screening for gastric cancer tumor markers. However, the specificity and detection rate of existing tumor markers are low. Aptamer, also known as chemical antibody, is a kind of single‐stranded oligonucleotide (DNA or RNA) obtained by SELEX. It is widely used in the diagnosis of new diseases and as a molecular probe for treatment. This study used Med‐SELEX technology, using agarose magnetic beads as the carrier, and cancer culture medium as the screening target. APT‐1 specific to gastric cancer cell culture medium was screened out from random library through q‐PCR real‐time monitoring and screening process, and its affinity and specificity for gastric cancer serum were verified by subsequent q‐PCR and quantum dot methods. Serum stability test and cytotoxicity test proved its practicability. APT‐1 is not only of great significance for the early diagnosis of gastric cancer and the construction of targeted drug delivery system for gastric cancer, but also may be used as a probe to discover new tumor markers for gastric cancer. [ABSTRACT FROM AUTHOR]

Low-density lipoprotein receptor-related protein 6 (LRP6), a member of the low-density lipoprotein receptor (LDLR) family, displays a unique structure and ligand-binding function. As a co-receptor of the Wnt/β-catenin signaling pathway, LRP6 is a novel therapeutic target that plays an important role in the regulation of cardiovascular disease, lipid metabolism, tumorigenesis, and some classical signals. By using capillary electrophoresis–systematic evolution of ligands by exponential enrichment (CE-SELEX), with recombinant human LRP-6 as the target, four candidate aptamers with a stem-loop structure were selected from an ssDNA library—AptLRP6-A1, AptLRP6-A2, AptLRP6-A3, and AptLRP6-A4. The equilibrium dissociation constant KD values between these aptamers and the LRP6 protein were in the range of 0.105 to 1.279 μmol/L, as determined by CE-LIF analysis. Their affinities and specificities were further determined by the gold nanoparticle (AuNP) colorimetric method. Among them, AptLRP6-A3 showed the highest affinity with LRP6-overexpressed human breast cancer cells. Therefore, the LRP6 aptamer identified in this study constitutes a promising modality for the rapid diagnosis and treatment of LRP6-related diseases. [ABSTRACT FROM AUTHOR]

Recent innovations in point-of-care (POC) diagnostic technologies have paved a critical road for the improved application of biomedicine through the deployment of accurate and affordable programs into resource-scarce settings. The utilization of antibodies as a bio-recognition element in POC devices is currently limited due to obstacles associated with cost and production, impeding its widespread adoption. One promising alternative, on the other hand, is aptamer integration, i.e., short sequences of single-stranded DNA and RNA structures. The advantageous properties of these molecules are as follows: small molecular size, amenability to chemical modification, low- or nonimmunogenic characteristics, and their reproducibility within a short generation time. The utilization of these aforementioned features is critical in developing sensitive and portable POC systems. Furthermore, the deficiencies related to past experimental efforts to improve biosensor schematics, including the design of biorecognition elements, can be tackled with the integration of computational tools. These complementary tools enable the prediction of the reliability and functionality of the molecular structure of aptamers. In this review, we have overviewed the usage of aptamers in the development of novel and portable POC devices, in addition to highlighting the insights that simulations and other computational methods can provide into the use of aptamer modeling for POC integration. [ABSTRACT FROM AUTHOR]

Aptamers are single-stranded DNA or RNA sequences that can specifically bind with the target protein or molecule via specific secondary structures. Compared to antibody-drug conjugates (ADC), aptamer‒drug conjugate (ApDC) is also an efficient, targeted drug for cancer therapy with a smaller size, higher chemical stability, lower immunogenicity, faster tissue penetration, and facile engineering. Despite all these advantages, several key factors have delayed the clinical translation of ApDC, such as in vivo off-target effects and potential safety issues. In this review, we highlight the most recent progress in the development of ApDC and discuss solutions to the problems noted above. Aptamer-drug conjugate (ApDC) represents an efficient, targeted delivery system. Compared to antibody-drug conjugates, ApDC is smaller and has increased chemical stability with lower immunogenicity, faster tissue penetration, and facile engineering. [Display omitted] [ABSTRACT FROM AUTHOR]

As a type II transmembrane serine protease, fibroblast activation protein (FAP) is specifically expressed on the surface of fibroblasts associated with a variety of epithelial-derived malignancies such as pancreatic cancer, breast cancer, and colon cancer. It participates in the processes of tumorigenesis, progression, and immunosuppression. FAP constitutes an important target for tumor treatment; however, the current studies on FAP are mainly related to structural characteristics, enzymatic properties, and biological functions, and aptamers of FAP have not been investigated. In this work, by using recombinant human FAP as the target, five candidate aptamers, which are AptFAP-A1, AptFAP-A2, AptFAP-A3, AptFAP-A4, and AptFAP-A5, were selected by capillary electrophoresis–systematic evolution of ligands by exponential enrichment (CE-SELEX), and their secondary structures were predicted to be mainly stem-loop. Moreover, the CE-laser-induced fluorescence (LIF) method was used to determine the equilibrium dissociation constant KD values between the FAP protein and candidate aptamers, and the KD value was in the low molar range. Finally, Cy5-labeled aptamers were co-incubated with human pancreatic cancer-associated fibroblasts highly expressing FAP protein, and confocal microscopy imaging showed that aptamer AptFAP-A4 had the highest affinities with the cells. The FAP aptamers screened in this study provide a promising direction for the development of rapid tumor diagnosis and targeted therapy. [ABSTRACT FROM AUTHOR]

A DNA structure, known as triple-stranded DNA, is made up of three oligonucleotide chains that wind around one another to form a triple helix (TFO). Hoogsteen base pairing describes how triple-stranded DNA may be built at certain conditions by the attachment of the third strand to an RNA, PNA, or DNA, which might all be employed as oligonucleotide chains. In each of these situations, the oligonucleotides can be employed as an anchor, in conjunction with a specific bioactive chemical, or as a messenger that enables switching between transcription and replication through the triplex-forming zone. These data are also considered since various illnesses have been linked to the expansion of triplex-prone sequences. In light of metabolic acidosis and associated symptoms, some consideration is given to the impact of several low-molecular-weight compounds, including pH on triplex production in vivo. The review is focused on the development of biomedical oligonucleotides with triplexes. [ABSTRACT FROM AUTHOR]

Inhibitory oligodeoxynucleotides (ODNs) are short single-stranded DNA, which capable of folding into complex structures, enabling them to bind to a large variety of targets. With appropriate modifications, the inhibitory oligodeoxynucleotides exhibited many features of long half-life time, simple production, low toxicity and immunogenicity. In recent years, inhibitory oligodeoxynucleotides have received considerable attention for their potential therapeutic applications in immune-mediated inflammatory diseases (IMIDs). Inhibitory oligodeoxynucleotides could be divided into three categories according to its mechanisms and targets, including antisense ODNs (AS-ODNs), DNA aptamers and immunosuppressive ODNs (iSup ODNs). As a synthetic tool with immunomodulatory activity, it can target RNAs or proteins in a specific way, resulting in the reduction, increase or recovery of protein expression, and then regulate the state of immune activation. More importantly, inhibitory oligodeoxynucleotides have been used to treat immune-mediated inflammatory diseases, including inflammatory disorders and autoimmune diseases. Several inhibitory oligodeoxynucleotide drugs have been developed and approved on the market already. These drugs vary in their chemical structures, action mechanisms and cellular targets, but all of them could be capable of inhibiting excessive inflammatory responses. This review summarized their chemical modifications, action mechanisms and applications of the three kinds of inhibitory oligodeoxynucleotidesin the precise treatment of immune-mediated inflammatory diseases. [ABSTRACT FROM AUTHOR]

The complexity of genetic circuits has not seen a significant increase over the last decades, even with the rapid development of synthetic biology tools. One of the bottlenecks is the limited number of orthogonal transcription factor–operator pairs. Researchers have tried to use aptamer–ligand pairs as genetic parts to regulate transcription. However, most aptamers selected using traditional methods cannot be directly applied in gene circuits for transcriptional regulation. To that end, we report a new method called CIVT-SELEX to select DNA aptamers that can not only bind to macromolecule ligands but also undergo significant conformational changes, thus affecting transcription. The single-stranded DNA library with affinity to our example ligand human thrombin protein is first selected and enriched. Then, these ssDNAs are inserted into a genetic circuit and tested in the in vitro transcription screening to obtain the ones with significant inhibitory effects on downstream gene transcription when thrombins are present. These aptamer–thrombin pairs can inhibit the transcription of downstream genes, demonstrating the feasibility and robustness of their use as genetic parts in both linear DNAs and plasmids. We believe that this method can be applied to select aptamers of any target ligands and vastly expand the genetic part library for transcriptional regulation. [ABSTRACT FROM AUTHOR]

An aptamer is a single-stranded DNA or RNA that binds to a specific target with high binding affinity. Aptamers are developed through the process of systematic evolution of ligands by exponential enrichment (SELEX), which is repeated to increase the binding power and specificity. However, the SELEX process is time-consuming, and the characterization of aptamer candidates selected through it requires additional effort. Here, we describe in silico methods in order to suggest the most efficient way to develop aptamers and minimize the laborious effort required to screen and optimise aptamers. We investigated several methods for the estimation of aptamer-target molecule binding through conformational structure prediction, molecular docking, and molecular dynamic simulation. In addition, examples of machine learning and deep learning technologies used to predict the binding of targets and ligands in the development of new drugs are introduced. This review will be helpful in the development and application of in silico aptamer screening and characterization. [ABSTRACT FROM AUTHOR]

Interleukin-17A (IL-17A) is an important pro-inflammatory cytokine observed in the development of many disorders, such as psoriasis, rheumatoid arthritis, and multiple sclerosis. The anti-IL-17A biological drugs, including Secukinumab, Ixekizumab, and Brodalumab, are monoclonal antibodies approved for several disease treatments. Due to the disadvantages of biological therapies, including their immunogenicity, difficulties in scale generation, and high production costs and time, it is necessary to find new alternative anti- IL-17A agents for these monoclonal antibodies. Our study aimed to identify ssDNA aptamers that block IL-17A activity using the protein-SELEX procedure. The hIL-17A was expressed in codon plus E. coli , and after 14 rounds of the SELEX process, monitoring of aptamer pools was done using the dot blot method. Three families of aptamers were obtained from the selected round 9 aptamer pool, and seven truncates were created. Inhibitory effects of aptamer truncate on IL-17-induced CCL20 expression in HaCaT keratinocytes were evaluated. All aptamer truncates had a significant inhibitory effect compared to the library, but the inhibitory effect of M2 and M7 truncates was more than 80%. Moreover, we evaluated the potential binding site of selected aptamers by ELISA. We introduced a new small 17-nucleotide DNA aptamer that efficiently binds and blocks hIL-17A with a 0.3 nM kd, a potential anti-IL-17A therapeutic agent. • Several stringency were considered in SELEX process for improve affinity of selected aptamers. • The selected aptamer families were truncated to shorter sequences. • Two ssDNA aptamers (M2 &M7) with high affinities were obtained against human IL17A. • The selected DNA aptamer (M2) had more than 85% IL17A blocking efficacy with 0.3 nM kd. [ABSTRACT FROM AUTHOR]

Background: The paucity of SARS-CoV-2-specific virulence factors has greatly hampered the therapeutic management of patients with COVID-19 disease. Although available vaccines and approved therapies have shown tremendous benefits, the continuous emergence of new variants of SARS-CoV-2 and side effects of existing treatments continue to challenge therapy, necessitating the development of a novel effective therapy. We have previously shown that our developed novel single-stranded DNA aptamers not only target the trimer S protein of SARS-CoV-2, but also block the interaction between ACE2 receptors and trimer S protein of Wuhan origin, Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants of SARS-CoV-2. We herein performed in vivo experiments that administer the aptamer to the lungs by intubation as well as in vitro studies utilizing PBMCs to prove the efficacy and safety of our most effective aptamer, AYA2012004_L. Methods: In vivo studies were conducted in transgenic mice expressing human ACE2 (K18hACE2), C57BL/6J, and Balb/cJ. Flow cytometry was used to check S-protein expressing pseudo-virus-like particles (VLP) uptake by the lung cells and test the immuogenicity of AYA2012004_L. Ames test was used to assess mutagenicity of AYA2012004_L. RT-PCR and histopathology were used to determine the biodistribution and toxicity of AYA2012004_L in vital organs of mice. Results: We measured the in vivo uptake of VLPs by lung cells by detecting GFP signal using flow cytometry. AYA2012004_L specifically neutralized VLP uptake and also showed no inflammatory response in mice lungs. In addition, AYA2012004_L did not induce inflammatory response in the lungs of Th1 and Th2 mouse models as well as human PBMCs. AYA2012004_L was detectable in mice lungs and noticeable in insignificant amounts in other vital organs. Accumulation of AYA2012004_L in organs decreased over time. AYA2012004_L did not induce degenerative signs in tissues as seen by histopathology and did not cause changes in the body weight of mice. Ames test also certified that AYA2012004_L is non-mutagenic and proved it to be safe for in vivo studies. Conclusions: Our aptamer is safe, effective, and can neutralize the uptake of VLPs by lung cells when administered locally suggesting that it can be used as a potential therapeutic agent for COVID-19 management. [ABSTRACT FROM AUTHOR]

A sensitive and selective method for determining chloramphenicol (CAP) based on RecJf exonuclease-assisted signal amplification and graphdiyne (GDY) is reported. GDY possesses a strong absorptivity for single-stranded DNA and is proposed for the first time as a new specific and eminently sensitive type of fluorescence sensing platform for chloramphenicol. Under the optimized conditions, the assay operates from 10 to 80 µg/L chloramphenicol with a 43.6 ng/L detection limit. The procedure was employed to determine chloramphenicol in food samples, with recoveries from 93.60% to 107.90%. The proposed technique offers a novel development in food safety using sensitive aptasensors for detecting small molecules. [ABSTRACT FROM AUTHOR]

The high failure rate of the in vitro aptamer selection process by SELEX (Systematic Evolution of Ligands by EXponential enrichment) limits the production of these innovative oligonucleotides and, consequently, limits their potential applications. The generation of single-stranded DNA (ssDNA) is a critical step of SELEX, directly affecting the enrichment and the selection of potential binding sequences. The main goal of this study was to confirm the best method for generating ssDNA by comparing the purification of ssDNA, using streptavidin-coated beads, and lambda exonuclease digestion, and by improving ssDNA recovery through protocol improvements. In addition, three techniques for quantifying the ssDNA generated (Qubit vs. NanodropTM vs. gel quantification) were compared, and these demonstrated the accuracy of the gel-based quantification method. Lambda exonuclease digestion was found to be more efficient for ssDNA recovery than purification using streptavidin-coated beads, both quantitatively and qualitatively. In conclusion, this work provides a detailed and rigorous protocol for generating ssDNA, improving the chances of a successful aptamer selection process. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two structure-switching aptamer-based fluorescent probes were designed. • Target induced structural transformation activated exonuclease-assisted signal amplification strategies were proposed and implemented. • Turn-on sensing methods were successfully applied to the selective and sensitive detection of lead ions in water and food matrices. Herein, two simple fluorescent signal-on sensing strategies for detecting lead ions (Pb2+) were established based on structure-switching aptamer probes and exonuclease-assisted signal amplification strategies. Two hairpin-structure fluorescent probes with blunt-ended stem arms were designed by extending the base sequence of Pb2+ aptamer (PS2.M) and labelling the probes with FAM (in probe 1) and 2-aminopurine (2-AP) (in probe 2), respectively. In method 1, graphene oxide (GO) was added to adsorb probe 1 and quench the fluorescence emission of FAM to achieve low fluorescent background. In method 2, fluorescent 2-AP molecule inserted into the double-stranded DNA of probe 2 was quenched as a result of base stacking interactions, leading to a simplified, quencher-free approach. The addition of Pb2+ can induce the probes to transform into G-quadruplex structures, exposing single DNA strands at the 3′ end (the extended sequences). This exposure enables the activation of exonuclease I (Exo I) on the probes, leading to the cleavage effect and subsequent release of free bases and fluorophores, thereby resulting in amplified fluorescence signals. The two proposed methods exhibit good specificity and sensitivity, with detection limits of 0.327 nM and 0.049 nM Pb2+ for method 1 and method 2, respectively, and have been successfully applied to detect Pb2+ in river water and fish samples. Both detection methods employ the structure-switching aptamer probes and can be completed in two or three steps without the need for complex analytical instruments. Therefore, they have a broad prospect in the sensitive and simple detection of lead ion contamination in food and environmental samples. [ABSTRACT FROM AUTHOR]

Virus-induced infectious diseases have a detrimental effect on public health and exert significant influence on the global economy. Therefore, the rapid and accurate detection of viruses is crucial for effectively preventing and diagnosing infections. Aptamer-based detection technologies have attracted researchers' attention as promising solutions. Aptamers, small single-stranded DNA or RNA screened via systematic evolution of ligands by exponential enrichment (SELEX), possess a high affinity towards their target molecules. Numerous aptamers targeting viral marker proteins or virions have been developed and widely employed in aptamer-based biosensors (aptasensor) for virus detection. This review introduces SELEX schemes for screening aptamers and discusses distinctive SELEX strategies designed explicitly for viral targets. Furthermore, recent advances in aptamer-based biosensing methods for detecting common viruses using different virus-specific aptamers are summarized. Finally, limitations and prospects associated with developing of aptamer-based biosensors are discussed. [ABSTRACT FROM AUTHOR]

Endocrine-disrupting chemicals (EDCs) are substances that can disrupt the normal functioning of hormones.Using aptamers, which are biological recognition elements, biosensors can quickly and accurately detect EDCs in environmental samples. However, the elucidation of aptamer structures by conventional methods is highly challenging due to their complexity. This has led to the development of three-dimensional aptamer structures based on different models and techniques. To do this, we developed a way to predict the 3D structures of the SS DNA needed for this sequence by starting with an aptamer sequence that has biosensor properties specific to bisphenol-A (BPA), one of the chemicals found in water samples that can interfere with hormones. In addition, we will elucidate the intermolecular mechanisms and binding affinity between aptamers and endocrine disruptors using bioinformatics techniques such as molecular docking, molecular dynamics simulation, and binding energies. The outcomes of our study are to compare modeling programs and force fields to see how reliable they are and how well they agree with results found in the existing literature, to understand the intermolecular mechanisms and affinity of aptamer-based biosensors, and to find a new way to make aptamers that takes less time and costs less. [Display omitted] • Aptamer-based biosensors detect endocrine disruptors, offering a rapid and accurate environmental monitoring solution. • Predicting 3D structures of aptamers aids in understanding their binding affinity with endocrine disruptors. • Primary single-stranded DNA was transformed into two-dimensional and three-dimensional structures using the Vfold2D and Vfold3D servers. • The molecular dynamics simulation of the modeled aptamer revealed that the CHARMM force fields displayed less strong interactions when binding with the target structure compared to the OL15 and BSC1 force fields. • A computational approach was devised to enhance the binding strength and specificity of single-stranded DNA aptamers towards bisphenol A. [ABSTRACT FROM AUTHOR]

To date, numerous aptamer-based biosensing platforms have been developed for sensitive and selective monitoring of target analytes, relying on analyte-induced conformational changes in the aptamer for the quantification of the analyte and the conversion of the binding event into a measurable signal. Despite the impact of these conformational rearrangements on sensor performance, the influence of the environment on the structural conformations of aptamers has rarely been investigated, so the link between parameters directly influencing aptamer folding and the ability of the aptamer to bind to the target analyte remains elusive. Herein, the effect a number of variables have on an aptamer’s 3D structure was examined, including the pH of the buffering medium, as well as the anchoring of the aptamer on a solid support, with the use of two label-free techniques. Circular dichroism spectroscopy was utilized to study the conformation of an aptamer in solution along with any changes induced to it by the environment (analyte binding, pH, composition and ionic strength of the buffer solution), while quartz crystal microbalance with dissipation monitoring was employed to investigate the surface-bound aptamer’s behavior and performance. Analysis was performed on an aptamer against oxytetracycline, serving as a model system, representative of aptamers selected against small molecule analytes. The obtained results highlight the influence of the environment on the folding and thus analyte-binding capacity of an aptamer and emphasize the need to deploy appropriate surface functionalization protocols in sensor development as a means to minimize the steric obstructions and undesirable interactions of an aptamer with a surface onto which it is tethered.

Controlling food safety and preventing the growing spread of antibiotics into food products have been challenging problems for the protection of human health. Hence, the development of easy-to-use, fast, and sensitive analytical methods for the detection of antibiotics in food products has become one of the priorities in the food industry. In this paper, an electrochemical platform based on the ssDNA aptamer for the selective detection of tetracycline has been proposed. The aptasensor is based on a thiolated aptamer, labelled with ferrocene, which has been covalently co-immobilized onto a gold electrode surface with 6-mercaptohexan-1-ol. The changes in the redox activity of ferrocene observed on the aptamer–antibiotics interactions have been the basis of analytical signal generation registered by square-wave voltammetry. Furthermore, the detection of tetracycline-spiked cow milk samples has been successfully demonstrated. The limits of detection (LODs) have been obtained of 0.16 nM and 0.20 nM in the buffer and spiked cow milk, respectively, which exceed the maximum residue level (225 nM) more than 1000 times. The proposed aptasensor offers high selectivity for tetracycline against other structurally related tetracycline derivatives. The developed biosensor characterized by simplicity, a low detection limit, and high reliability shows practical potential for the detection of tetracycline in animal-origin milk. [ABSTRACT FROM AUTHOR]

An ultrasensitive biosensor was designed and constructed for tobramycin detection. As a target recognition component, the DNA probe consists of an aptamer region for tobramycin binding and a template for amplification. In the absence of tobramycin, the probe was locked to form a stem–loop structure. In the presence of the target, the binding of tobramycin led to a conformational change in the probe. The released 3′ end was used as a primer for the strand displacement amplification (SDA) to produce a large amount of single-stranded trigger DNA, which then efficiently initiated the following hybridization chain reaction (HCR) to produce a long duplex DNA with many fluorophores. The signals were detected after the addition of graphene oxide (GO) to quench the fluorescence from excess hairpin DNA. Through sequence and reaction condition optimization, the biosensor exhibited high selectivity for tobramycin. The linearity range and limit of detection (LOD) were 0.5–30 nM and 0.06 nM, respectively. Moreover, the application of detecting tobramycin in milk and lake water samples showed that this method is reliable and could be further used in food safety control and environmental monitoring. [ABSTRACT FROM AUTHOR]

Meningitis, acute infection of the meninges, is the 10th leading cause of mortality among infectious diseases. Although many different causes for meningitis (viruses and bacteria) have been diagnosed, the most common ones are Neisseria meningitidis , Haemophilus influenzae , and Streptococcus pneumoniae. The effort to find a new method for detection of bacterial meningitis is an urgent need for clinical treatment. DNA aptamers generated by cell-systematic evolution of ligands by exponential enrichment (SELEX) against bacterial cells provide a novel cell labeling and biosensing technique. Here, we isolated single-stranded DNA aptamers during the SELEX method with a high affinity for different bacterial genera. This approach was demonstrated on H. influenzae type B, N. meningitidis serogroups A, B, C, and Y, and Streptococcus pneumoniae serotypes 18, 14, 19A, 6A, and 6B which served as targets in 20 rounds of cell-SELEX. After 20 rounds of SELEX, a total of 93 aptamers were identified. Among these, aptamers C65 and C50 showed the highest affinity toward targets with a dissociation constant of 6.98 and 15.79, respectively. Selected aptamers were able to successfully detect clinical bacterial strains isolated from cerebrospinal fluid samples of meningitis patients by double-aptamer sandwich enzyme-linked oligonucleotide assay (ELONA). Our findings demonstrated that aptamers with broad affinity to bacterial taxa in different genera can be isolated for the development of diagnostic tools for multiple targets. We further showed that sandwich ELONA based on single-stranded DNA aptamer is sensitive and specific enough for detection of the superior cause of bacterial meningitis. [ABSTRACT FROM AUTHOR]

Aptamers are a group of synthetic single-stranded nucleic acids. They are generated from a random library of single-stranded DNA or RNA by a technology named systematic evolution of ligands by exponential enrichment (SELEX). SELEX is a repetitive process to select and identify suitable aptamers that show high affinity and specificity towards target cells. Great strides have been achieved in the design, construction, and use of aptamers up to this point. However, only a small number of aptamer-based applications have achieved widespread commercial and clinical acceptance. Additionally, finding more effective ways to acquire aptamers with high affinity remains a challenge. Therefore, it is crucial to thoroughly examine the existing dearth and advancement in aptamer-related technologies. This review focuses on aptamers that are generated by SELEX to detect pathogenic microorganisms and mammalian cells, as well as in cell-internalizing SELEX for diagnostic and therapeutic purposes. The development of novel aptamer-based biosensors using optical and electrical methods for microbial detection is reported. The applications and limitations of aptamers are also discussed. [ABSTRACT FROM AUTHOR]

Simple Summary: Carcinoembryonic antigen (CEA), a well-known cancer biomarker that is used to diagnose various cancers, notably colorectal cancer, was used as a target to select and characterize single-stranded DNA aptamers. These short and compact nucleic acid sequences, known as aptamers, are becoming ever more popular in both therapeutics and diagnostics. In this study, SELEX and NGS techniques were employed in the aptamer selection processes. Aptamer characterization techniques, such as enzyme-linked oligonucleotide assay (ELONA), dot blot, and bioinformatics assays, were conducted to observe the affinity binding of the selected aptamer to the target protein. Confocal microscopy results demonstrated how the fluorescently labelled aptamer sequence recognized CEA, which is expressed in HT-29 human colon adenocarcinoma cell line. These findings show that the selected aptamer has the potential to be employed as a recognition component in rapid detection systems, such as aptasensors. One of the major causes of a drastically shorter life expectancy and one of the most prevalent diseases in the world today is cancer. Given the data on the rise in cancer cases throughout the world, it is obvious that, despite the diagnostic techniques currently being used, there is a pressing need to develop precise and sensitive techniques for early diagnosis of the disease. A high degree of affinity and specificity towards particular targets is maintained by the short nucleic acid molecules known as aptamers. Aptamers outperform antibodies due to their unique benefits, such as their simplicity in synthesis and modification, lack of toxicity, and long-term stability. Utilizing an accurate recognition element and a robust signal transduction mechanism, molecular diagnostics can be extremely sensitive and specific. In this study, development of new single-stranded DNA aptamers against CEA for use in cancer diagnostics was accomplished using SELEX and NGS methods. As a result of 12 iterative SELEX rounds, nine aptamer candidates against CEA were developed. NGS comparative analysis revealed that round twelve had an enriched number of aptamers that were specifically bound, as opposed to round eight. Among the selected nine sequences characterized by bioinformatics analysis and ELONA, an aptamer sequence with the highest specificity and affinity for the target protein was identified and further examined. Aptamer sequence (6) was screened in a concentration-dependent assay, specificity analysis was performed, and its potential secondary and tertiary structures were predicted, which enabled us to test one of the possible putative interactions with CEA. Finally, aptamer sequence (6) labelled with a Cy5 fluorescent tag was used in confocal microscopy to observe its binding towards the CEA expressed in HT-29 human colon adenocarcinoma cell line. [ABSTRACT FROM AUTHOR]

Within the last decade, the application range of aptamers in biochemistry and medicine has expanded rapidly. More than just a replacement for antibodies, these intrinsically structured RNA‐ or DNA‐oligonucleotides show great potential for utilization in diagnostics, specific drug delivery, and treatment of certain medical conditions. However, what is analyzed less frequently is the process of aptamer identification known as systematic evolution of ligands by exponential enrichment (SELEX) and the functional mechanisms that lie at its core. SELEX involves numerous singular processes, each of which contributes to the success or failure of aptamer generation. In this review, critical steps during aptamer selection are discussed in‐depth, and specific problems are presented along with potential solutions. The discussed aspects include the size and molecule type of the selected target, the nature and stringency of the selection process, the amplification step with its possible PCR bias, the efficient regeneration of RNA or single‐stranded DNA, and the different sequencing procedures and screening assays currently available. Finally, useful quality control steps and their role within SELEX are presented. By understanding the mechanisms through which aptamer selection is influenced, the design of more efficient SELEX procedures leading to a higher success rate in aptamer identification is enabled. [ABSTRACT FROM AUTHOR]

The global pandemic of COVID-19 has created an unrivalled need for sensitive and rapid point-of-care testing (POCT) methods for the detection of infectious viruses. For the novel coronavirus SARS-CoV-2, the nucleocapsid protein (N-protein) is one of the most abundant structural proteins of the virus and it serves as a useful diagnostic marker for detection. Herein, we report a fiber optic particle plasmon resonance (FOPPR) biosensor which employed a single-stranded DNA (ssDNA) aptamer as the recognition element to detect the SARS-CoV-2 N-protein in 15 min with a limit of detection (LOD) of 2.8 nM, meeting the acceptable LOD of 106 copies/mL set by the WHO target product profile. The sensor chip is a microfluidic chip based on the balance between the gravitational potential and the capillary force to control fluid loading, thus enabling the power-free auto-flowing function. It also has a risk-free self-contained design to avoid the risk of the virus leaking into the environment. These findings demonstrate the potential for designing a low-cost and robust POCT device towards rapid antigen detection for early screening of SARS-CoV-2 and its related mutants. [ABSTRACT FROM AUTHOR]

In this work, the single-stranded DNA (ssDNA) aptamers specific to florfenicol (FF) and having a high binding affinity were prepared using the magnetic bead-based systematic evolution of ligands by the exponential enrichment technique (MB-SELEX). After 10 rounds of the MB-SELEX screening, aptamers that can simultaneously recognize FF and its metabolite florfenicol amine (FFA) were obtained. The aptamer with the lowest dissociation constant (Kd) was truncated and optimized based on a secondary structure analysis. The optimal aptamer selected was Apt-14t, with a length of 43 nt, and its dissociation constant was 4.66 ± 0.75 nM, which was about 7 times higher than that of the full-length sequence. The potential binding sites and interactions with FF were demonstrated by molecular docking simulations. In addition, a colorimetric strategy for nanogold aptamers was constructed. The linear detection range of this method was 0.00128–500 ng/mL and the actual detection limit was 0.00128 ng/mL. Using this strategy to detect florfenicol in actual milk and eggs samples, the spiked recoveries were 88.9–123.1% and 84.0–112.2%, respectively, and the relative standard deviation was less than 5.6%, showing high accuracy. [ABSTRACT FROM AUTHOR]

Aptamers are chemically synthesized single-stranded DNA or RNA oligonucleotides widely used nowadays in sensors and nanoscale devices as highly sensitive biorecognition elements. With proper design, aptamers are able to bind to a specific target molecule with high selectivity. To date, the systematic evolution of ligands by exponential enrichment (SELEX) process is employed to isolate aptamers. Nevertheless, this method requires complex and time-consuming procedures. In silico methods comprising machine learning models have been recently proposed to reduce the time and cost of aptamer design. In this work, we present a new in silico approach allowing the generation of highly sensitive and selective RNA aptamers towards a specific target, here represented by ammonium dissolved in water. By using machine learning and bioinformatics tools, a rational design of aptamers is demonstrated. This "smart" SELEX method is experimentally proved by choosing the best five aptamer candidates obtained from the design process and applying them as functional elements in an electrochemical sensor to detect, as the target molecule, ammonium at different concentrations. We observed that the use of five different aptamers leads to a significant difference in the sensor's response. This can be explained by considering the aptamers' conformational change due to their interaction with the target molecule. We studied these conformational changes using a molecular dynamics simulation and suggested a possible explanation of the experimental observations. Finally, electrochemical measurements exposing the same sensors to different molecules were used to confirm the high selectivity of the designed aptamers. The proposed in silico SELEX approach can potentially reduce the cost and the time needed to identify the aptamers and potentially be applied to any target molecule. [ABSTRACT FROM AUTHOR]

The ability to selectively capture biomacromolecules and other components from solution has many important applications in biotechnology. However, capturing targets from solution while minimizing interference with the sample solution is still challenging. Here, we describe the design and assembly of a group of DNA hydrogels consisting of long single-stranded DNA produced by rolling amplification reaction (RCA) and crosslinked by DNA duplexes. The developed DNA hydrogels can selectively capture and separate oligonucleotides, proteins and bacteria from solution in situ without complex separation processes. Since such DNA hydrogels can capture their targets in the solution independently, multiple DNA hydrogels that target different compounds can be employed to separate different compounds in the solution at the same time. The work not only expands the application of DNA hydrogels, but also paves the way for developing novel selective biomaterials. Biomaterials capable of selectively capturing various components have great potential in the field of biotechnology. Here, we proposed a new class of hydrogel composed of crosslinked long DNA strands for selectively capturing DNA, protein and bacteria. Unlike traditional polymeric hydrogels that have small meshes and limit macromolecule diffusion owing to the short distance between two adjacent crosslinks, the described DNA hydrogel has a much larger distance between its crosslinks because of the sequence designability of DNA, which allows easy diffusion of biomacromolecules through its networks and greatly expand its specific surface area. Moreover, the developed DNA hydrogel can also easily combine different aptamers to target different components via the Watson-Crick base pairing without making significant changes in its original design. [Display omitted] [ABSTRACT FROM AUTHOR]

Legionella pneumophila is the major causative agent of Legionnaires' disease and Pontiac fever, which pose major public health problems. Rapid detection of L. pneumophila is important for global control of these diseases. Aptamers, short oligonucleotides that bind to targets with high affinity and specificity, have great potential for use in pathogenic bacterium detection, diagnostics, and therapy. Here, we used a whole-cell SELEX (systematic evolution of ligands by exponential enrichment) method to isolate and characterize single-stranded DNA (ssDNA) aptamers against L. pneumophila. A total of 60 ssDNA sequences were identified after 17 rounds of selection. Other bacterial species (Escherichia coli, Bacillus subtilis, Pseudomonas syringae, Staphylococcus aureus, Legionella quateirensis, and Legionella adelaidensis) were used for counterselection to enhance the specificity of ssDNA aptamers against L. pneumophila. Four ssDNA aptamers showed strong affinity and high selectivity for L. pneumophila, with Kd values in the nanomolar range. Bioinformatic analysis of the most specific aptamers revealed predicted conserved secondary structures that might bind to L. pneumophila cell walls. In addition, the binding of these four fluorescently labeled aptamers to the surface of L. pneumophila was observed directly by fluorescence microscopy. These aptamers identified in this study could be used in the future to develop medical diagnostic tools and public environmental detection assays for L. pneumophila. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus （ V. parahaemolyticus ）is a widespread food-borne pathogen in seafood, which poses a serious threat to human health and public safety. However, sensor platform projects that do not require complex sample processing are rare. Hence, we based on CRISPR/Cas14a and Exonuclease III (Exo III) to build a novel fluorescent biosensor that did not require nucleic acid extraction and amplification. A locked single-stranded DNA probe to a target-pathogen specific aptamer was designed to be opened as free single-stranded DNA (ssDNA) upon aptamer binding to the target pathogen. Exo III then cleaves hairpin B (HP B) from the 3′-terminus, releasing ssDNA probe A and ssDNA C. Then, ssDNA probe A hybridizes with another HP B to start a new cycle, and ssDNA C acts as a substrate to activate Cas14a/sgRNA trans-cleavage activity. By exploiting this novel design principle, we detected V. parahaemolyticus over a wide linear range with a limit of detection (LOD) of 6 CFU/mL. • Novel programmable Cas14a-Exo III ligand biosensor. • No need to extract Vibrio parahaemolyticus nucleic acid. • The ligand biosensor exhibited a lower limit of detection. [ABSTRACT FROM AUTHOR]

Berberine (BBR), a key component in Kampo medicine, is a cationic benzylisoquinoline alkaloid whose detection plays a critical role in the quality control of these traditional remedies. Traditional methods for detecting BBR often involve complex procedures, which can be time-consuming and costly. To address this challenge, our study focuses on developing a simpler, faster, and more efficient detection method for BBR in Kampo medicine formulations. We successfully developed a rapid fluorometric detection method for BBR using colloidal gold nanoparticle-based systematic evolution of ligands by exponential enrichment (GOLD-SELEX). Initially, specific single-stranded DNA (ssDNA) sequences were selected for their ability to enhance BBR's fluorescence intensity. The optimal ssDNA sequence, identified as BBR38, was further truncated to produce BBR38S, a stem-loop ssDNA that improved fluorescence upon interaction with BBR. To further enhance the fluorescence, the BBR38S aptamer underwent additional modifications, including stem truncation and nucleotide mutations, resulting in the higher fluorescence variant BBR38S-3 A10C. The final product, TetBBR38S, a tetramer version of BBR38S-3 A10C, exhibited a linear detection range of 0.780–50.0 μg mL–1 and a limit of detection of 0.369 μg mL−1. The assay demonstrated sufficient selectivity and was successfully applied to analyze 128 different Kampo medicine formulations, accurately detecting BBR content with high precision. This study represents an advancement in Kampo medicine research, marking the first successful application of an aptamer-based approach for BBR detection in complex matrices. The developed method is not only simple and rapid (with a detection time of 5 min) but also cost-effective, which is crucial for widespread application. [Display omitted] • Developed a fluorometric-based detection method for Berberine (BBR). • Utilized GOLD-SELEX for specific ssDNA selection, leading to the discovery of BBR38. • Further modifications resulted in TetBBR38S, enhancing fluorogenic activity. • Rapid (5 min), cost-effective BBR detection method. • First aptasensor application for BBR in Kampo medicine. [ABSTRACT FROM AUTHOR]

• Self-assembled Eu-dipeptide microparticles own multi-fluorescent emission centers. • Eu-PMPs@cDNA and MNPs@aptamer is used as signal reporter and capture, respectively. • Noncompetitive fluorescence sensing strategy is reliable for SMZ determination. • The LOD and recoveries are 0.014 ng/mL and 81.78 %-119.46 %, respectively. Self-assembled Eu-dipeptide (tryptophan-phenylalanine) microparticles with multi-emission fluorescence was prepared and modified with a single-stranded DNA corresponding to the sulfamethazine (SMZ) adapter (Eu-PMPs@cDNA). Aptamer-functionalized magnetic Fe 3 O 4 (MNPs@aptamer) was used to specifically bind the target SMZ. Using Eu-PMPs@cDNA as fluorescent signal probe and MNPs@aptamer as catcher, a noncompetitive fluorescence sensing strategy was developed for determination of SMZ with good sensitivity, accuracy, selectivity, and stability. Under the optimized conditions, fluorescence increases linearly in the 0–20 ng/mL SMZ concentration range, and the detection limit is 0.014 ng/mL. The fluorescence sensing method was applied to analysis of water and fish muscle samples, and recoveries ranged from 81.78 to 119.46 % with relative standard deviations below 4.2 %. This study offered a reliable and sensitive fluorescence sensing strategy for SMZ determination in food samples, which owns great potential for wide-ranging application in harmful compounds assay by simply changing the type of aptamer and its complementary single-stranded DNA. [ABSTRACT FROM AUTHOR]

Current treatment of snakebite relies on immunoglobulin-rich antivenoms. However, production of these antivenoms is complicated and costly. Aptamers – single-stranded DNAs or RNAs with specific folding structures that bind to specific target molecules – represent excellent alternatives or complements to antibody-based therapeutics. However, no studies have systematically assessed the feasibility of using aptamers to mitigate venom-induced toxicity in vivo. β-bungarotoxin is the predominant protein responsible for the toxicity of the venom of Bungarus multicinctus , a prominent venomous snake inhabiting Taiwan. In this study, we reported the screening and optimization of a DNA aptamer against β-bungarotoxin and tested its utility in a mouse model. After 14 rounds of directed evolution of ligands by exponential enrichment, an aptamer, called BB3, displaying remarkable binding affinity and specificity for β-bungarotoxin was obtained. Following structural prediction and point-modification experiments, BB3 underwent truncation and was modified with 2'- O -methylation and a 3′-inverted dT. This optimized aptamer showed sustained, high-affinity binding for β-bungarotoxin and exhibited remarkable nuclease resistance in plasma. Importantly, administration of this optimized aptamer extended the survival time of mice treated with a lethal dose of β-bungarotoxin. Collectively, our data provide a compelling illustration of the potential of aptamers as promising candidates for development of recombinant antivenom therapies. [Display omitted] [ABSTRACT FROM AUTHOR]

• A biosensor with high-density aptamer is developed for sensitive thrombin detection. • Primer exchange reaction with simple hairpin design amplifys detection signal. • Label-free analysis of thrombin utilizes electrochemical impedance spectroscopy. We report an electrochemical aptamer-based (E-AB) sensor for label-free quantitative detection of thrombin. In-situ synthesis of high-density aptamer replicas on the electrode is accomplished based on primer exchange reaction (PER), by which a DNA primer is continuously extended with the presence of a catalytic hairpin template and DNA polymerase. The PER enables the synthesis of long single-stranded DNA with replicated aptamer sequence for thrombin binding. The PER products on the surface of a gold electrode increase the density of aptamers on a limited electrode area, which can provide more effective binding sites for thrombin and therefore improve the performance of the sensor based on electrochemical impedance spectroscopy. Compared with the conventional E-AB sensor based on a 15-mer aptamer immobilized on the electrode, our method has a lower detection limit of 0.91 pM, and a wider linear range of 1.0 pM to 50 nM. Therefore, we believe our method is promising for label-free electrochemical detection of biomarkers. [ABSTRACT FROM AUTHOR]

In recent years, the conventional preparation of silver nanoclusters (AgNCs) has attracted much attention due to their ultra-small size, tunable fluorescence, easy-to-engineer, as well as biocompatible material. Moreover, its great affinity towards cytosine bases on single-stranded DNA has led to the construction of biosensors, especially aptamers, for a broad variety of applications in food safety and environmental protection. In past years, numerous researchers paid attention to the construction of AgNCs aptasensor. Therefore, this review will be an effort to summarize the synthetic strategy along with the influences of factors on synthesis, categorize the sensing mechanism of aptamer-functionalized AgNCs biosensors, as well as their specific applications in food safety detection including heavy metal, toxin, and foodborne pathogenic bacteria. Furthermore, a brief conclusion and outlook regarding the prospects and challenges of their applications in food safety were drawn in line with the developments in DNA-AgNCs. [Display omitted] • Concise introduction of fluorescent DNA-AgNCs in food safety. • Brief classification of the detection mechanism and signal amplification strategy. • DNA-AgNCs advance food safety monitoring with versatile and potent sensing capabilities. • The summarization and outlook of way forward strategies, challenges and future prospect. [ABSTRACT FROM AUTHOR]

In Europe, tick-borne diseases (TBDs) cause significant morbidity and mortality, affecting both human and animal health. Ticks can transmit a wide variety of pathogens (bacteria, viruses, and parasites) and feed on many vertebrate hosts. The incidence and public health burden of TBDs are tending to intensify in Europe due to various factors, mainly anthropogenic and often combined. Early detection of tick-borne pathogens (TBPs), preventive measures and treatment are of great importance to control TBDs and their expansion. However, there are various limitations in terms of the sensitivity and/or specificity of detection and prevention methods, and even in terms of feasibility. Aptamers are single-stranded DNA or RNA that could address these issues as they are able to bind with high affinity and specificity to a wide range of targets (e.g., proteins, small compounds, and cells) due to their unique three-dimensional structure. To date, aptamers have been selected against TBPs such as tick-borne encephalitis virus, Francisella tularensis , and Rickettsia typhi. These studies have demonstrated the benefits of aptamer-based assays for pathogen detection and medical diagnosis. In this review, we address the applications of aptamers to TBDs and discuss their potential for improving prevention measures (use of chemical acaricides, vaccination), diagnosis and therapeutic strategies to control TBDs. • The incidence of tick-borne diseases is increasing in Europe, with a major impact on human and animal health. • Their control is hampered by the lack of sensitive and specific detection and prevention tools. • Aptamers are promising tools, as they can bind with high affinity and specificity to their target. • This review compares aptamers with the current approach to tick-borne disease prevention and control. [ABSTRACT FROM AUTHOR]

NELL1 (Neural epidermal growth factor-like (EGFL)-like protein) is an important biomarker associated with tissue and bone development and regeneration. NELL1 upregulation has been linked with metastasis and negative prognosis in rhabdomyosarcoma (RMS). Furthermore, multiple recent studies have also shown the importance of NELL1 in inflammatory bowel disease and membranous nephropathy, amongst other diseases. In this study, several anti-NELL1 DNA aptamers were selected from a randomized ssDNA pool using a fluorescence-guided method and evaluated for their binding affinity and selectivity. Several other methods such as a metabolic assay and confocal microscopy were also applied for the evaluation of the selected aptamers. The top three candidates were evaluated further, and AptNCan3 was shown to have a binding affinity up to 959.2 nM. Selectivity was examined in the RH30 RMS cells that overexpressed NELL1. Both AptNCan2 and AptNCan3 could significantly suppress metabolic activity in RMS cells. AptNCan3 was found to locate on the cell membrane and also on intracellular vesicles, which matched the location of NELL1 shown by antibodies in previous research. These results indicate that the selected anti-NELL1 aptamer showed strong and highly specific binding to NELL1 and therefore has potential to be used for in vitro or in vivo studies and treatments. [ABSTRACT FROM AUTHOR]

Bacterial kidney disease (BKD) is a major health problem of salmonids, affecting both wild and cultured salmon. The disease is caused by Renibacterium salmoninarum (Rs), a fastidious, slow-growing and strongly Gram-positive diplobacillus that produces chronic, systemic infection characterized by granulomatous lesions in the kidney and other organs, often resulting in death. Fast detection of the pathogen is important to limit the spread of the disease, particularly in hatcheries or aquaculture facilities. Aptamers are increasingly replacing conventional antibodies as platforms for the development of rapid diagnostic tools. In this work, we describe the first instance of isolating and characterizing a ssDNA aptamer that binds with high affinity to p57 or major soluble antigen (MSA), the principal antigen found on the cell wall surface of Rs. Specifically, in this study a construct of the full-length protein containing a DNA binding domain (MSA-R2c) was utilized as target. Aptamers were isolated from a pool of random sequences using GO-SELEX (graphene oxide-systematic evolution of ligands by exponential enrichment) protocol. The selection generated multiple aptamers with conserved motifs in the random region. One aptamer with high frequency of occurrence in different clones was characterized and found to display a strong binding affinity to MSA-R2c with a Kd of 3.0 ± 0.6 nM. The aptamer could be potentially utilized for the future development of a sensor for rapid and onsite detection of Rs in water or in infected salmonids, replacing time-consuming and costly lab analyses. [ABSTRACT FROM AUTHOR]

In recent years, aza‐boron dipyrromethene (aza‐BODIPY) derivatives have attracted considerable attention as fluorescent imaging agents. In this work, two polyethylene glycol functionalized water soluble aza‐BODIPY derivatives (a mono cycloaddition product 1PEG‐BOD and a double cycloaddition product 2PEG‐BOD) were synthesized through a cycloaddition click reaction between aza‐BODIPY and N3‐PEG2000‐COOH. The absorption and fluorescence profiles of these two compounds were in near‐infrared region. The fluorescence quantum yield of 2PEG‐BOD was 20 times higher than 1PEG‐BOD in water. The attachment of an A549 lung cancer cell targeting aptamer with 2PEG‐BOD afforded APT‐PEG‐BOD. The average fluorescence intensity of APT‐PEG‐BOD (5 μmol/L) in A549 lung cancer cells calculated from confocal microscopy images was 25–33 times stronger than that in other cell lines, such as MDA‐MB‐231, HT1080 and LO2. These results indicated that APT‐PEG‐BOD shows high affinity to A549 lung cancer cells. [ABSTRACT FROM AUTHOR]

A colorimetric biosensor assay has been developed for Cd2+ and Hg2+ detection based on Cd2+-dependent DNAzyme cleavage and Hg2+-binding-induced conformational switching of the G-quadruplex fragment. Two types of multifunctional magnetic beads (Cd-MBs and Hg-MBs) were synthesized by immobilizing two functionalized DNA sequences on magnetic beads via avidin-biotin chemistry. For Cd2+ detection, Cd-MBs are used as recognition probes, which are modified with a single phosphorothioate ribonucleobase (rA) substrate (PS substrate) and a Cd2+-specific DNAzyme (Cdzyme). In the presence of Cd2+, the PS substrate is cleaved by Cdzyme, and single-stranded DNA is released as the signal transduction sequence. After molecular assembly with the other two oligonucleotides, duplex DNA is produced, and it can be recognized and cleaved by FokI endonuclease. Thus, a signal output component consisting of a G-quadruplex fragment is released, which catalyzes the oxidation of ABTS with the addition of hemin and H2O2, inducing a remarkably amplified colorimetric signal. To rule out false-positive results and reduce interference signals, Hg-MBs modified with poly-T fragments were used as Hg2+ accumulation probes during the course of Cd2+ detection. On the other hand, Hg-MBs can perform their second function in Hg2+ detection by changing the catalytic activity of the G-quadruplex/hemin DNAzyme. In the presence of Hg2+, the G-quadruplex structure in Hg-MBs is disrupted upon Hg2+ binding. In the absence of Hg2+, an intensified color change can be observed by the naked eye for the formation of intact G-quadruplex/hemin DNAzymes. The biosensor assay exhibits excellent selectivity and high sensitivity. The detection limits for Cd2+ and Hg2+ are 1.9 nM and 19.5 nM, respectively. Moreover, the constructed sensors were used to detect environmental water samples, and the results indicate that the detection system is reliable and could be further used in environmental monitoring. The design strategy reported in this study could broadly extend the application of metal ion-specific DNAzyme-based biosensors. [ABSTRACT FROM AUTHOR]

Cardiac troponin I (cTnI) is a specific biomarker of acute myocardial infarction (AMI). However, cTnI detection kits prepared with antibodies have many defects. Nucleic acid aptamers are sequences of single-strand DNA or RNA that can overcome the deficiency of antibodies. Herein, sandwich ELONA methods were established based on aptamers. Two selected ssDNA aptamers (Apt3 and Apt6) showed high binding affinity and sensibility (Apt3: Kd = 1.01 ± 0.07 nM, Apt6: k = 0.68 ± 0.05) and did not bind to the same domain of cTnI. Therefore, these two aptamers can be applied to the ELONA methods. The detection range of cTnI using the dual-aptamer sandwich ELONA method was 0.05–200 ng/mL, and the bioanalytical method verification results can meet the national standard of Chinese Pharmacopoeia (2020 Edition). There was no difference between results of the dual-aptamer sandwich ELONA method and the diagnostic results of serum obtained from 243 people (P = 0.39, P ˃ 0.05). The sensitivity and specificity of the ELONA with cTnI in serum were 96.46% and 93.85%, respectively. Compared with the FICA kit, which is clinically used, the consequences of ELONA method are closer to the diagnostic results. This study suggests that the aptamers Apt3 and Apt6 have high affinity and strong specificity and that the dual-aptamer sandwich ELONA method has a wide detection range and can be used to determine cTnI in serum, with potential applications in the diagnosis of AMIs. [ABSTRACT FROM AUTHOR]