Search Results (44)

The urgent need for advanced analytical tools for environmental monitoring and food safety drives the development of novel biosensing approaches and solutions. A computationally driven workflow for the development of a rapid electrochemical aptasensor for okadaic acid (OA), a critical marine biotoxin, is reported. The core of this strategy is a rational design process, where in silico modeling was employed to optimize the biological recognition element. A 63-nucleotide aptamer was successfully truncated to a highly efficient 31-nucleotide variant. Molecular docking simulations confirmed the high binding affinity of the minimized aptamer and guided the design of the surface immobilization chemistry to ensure robust performance. The fabricated sensor, which utilizes a ferrocene-labeled aptamer, delivered a sensitive response with a detection limit of 2.5 nM (n = 5) over a linear range of 5–200 nM. A significant advantage for practical applications is the remarkably short assay time of 5 min. The sensor’s applicability was successfully validated in complex food matrices, achieving excellent recovery rates of 82–103% in spiked mussel samples. This study establishes an integrated computational–experimental methodology that streamlines the development of high-performance biosensors for critical food safety and environmental monitoring challenges. Full article

Nucleic acid aptamers are single-stranded DNA or RNA molecules that can bind to a target with high specificity and affinity, as screened by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). In recent years, SELEX technologies have been significantly advanced for the screening of aptamers for a variety of target molecules, cells, and even bacteria and viruses. By integrating recent advances of emerging technologies with SELEX, novel screening technologies for nucleic acid aptamers have emerged with improved screening efficiency, reduced production costs and enhanced aptamer performance for a wide range of applications in medical diagnostics, drug delivery, and environmental monitoring. Aptasensors utilize aptamers to detect a wide range of analytes, allowing for the accurate identification and determination of small molecules, proteins, and even whole cells with remarkable specificity and sensitivity. Further optimization of the aptasensor can be achieved by aptamer truncation, which not only maintains the high specificity and affinity of the aptamer binding with the target analytes, but also reduces the manufacturing cost. Predictive models also demonstrate the powerful capability of determination of the minimal functional sequences by simulation of aptamer–target interaction processes, thus effectively shortening the aptamer screening procedure and reducing the production costs. This paper summarizes the research progress of protein-targeted aptamer screening in recent years, introduces several typical aptasensors at present, discusses the optimization methods of aptasensors by combining efficient SELEX with advanced predictive algorithms or post-SELEX processes, as well as the challenges and opportunities faced by aptasensors. Full article

Access to safe water remains a vital public health challenge, especially in low- and middle-income countries like Colombia, where untreated sources lead to severe diarrheal diseases in children under five. Escherichia coli (E. coli), a key indicator of fecal contamination, is often detected using culture-based methods that are time-consuming and rely on specialized infrastructure. To overcome these limitations, we developed an aptamer-based isolation system targeting environmental E. coli. Aptamers were obtained using a Cell-SELEX protocol, and after six enrichment rounds, two candidates—APT-EC-1 and its truncated version APT-EC-MUT—were synthesized and attached to carboxyl-functionalized magnetic nanoparticles (MNP-COOH). Both complexes demonstrated a strong binding affinity and high specificity, successfully isolating E. coli from environmental and ATCC reference strains in the laboratory. Sensitivity tests detected E. coli at dilutions up to 1:10,000, showing reliable performance. In early in-field testing with environmental water samples, APT-EC-1 consistently identified E. coli colonies, while APT-EC-MUT struggled with low bacterial levels, illustrating performance differences. These findings demonstrate the promise of aptamer-functionalized MNPs as the basis for quick, affordable, and portable biosensors for water quality testing, especially in resource-scarce areas. Future efforts will add colorimetric or electrochemical readouts to allow real-time, on-site detection of fecal contamination. Full article

The N-glycolylneuraminic acid (Neu5Gc), a major salivary acid molecule found on the cell surface of animals such as pigs, cows, and sheep, can be metabolically incorporated into the body through consumption of animal-derived foods like red meat. This leads to an immune response and chronic inflammation in individuals who do not naturally produce Neu5Gc, including humans and poultry, further increasing the risk of cancer. The trans-cleavage activity of Cas12a is activated by the recognition of the target aptamer by the crRNA, resulting in the cleavage of the dual-labeled probe. By combining this with immunochromatographic techniques, we established a chromatographic test strip assay that allows immediate on-site detection of Neu5Gc contamination in non-red meat samples devoid of Neu5Gc. Further optimization enabled specific detection within 25 min with a minimum detectable limit of 10 ng/mL. These analyses successfully detected the spiked samples and actual samples containing Neu5Gc. The developed lateral flow test strips based on aptamer-Cas12a can be utilized for detecting Neu5Gc contamination in non-red meat food products, animal bioproducts, and poultry feeds. Full article

Aptamers are single-stranded DNA or RNA oligonucleotides screened by systematic evolution of ligands by exponential enrichment (SELEX) methods, which are widely used in food analysis. Aptamers have the advantages of low molecular weight, ease of preparation, simplicity of chemical modification, and structural stability. Aptamers generated by SELEX are typically 80–100 bases in length, and the affinity of the aptamer can be improved by sequence optimization. Methods of aptamer optimization commonly include truncation, mutation, and chemical modification, and molecular docking, molecular dynamics, circular dichroism, and isothermal titration to assess often the binding performance of the aptamer to the target. Optimized aptamers usually enhance the affinity of the aptamer for the target and increase its sensitivity in the detection of pesticides, heavy metals, fungal toxins, pathogenic bacteria, and other objects. This paper focuses on truncation, mutation, chemical modification, the introduction of rare nucleotides, and computer-aided design. It provides an overview of non-immobilized optimization metrics. Full article

With the goal of fast and accurate diagnosis of infectious diseases, this study presents a novel electrochemical biosensor that employs a refined aptamer (C9t) for the detection of spike (S) protein SARS-CoV-2 variants in a flexible multielectrode aptasensor array with PoC capabilities. Two aptamer modifications were employed: removing the primer binding sites and including two dithiol phosphoramidite anchor molecules. Thus, reducing fabrication time from 24 to 3 h and increasing the stability and sparseness for multi-thiol aptasensors compared to a standard aptasensor using single thiols, without a reduction in aptamer density. The biosensor fabrication, optimization, and detection were verified in detail by electrochemistry, QCM-D, SPR, and XPS. The analyte–receptor binding was further confirmed spectroscopically at the level of individual molecules by AFM-IR. The aptasensor possesses a low limit of detection (8.0 fg/mL), the highest sensitivity reported for S protein (209.5 signal per concentration decade), and a wide dynamic detection range (8.0 fg/mL–38 ng/mL) in nasopharyngeal samples, covering the clinically relevant range. Furthermore, the C9t aptasensor showed high selectivity for SARS-CoV-2 S proteins over biomarkers for MERS-CoV, RSV, and Influenza. Even more, it showed a three times higher sensitivity for the Omicron in comparison to the Wuhan strain (wild type), alpha, and beta variants of the SARS-CoV-2 virus. Those results demonstrate the creation of an affordable and variant-selective refined C9t aptasensor that outperformed current rapid diagnosis tests. Full article

Background: Oncological diseases are a major focus in medicine, with millions diagnosed each year, leading researchers to seek new diagnostic and treatment methods. One promising avenue is the development of targeted therapies and rapid diagnostic tests using recognition molecules. The pharmaceutical industry is increasingly exploring nucleic acid-based therapeutics. However, producing long oligonucleotides, especially aptamers, poses significant production challenges. Objectives: This study aims to demonstrate the efficacy of using molecular modeling, supported by experimental procedures, for altering aptamer nucleotide sequences while maintaining their binding capabilities. The focus is on reducing production costs and enhancing binding dynamics by removing nonfunctional regions and minimizing nonspecific binding. Methods: A molecular modeling approach was employed to elucidate the structure of a DNA aptamer, Gli-55, facilitating the truncation of nonessential regions in the Gli-55 aptamer, which selectively binds to glioblastoma (GBM). This process aimed to produce a truncated aptamer, Gli-35, capable of forming similar structural elements to the original sequence with reduced nonspecific binding. The efficiency of the truncation was proved by flow cytometry, fluorescence polarization (FP), and confocal microscopy. Results: The molecular design indicated that the new truncated Gli-35 aptamer retained the structural integrity of Gli-55. In vitro studies showed that Gli-35 had a binding affinity comparable to the initial long aptamer while the selectivity increased. Gli-35 internalized inside the cell faster than Gli-55 and crossed the blood–brain barrier (BBB), as demonstrated in an in vitro model. Conclusions: The success of this truncation approach suggests its potential applicability in scenarios where molecular target information is limited. The study highlights a strategic and resource-efficient methodology for aptamer development. By employing molecular modeling and truncation, researchers can reduce production costs and avoid trial and error in sequence selection. This approach is promising for enhancing the efficiency of therapeutic agent development, particularly in cases lacking detailed molecular target insights. Full article

Chronic immunoinflammatory rheumatic diseases, such as axial spondyloarthritis (AxSpA), are accompanied by a dysregulation of bone remodeling. Among potential biomarkers of bone metabolism, the Wnt pathway antagonist, Dickkopf-1 (DKK-1), is of particular interest because of its potential to reflect a shift towards joint ossification or osteoporosis, but its diagnostic value needs validation. There is still a lack of stable and efficient methods of measuring serum DKK-1 levels suitable for longitude studies. The use of aptamer-based diagnostic assays could be very promising for this purpose. We generated novel anti-DKK-1 DNA aptamers from a combinatorial library with a pre-defined sequence pattern in the randomized region. This approach showed high efficacy, as only four SELEX rounds of selection produced high-affinity aptamers with dissociation constants ranging from 1.3 to 3.7 nM. A family of their truncated versions was also developed by rational design. Novel DNA aptamers functioned as capture components in a microplate ELISA-like assay with HRP-conjugated anti-DKK-1 antibody as a reporter component. We succeeded in revealing the aptamer/aptamer sandwich pairs that provided an aptamer-only sandwich colorimetric assay. The aptamer/antibody colorimetric test systems were also examined in the analyses of blood serum from AxSpA patients and shown sufficient workability. However, in a number of cases we registered significant differences between assays based on TD10 and DK4 aptamers and made some suggestions about the origin of this effect. Full article

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. Full article

Brevetoxins (PbTxs) are very potent marine neurotoxins that can cause an illness clinically described as neurologic shellfish poisoning (NSP). These toxins are cyclic polyether in chemistry and have increased their geographical distribution in the past 2 decades. However, the ethical problems as well as technical difficulties associated with currently employed analysis methods for marine toxins have spurred the quest for suitable alternatives to be applied in a regulatory monitoring regime. In this work, we reported the first instance of concurrent aptamer selection of Brevetoxin-1 (PbTx-1) and Brevetoxin-2 (PbTx-2) and constructed a biolayer interferometry (BLI) biosensor utilizing PbTx-1 aptamer as a specific recognition element. Through an in vitro selection process, we have, for the first time, successfully selected DNA aptamers with high affinity and specificity to PbTx-1 and PbTx-2 from a vast pool of random sequences. Among the selected aptamers, aptamer A5 exhibited the strongest binding affinity to PbTx-1, with an equilibrium dissociation constant (K_D) of 2.56 μM. Subsequently, we optimized aptamer A5 by truncation to obtain the core sequence (A5-S3). Further refinement was achieved through mutations based on the predictions of a QGRS mapper, resulting in aptamer A5-S3G, which showed a significant increase in the K_D value by approximately 100-fold. Utilizing aptamer A5-S3G, we fabricated a label-free, real-time optical BLI aptasensor for the detection of PbTx-1. This aptasensor displayed a broad detection range from 100 nM to 4000 nM PbTx-1, with a linear range between 100 nM and 2000 nM, and a limit of detection (LOD) as low as 4.5 nM. Importantly, the aptasensor showed no cross-reactivity to PbTx-2 or other marine toxins, indicating a high level of specificity for PbTx-1. Moreover, the aptasensor exhibited excellent reproducibility and stability when applied for the detection of PbTx-1 in spiked shellfish samples. We strongly believe that this innovative aptasensor offers a promising alternative to traditional immunological methods for the specific and reliable detection of PbTx-1. Full article

Esophageal cancer ranks the seventh in cancer incidence and the sixth in cancer death. Esophageal squamous cell carcinoma (ESCC) accounts for approximately 90% of the total cases of esophageal cancer. Chemotherapy is the most effective drug-based method for treatment of esophageal cancer. However, severe side effects of traditional chemotherapy limit its treatment efficacy. Targeted chemotherapy can deliver chemotherapeutic drugs to cancer cells and specifically kill these cells with reduced side effects. In the work, the bivalent aptamer-DNA carrier (BAD) was designed by using an ESCC cell-specific aptamer as the recognition molecule and a GC base-rich DNA sequence as the drug carrier. With doxorubicin (Dox) as chemotherapeutic drugs, the bivalent aptamer-DNA-Dox conjugate (BADD) was constructed for targeted killing of ESCC cells. Firstly, the truncated A2(35) aptamer with a retained binding ability was obtained through optimization of an intact A2(80) aptamer and was used to fuse with DNA carrier sequences for constructing the BAD through simple DNA hybridization. The results of gel electrophoresis and flow cytometry analysis showed that the BAD was successfully constructed and had a stronger binding affinity than monovalent A2(35). Then, the BAD was loaded with Dox drugs to construct the BADD through noncovalent intercalation. The results of fluorescence spectra and flow cytometry assays showed that the BADD was successfully constructed and can bind to target cells strongly. Confocal imaging further displayed that the BADD can be specifically internalized into target cells and release Dox. The results of CCK-8 assays, Calcein AM/PI staining, and wound healing assays demonstrated that the BADD can specifically kill target cells, but not control cells. Our results demonstrate that the developed BADD can specifically deliver doxorubicin to target ESCC cells and selectively kill these cells, offering a potentially effective strategy for targeted chemotherapy of ESCC. Full article

A myogenetic oligodeoxynucleotide (myoDN), iSN04 (5′-AGA TTA GGG TGA GGG TGA-3′), is a single-stranded 18-base telomeric DNA that serves as an anti-nucleolin aptamer and induces myogenic differentiation, which is expected to be a nucleic acid drug for the prevention of disease-associated muscle wasting. To improve the drug efficacy and synthesis cost of myoDN, shortening the sequence while maintaining its structure-based function is a major challenge. Here, we report the novel 12-base non-telomeric myoDN, iMyo01 (5′-TTG GGT GGG GAA-3′), which has comparable myogenic activity to iSN04. iMyo01 as well as iSN04 promoted myotube formation of primary-cultured human myoblasts with upregulation of myogenic gene expression. Both iMyo01 and iSN04 interacted with nucleolin, but iMyo01 did not bind to berberine, the isoquinoline alkaloid that stabilizes iSN04. Nuclear magnetic resonance revealed that iMyo01 forms a G-quadruplex structure despite its short sequence. Native polyacrylamide gel electrophoresis and a computational molecular dynamics simulation indicated that iMyo01 forms a homodimer to generate a G-quadruplex. These results provide new insights into the aptamer truncation technology that preserves aptamer conformation and bioactivity for the development of efficient nucleic acid drugs. Full article

Aptamers are currently being investigated for their potential to improve virotherapy. They offer several advantages, including the ability to prevent the aggregation of viral particles, enhance target specificity, and protect against the neutralizing effects of antibodies. The purpose of this study was to comprehensively investigate an aptamer capable of enhancing virotherapy. This involved characterizing the previously selected aptamer for vaccinia virus (VACV), evaluating the aggregation and molecular interaction of the optimized aptamers with the recombinant oncolytic virus VV-GMCSF-Lact, and estimating their immunoshielding properties in the presence of human blood serum. We chose one optimized aptamer, NV14t_56, with the highest affinity to the virus from the pool of several truncated aptamers and built its 3D model. The NV14t_56 remained stable in human blood serum for 1 h and bound to VV-GMCSF-Lact in the micromolar range (Kd ≈ 0.35 μM). Based on dynamic light scattering data, it has been demonstrated that aptamers surround viral particles and inhibit aggregate formation. In the presence of serum, the hydrodynamic diameter (by intensity) of the aptamer–virus complex did not change. Microscale thermophoresis (MST) experiments showed that NV14t_56 binds with virus (EC50 = 1.487 × 10⁹ PFU/mL). The analysis of the amplitudes of MST curves reveals that the components of the serum bind to the aptamer–virus complex without disrupting it. In vitro experiments demonstrated the efficacy of VV-GMCSF-Lact in conjunction with the aptamer when exposed to human blood serum in the absence of neutralizing antibodies (Nabs). Thus, NV14t_56 has the ability to inhibit virus aggregation, allowing VV-GMCSF-Lact to maintain its effectiveness throughout the storage period and subsequent use. When employing aptamers as protective agents for oncolytic viruses, the presence of neutralizing antibodies should be taken into account. Full article

Accurate determination of serotonin (ST) provides insight into neurological processes and enables applications in clinical diagnostics of brain diseases. Herein, we present an electrochemical aptasensor based on truncated DNA aptamers and a polyethylene glycol (PEG) molecule-functionalized sensing interface for highly sensitive and selective ST detection. The truncated aptamers have a small size and adopt a stable stem-loop configuration, which improves the accessibility of the aptamer for the analyte and enhances the sensitivity of the aptasensor. Upon target binding, these aptamers perform a conformational change, leading to a variation in the Faraday current of the redox tag, which was recorded by square wave voltammetry (SWV). Using PEG as blocking molecules minimizes nonspecific adsorption of other interfering molecules and thus endows an enhanced antifouling ability. The proposed electrochemical aptamer sensor showed a wide range of detection lasting from 0.1 nM to 1000 nM with a low limit of detection of 0.14 nM. Owing to the unique properties of aptamer receptors, the aptasensor also exhibits high selectivity and stability. Furthermore, with the reduced unspecific adsorption, assaying of ST in human serum and artificial cerebrospinal fluid (aCSF) showed excellent performance. The reported strategy of utilizing antifouling PEG describes a novel approach to building antifouling aptasensors and holds great potential for neurochemical investigations and clinical diagnosis. Full article

Aptamers are an excellent choice for the selective detection of small molecules. However, the previously reported aptamer for chloramphenicol suffers from low affinity, probably as a result of steric hindrance due to its bulky nature (80 nucleotides) leading to lower sensitivity in analytical assays. The present work was aimed at improving this binding affinity by truncating the aptamer without compromising its stability and three-dimensional folding. Shorter aptamer sequences were designed by systematically removing bases from each or both ends of the original aptamer. Thermodynamic factors were evaluated computationally to provide insight into the stability and folding patterns of the modified aptamers. Binding affinities were evaluated using bio-layer interferometry. Among the eleven sequences generated, one aptamer was selected based on its low dissociation constant, length, and regression of model fitting with association and dissociation curves. The dissociation constant could be lowered by 86.93% by truncating 30 bases from the 3′ end of the previously reported aptamer. The selected aptamer was used for the detection of chloramphenicol in honey samples, based on a visible color change upon the aggregation of gold nanospheres caused by aptamer desorption. The detection limit could be reduced 32.87 times (1.673 pg mL⁻¹) using the modified length aptamer, indicating its improved affinity as well as its suitability in real-sample analysis for the ultrasensitive detection of chloramphenicol. Full article