Pool Of Random Sequences Research Articles

Brevetoxin Aptamer Selection and Biolayer Interferometry Biosensor Application.

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 (KD) 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 KD 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.

Electrochemical aptasensors for pathogenic detection toward point-of-care diagnostics.

A biosensor system refers to a biomedical device, which detects biological, chemical, or biochemical components by converting those signals to an electrical signal by utilizing and uniting physical or chemical transducer with biorecognition elements. An electrochemical biosensor is generally based on the reaction of either production or consumption of electrons under a three-electrode system. Biosensor systems are exploited in a wide range of areas, such as medicine, agriculture, husbandry, food, industry, environment protection, quality control, waste disposal, and the military. Pathogenic infections are the third leading cause of death worldwide after cardiovascular diseases and cancer. Therefore, there is an urgent need for effective diagnostic tools to control food, water, and soil contamination result in protecting human life and health. Aptamers are peptide or oligonucleotide-based molecules that show very high affinity to their targets that are produced from large pools of random amino acid or oligonucleotide sequences. Generally, aptamers have been utilized for fundamental sciences and clinical implementations for their target-specific affinity and have been intensely exploited for different kinds of biosensor applications for approximately 30 years. The convergence of aptamers with biosensor systems enabled the construction of voltammetric, amperometric, and impedimetric biosensors for the detection of specific pathogens. In this review, electrochemical aptamer biosensors were evaluated by discussing the definition, types, and production techniques of aptamers, the advantages of aptamers as a biological recognition element against their alternatives, and a wide range of aptasensor examples from literature in the detection of specific pathogens.

Toehold-Mediated Strand Displacement in Random Sequence Pools.

Toehold-mediated strand displacement (TMSD) has been used extensively for molecular sensing and computing in DNA-based molecular circuits. As these circuits grow in complexity, sequence similarity between components can lead to cross-talk, causing leak, altered kinetics, or even circuit failure. For small non-biological circuits, such unwanted interactions can be designed against. In environments containing a huge number of sequences, taking all possible interactions into account becomes infeasible. Therefore, a general understanding of the impact of sequence backgrounds on TMSD reactions is of great interest. Here, we investigate the impact of random DNA sequences on TMSD circuits. We begin by studying individual interfering strands and use the obtained data to build machine learning models that estimate kinetics. We then investigate the influence of pools of random strands and find that the kinetics are determined by only a small subpopulation of strongly interacting strands. Consequently, their behavior can be mimicked by a small collection of such strands. The equilibration of the circuit with the background sequences strongly influences this behavior, leading to up to 1 order of magnitude difference in reaction speed. Finally, we compare two established and one novel technique that speed up TMSD reactions in random sequence pools: a three-letter alphabet, protection of toeholds by intramolecular secondary structure, or by an additional blocking strand. While all of these techniques were useful, only the latter can be used without sequence constraints. We expect that our insights will be useful for the construction of TMSD circuits that are robust to molecular noise.

Isolation and Characterization of a ssDNA Aptamer against Major Soluble Antigen of Renibacterium salmoninarum.

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.

Diverse high-affinity DNA aptamers for wild-type and B.1.1.7 SARS-CoV-2 spike proteins from a pre-structured DNA library.

We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.

Structured sequences emerge from random pool when replicated by templated ligation

The central question in the origin of life is to understand how structure can emerge from randomness. The Eigen theory of replication states, for sequences that are copied one base at a time, that the replication fidelity has to surpass an error threshold to avoid that replicated specific sequences become random because of the incorporated replication errors [M. Eigen, Naturwissenschaften 58 (10), 465-523 (1971)]. Here, we showed that linking short oligomers from a random sequence pool in a templated ligation reaction reduced the sequence space of product strands. We started from 12-mer oligonucleotides with two bases in all possible combinations and triggered enzymatic ligation under temperature cycles. Surprisingly, we found the robust creation of long, highly structured sequences with low entropy. At the ligation site, complementary and alternating sequence patterns developed. However, between the ligation sites, we found either an A-rich or a T-rich sequence within a single oligonucleotide. Our modeling suggests that avoidance of hairpins was the likely cause for these two complementary sequence pools. What emerged was a network of complementary sequences that acted both as templates and substrates of the reaction. This self-selecting ligation reaction could be restarted by only a few majority sequences. The findings showed that replication by random templated ligation from a random sequence input will lead to a highly structured, long, and nonrandom sequence pool. This is a favorable starting point for a subsequent Darwinian evolution searching for higher catalytic functions in an RNA world scenario.

Cofactors as Molecular Fossils To Trace the Origin and Evolution of Proteins.

Due to their early origin and extreme conservation, cofactors are valuable molecular fossils for tracing the origin and evolution of proteins. First, as the order of protein folds binding with cofactors roughly coincides with protein-fold chronology, cofactors are considered to have facilitated the origin of primitive proteins by selecting them from pools of random amino acid sequences. Second, in the subsequent evolution of proteins, cofactors still played an important role. More interestingly, as metallic cofactors evolved with geochemical variations, some geochemical events left imprints in the chronology of protein architecture; this provides further evidence supporting the coevolution of biochemistry and geochemistry. In this paper, we attempt to review the molecular fossils used in tracing the origin and evolution of proteins, with a special focus on cofactors.

In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole–activated substrates

The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5'-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide-activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3'-5' phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.

Lab-on-a-Chip Systems for Aptamer-Based Biosensing.

Aptamers are oligonucleotides or peptides that are selected from a pool of random sequences that exhibit high affinity toward a specific biomolecular species of interest. Therefore, they are ideal for use as recognition elements and ligands for binding to the target. In recent years, aptamers have gained a great deal of attention in the field of biosensing as the next-generation target receptors that could potentially replace the functions of antibodies. Consequently, it is increasingly becoming popular to integrate aptamers into a variety of sensing platforms to enhance specificity and selectivity in analyte detection. Simultaneously, as the fields of lab-on-a-chip (LOC) technology, point-of-care (POC) diagnostics, and personal medicine become topics of great interest, integration of such aptamer-based sensors with LOC devices are showing promising results as evidenced by the recent growth of literature in this area. The focus of this review article is to highlight the recent progress in aptamer-based biosensor development with emphasis on the integration between aptamers and the various forms of LOC devices including microfluidic chips and paper-based microfluidics. As aptamers are extremely versatile in terms of their utilization in different detection principles, a broad range of techniques are covered including electrochemical, optical, colorimetric, and gravimetric sensing as well as surface acoustics waves and transistor-based detection.

Large Phenotypic Enhancement of Structured Random RNA Pools.

Laboratory evolution of functional RNAs has applications in many areas of chemical and synthetic biology. In vitro selections critically depend on the presence of functional molecules, such as aptamers and ribozymes, in the starting sequence pools. For selection of novel functions the pools are typically transcribed from random-sequence DNA templates, yielding a highly diverse set of RNAs that contain a multitude of folds and biochemical activities. The phenotypic potential, the frequency of functional RNAs, is very low, requiring large complexity of starting pools, surpassing 1015 different sequences, to identify highly active isolates. Furthermore, the majority of random sequences is not structured and has a high propensity for aggregation; the in vitro selection process thus involves not just enrichment of functional RNAs, but also their purification from aggregation-prone "free-riders". We reasoned that purification of the nonaggregating, monomeric subpopulation of a random-sequence RNA pool will yield pools of folded, functional RNAs. We performed six rounds of selection for monomeric sequences and show that the enriched population is compactly folded. In vitro selections originating from various mixtures of the compact pool and a fully random pool showed that sequences from the compact pool always dominate the population once a biochemical activity is detectable. A head-to-head competition of the two pools starting from a low (5 × 1012) sequence diversity revealed that the phenotypic potential of the compact pool is about 1000-times higher than the fully random pool. A selection for folded and monomeric RNA pools thus greatly increases the frequency of functional RNAs from that seen in random-sequence pools, providing a facile experimental approach to isolation of highly active functional RNAs from low-diversity populations.

Structure of an RNA aptamer in complex with the fluorophore tetramethylrhodamine.

RNA aptamers—artificially created RNAs with high affinity and selectivity for their target ligand generated from random sequence pools—are versatile tools in the fields of biotechnology and medicine. On a more fundamental level, they also further our general understanding of RNA-ligand interactions e. g. in regard to the relationship between structural complexity and ligand affinity and specificity, RNA structure and RNA folding. Detailed structural knowledge on a wide range of aptamer–ligand complexes is required to further our understanding of RNA–ligand interactions. Here, we present the atomic resolution structure of an RNA–aptamer binding to the fluorescent xanthene dye tetramethylrhodamine. The high resolution structure, solved by NMR-spectroscopy in solution, reveals binding features both common and different from the binding mode of other aptamers with affinity for ligands carrying planar aromatic ring systems such as the malachite green aptamer which binds to the tetramethylrhodamine related dye malachite green or the flavin mononucleotide aptamer.

Detection of Gram-negative bacterial outer membrane vesicles using DNA aptamers

Infection of various pathogenic bacteria causes severe illness to human beings. Despite the research advances, current identification tools still exhibit limitations in detecting Gram-negative bacteria with high accuracy. In this study, we isolated single-stranded DNA aptamers against multiple Gram-negative bacterial species using Toggle-cell-SELEX (systemic evolution of ligands by exponential enrichment) and constructed an aptamer-based detection tool towards bacterial secretory cargo released from outer membranes of Gram-negative bacteria. Three Gram-negative bacteria, Escherichia coli DH5α, E. coli K12, and Serratia marcescens, were sequentially incubated with the pool of random DNA sequences at each SELEX loop. Two aptamers selected, GN6 and GN12, were 4.2-times and 3.6-times higher binding to 108 cells of Gram-negative bacteria than to Gram-positive bacteria tested, respectively. Using GN6 aptamer, we constructed an Enzyme-linked aptamer assay (ELAA) to detect bacterial outer membrane vesicles (OMVs) of Gram-negative bacteria, which contain several outer membrane proteins with potent immunostimulatory effects. The GN6-ELAA showed high sensitivity to detect as low as 25 ng/mL bacterial OMVs. Aptamers developed in this study show a great potential to facilitate medical diagnosis and early detection of bacterial terrorism, based on the ability to detect bacterial OMVs of multiple Gram-negative bacteria.

In Silico Engineering of Synthetic Binding Proteins from Random Amino Acid Sequences

SummarySynthetic proteins with high affinity and selectivity for a protein target can be used as research tools, biomarkers, and pharmacological agents, but few methods exist to design such proteins de novo. To this end, the In-Silico Protein Synthesizer (InSiPS) was developed to design synthetic binding proteins (SBPs) that bind pre-determined targets while minimizing off-target interactions. InSiPS is a genetic algorithm that refines a pool of random sequences over hundreds of generations of mutation and selection to produce SBPs with pre-specified binding characteristics. As a proof of concept, we design SBPs against three yeast proteins and demonstrate binding and functional inhibition of two of three targets in vivo. Peptide SPOT arrays confirm binding sites, and a permutation array demonstrates target specificity. Our foundational approach will support the field of de novo design of small binding polypeptide motifs and has robust applicability while offering potential advantages over the limited number of techniques currently available.

Single-round isolation of diverse RNA aptamers from a random sequence pool.

Aptamers are oligonucleotide ligands with specific binding affinity to target molecules. Generally, RNA aptamers are selected from an RNA pool with random sequences, using the technique termed SELEX, in which the target-binding RNA molecules are repeatedly isolated and exponentially amplified. Despite several advantages, SELEX often produces uncertain results during the iterative amplifications of the rare target-binding RNA molecules. Here, we develop a non-repeated, primer-less and target immobilization-free isolation method for generating RNA aptamers, which is robust to experimental noise. Uniquely, this method focuses on finding and removal of non-aptamer sequences from the RNA pool by RNase digestion leaving target-bound aptamer molecules, and thus is independent of aptamer types. The undigested RNA sequences remaining are so few in number that they must be mixed with a large excess of a known sequence for further manipulations and this sequence is then removed by restriction digestion followed by high-throughput sequencing analysis to identify aptamers. Using this method, we generated multiple RNA aptamers targeting α-thrombin and TGFβ1 proteins, independently. This method potentially generates thousands of sequences as aptamer candidates, which may enable us to predict a common average sequence or structural property of these aptamers that is different from input RNA.

Analysis of in vitro evolution reveals the underlying distribution of catalytic activity among random sequences.

The emergence of catalytic RNA is believed to have been a key event during the origin of life. Understanding how catalytic activity is distributed across random sequences is fundamental to estimating the probability that catalytic sequences would emerge. Here, we analyze the in vitro evolution of triphosphorylating ribozymes and translate their fitnesses into absolute estimates of catalytic activity for hundreds of ribozyme families. The analysis efficiently identified highly active ribozymes and estimated catalytic activity with good accuracy. The evolutionary dynamics follow Fisher's Fundamental Theorem of Natural Selection and a corollary, permitting retrospective inference of the distribution of fitness and activity in the random sequence pool for the first time. The frequency distribution of rate constants appears to be log-normal, with a surprisingly steep dropoff at higher activity, consistent with a mechanism for the emergence of activity as the product of many independent contributions.

Analysis of in vitro evolution reveals the underlying distribution of catalytic activity among random sequences.

The emergence of catalytic RNA is believed to have been a key event during the origin of life. Understanding how catalytic activity is distributed across random sequences is fundamental to estimating the probability that catalytic sequences would emerge. Here, we analyze the in vitro evolution of triphosphorylating ribozymes and translate their fitnesses into absolute estimates of catalytic activity for hundreds of ribozyme families. The analysis efficiently identified highly active ribozymes and estimated catalytic activity with good accuracy. The evolutionary dynamics follow Fisher's Fundamental Theorem of Natural Selection and a corollary, permitting retrospective inference of the distribution of fitness and activity in the random sequence pool for the first time. The frequency distribution of rate constants appears to be log-normal, with a surprisingly steep dropoff at higher activity, consistent with a mechanism for the emergence of activity as the product of many independent contributions.

Nucleic acid-based aptamers: applications, development and clinical trials.

Short single-stranded oligonucleotides called aptamers, often termed as chemical antibodies, have been developed as powerful alternatives to traditional antibodies with respect to their obvious advantages like high specificity and affinity, longer shelf-life, easier manufacturing protocol, freedom to introduce chemical modifications for further improvement, etc. Reiterative selection process of aptamers over 10-15 cycles starting from a large initial pool of random nucleotide sequences renders them with high binding affinity, thereby making them extremely specific for their targets. Aptamer-based detection systems are well investigated and likely to displace primitive detection systems. Aptamer chimeras (combination of aptamers with another aptamer or biomacromolecule or chemical moiety) have the potential activity of both the parent molecules, and thus hold the capability to perform diverse functions at the same time. Owing to their extremely high specificity and lack of immunogenicity or pathogenicity, a number of other aptamers have recently entered clinical trials and have garnered favorable attention from pharmaceutical companies. Promising results from the clinical trials provide new hope to change the conventional style of therapy. Aptamers have attained high therapeutic relevance in a short time as compared to synthetic drugs and/or other modes of therapy. This review follows the various trends in aptamer technology including production, selection, modifications and success in clinical fields. It focusses largely on the various applications of aptamers which mainly depend upon their selection procedures. The review also sheds light on various modifications and chimerizations that have been implemented in order to improve the stability and functioning of the aptamers, including introduction of locked nucleic acids (LNAs). The application of various aptamers in detection systems has been discussed elaborately in order to stress on their role as efficient diagnostic agents. The key aspect of this review is focused on success of aptamers on the basis of their performance in clinical trials for various diseases.

Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display.

Noncoding RNAs (ncRNAs) comprise an important class of regulatory molecules that mediate a vast array of biological processes. This broad functional capacity has also facilitated the design of artificial ncRNAs with novel functions. To further investigate and harness these capabilities, we developed CRISPR-Display (“CRISP-Disp”), a targeted localization method that uses Sp. Cas9 to deploy large RNA cargos to DNA loci. We demonstrate that exogenous RNA domains can be functionally appended onto the CRISPR scaffold at multiple insertion points, allowing the construction of Cas9 complexes with protein-binding cassettes, artificial aptamers, pools of random sequences, and RNAs up to 4.8 kilobases in length, including natural lncRNAs. Unlike most existing CRISPR methods, CRISP-Disp allows simultaneous multiplexing of distinct functions at multiple targets, limited only by the number of available functional RNA motifs. We anticipate that this technology will provide a powerful method with which to ectopically localize functional RNAs and ribonucleoprotein (RNP) complexes at specified genomic loci.

Aptamer-Based Electrochemical Biosensors for Marine Toxins

Marine toxins are naturally occurring chemicals that can contaminate certain seafood. They are usually tasteless, odourless and have no toxic effect on shellfish. However, they exhibit toxicity towards marine mammals, birds, fish and humans. The ethical problems as well as the technical difficulties associated with the currently employed analysis methods for marine toxins are encouraging the research for suitable alternatives to be applied in a regulatory monitoring regime. Here, we report electrochemical biosensing platforms for okadiac acid (OA) as well as brevetoxin-2 (BTX-2) detection using aptamers as specific receptors. Using in vitro selection, high affinity DNA aptamers to OA and BTX-2 were successfully selected for the first time from a large pool of random sequences. The binding of the toxins to aptamer pools/clones was monitored using fluorescence and electrochemical impedance spectroscopy (EIS). Most of the selected aptamers exhibited high binding affinity to their target toxins with a dissociation constants in the nanomolar range. The effects of the incubation time, pH and metal ions concentrations on the aptamers-toxins binding were studied. The highest affinity aptamers were then used to construct label-free impedimetric biosensors for both OA and BTX-2 detection showing good sensitivity. A high degree of cross reactivity of the selected BTX aptamers to the two similar congeners, BTX-2 and BTX-3 was observed, whereas no cross reactivity to other marine toxins was obtained. Moreover, the aptasensors were applied for the detection of OA and BTX-2 in spiked shellfish extracts showing very good recovery percentages. We believe that the continual emergence of novel, high-affinity aptamers will open the way to a variety of biosensing architectures, particularly for small-molecule toxin detection in complex samples.

Aptamer-based competitive electrochemical biosensor for brevetoxin-2.

Brevetoxins (BTXs) are very potent marine neurotoxins that increased in geographical distribution in the past decade causing the illness clinically described as neurological shellfish poisoning (NSP). The ethical problems as well as the technical difficulties associated with the currently employed analysis methods for marine toxins are encouraging the research for suitable alternatives to be applied in a regulatory monitoring regime. Here, we report an electrochemical biosensor platform for BTX-2 detection utilising aptamer as specific receptor. Using in vitro selection, high affinity DNA aptamers to BTX-2 were successfully selected for the first time from a large pool of random sequences. The binding of BTX-2 to aptamer pools/clones was monitored using fluorescence and electrochemical impedance spectroscopy (EIS). The aptamer BT10 exhibited the highest binding affinity to BTX-2, with a dissociation constant of 42nM. The effects of the incubation time, pH and metal ions concentrations on the aptamer-toxin binding were studied. The aptamer BT10 was used to construct a label-free competitive impedimetric biosensor for BTX-2 achieving a detection limit of 106pg/ml. We observed a high degree of cross reactivity of the selected aptamer to the two similar congeners, BTX-2 and -3, whereas no cross reactivity to other marine toxins was obtained. Moreover, the aptasensor was applied for the detection of BTX-2 in spiked shellfish extract showing a very high recovery percentage. We believe that the proposed aptasensor will facilitate the routine detection of BTX-2 in food samples.