Selection and Characterization of a Novel DNA Aptamer for Label-Free Fluorescence Biosensing of Ochratoxin A

Nucleic acid aptamers are emerging as useful molecular recognition tools for food safety monitoring. However, practical and technical challenges limit the number and diversity of available aptamer probes that can be incorporated into novel sensing schemes. This work describes the selection of novel DNA aptamers that bind to the important food contaminant ochratoxin A (OTA). Following 15 rounds of in vitro selection, sequences were analyzed for OTA binding. Two of the isolated aptamers demonstrated high affinity binding and selectivity to this mycotoxin compared to similar food adulterants. These sequences, as well as a truncated aptamer (minimal sequence required for binding), were incorporated into a SYBR® Green I fluorescence-based OTA biosensing scheme. This label-free detection platform is capable of rapid, selective, and sensitive OTA quantification with a limit of detection of 9 nM and linear quantification up to 100 nM

Aptamer based electrochemical sensors for emerging environmental pollutants

International audienceEnvironmental contaminants monitoring is one of the key issues in understanding and managing hazards to human health and ecosystems. In this context, aptamer based electrochemical sensors have achieved intense significance because of their capability to resolve a potentially large number of problems and challenges in environmental contamination. An aptasensor is a compact analytical device incorporating an aptamer (oligonulceotide) as the sensing element either integrated within or intimately associated with a physiochemical transducer surface. Nucleic acid is well known for the function of carrying and passing genetic information, however, it has found a key role in analytical monitoring during recent years. Aptamer based sensors represent a novelty in environmental analytical science and there are great expectations for their promising performance as alternative to conventional analytical tools. This review paper focuses on the recent advances in the development of aptamer based electrochemical sensors for environmental applications with special emphasis on emerging pollutants

Recent Achievements in Electrochemical and Surface Plasmon Resonance Aptasensors for Mycotoxins Detection

Mycotoxins are secondary metabolites of fungi that contaminate agriculture products. Their release in the environment can cause severe damage to human health. Aptasensors are compact analytical devices that are intended for the fast and reliable detection of various species able to specifically interact with aptamers attached to the transducer surface. In this review, assembly of electrochemical and surface plasmon resonance (SPR) aptasensors are considered with emphasis on the mechanism of signal generation. Moreover, the properties of mycotoxins and the aptamers selected for their recognition are briefly considered. The analytical performance of bio-sensors developed within last three years makes it possible to determine mycotoxin residues in water and agriculture/food products on the levels below their maximal admissible concentrations. Requirements for the development of sample treatment and future trends in aptasensors are also discussed. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Funding: T.K. acknowledges funding by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities (grant No 0671–2020−0063). T.H. acknowledges funding from the Science Grant Agency VEGA, project No.: 1/0419/20

A New Approach for Detection of Aflatoxin B1

Aflatoxin B1 (AFB1) is harmful to human health, mainly resulting from its toxic effects on the liver. AFB1 can lead to liver cell necrosis, hemorrhage, fibrosis, cirrhosis, etc. Acute AFB1 exposure at high levels can lead to hepatitis, whereas chronic exposure can result in liver cancer. In the past decades, a series of methods and techniques for detecting AFB1, including enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC), have been developed. This study reviewed the detection methods of AFB1 and the corresponding utilization and summarizes all methods for evaluating the toxification of AFB1

Identification and Biosensing Application of Molecular Recognition Elements

Molecular recognition elements (MREs) are biomolecules such as single-stranded DNA (ssDNA), RNA, small peptides and antibody fragments that can bind to user defined targets with high affinities and specificities. This binding property allows MREs to have a wide range of applications, including therapeutic, diagnostic, and biosensor applications. The identification of MREs can be achieved by using the process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This process begins with a large library of 109 to 1015 different random molecules, molecules that bind to the user defined target or positive target are enriched in the process. Subsequently, this process can be modified and tailored to direct the enriched library away from binding to related targets or negative targets, and thus increasing the specificity. Single-stranded DNA (ssDNA) MREs are particularly favorable for biosening applications due to their relative stability, reusability and low cost in production. This work investigated the identification and application of ssDNA MREs to detect different bacterial toxins and pesticide.;In Chapter 1, it begins by reviewing recent discovery and advancement in the SELEX technique for the identification and biosensing application of ssDNA MREs specific for bacteria, viruses, their related biomolecules, and selected environmental toxins. It is then followed by a brief discussion on major biosensing principles based upon ssDNA MREs. In Chapter 2, the pilot project of this work, ssDNA MRE specific for Pseudomonas aeruginosa exotoxin A was identified. In this chapter, a novel variation of SELEX called Decoy-SELEX, previously developed by our laboratory is described in greater detail. Additionally, the development of a ssDNA MRE modified enzyme-linked immunosorbent assay (ELISA) for the exotoxin A detection is also discussed. In Chapter 3, similar methodology was applied to identify a ssDNA MRE specific for the second target, Clostridium difficile toxin B. Subsequently, similar ssDNA MRE modified ELISA was developed for target detection in clinically relevant samples. In Chapter 4, ssDNA MRE specific for alpha toxin of Staphylococcus aureus was identified, and it was applied for sensitive detection of the target in clinically relevant samples. In Chapter 5, the overall conclusion and potential future studies as a result from this work is discussed. Lastly, in Appendix, the project of identifying and potential future application of ssDNA MREs specific for a pesticide, Fipronil is described.;Overall, this work has shown the proof-of-principle of using ssDNA MREs in biosensing application for target detections in clinically relevant samples. The work will be useful in the development of potential point-of-care diagnostic tools for rapid diagnosis of bacterial infections

Aptamer-Based Biosensor for Detection of Mycotoxins

Mycotoxins are a large type of secondary metabolites produced by fungi that pose a great hazard to and cause toxic reactions in humans and animals. A majority of countries and regulators, such as the European Union, have established a series of requirements for their use, and they have also set maximum tolerance levels. The development of high sensitivity and a specific analytical platform for mycotoxins is much in demand to address new challenges for food safety worldwide. Due to the superiority of simple, rapid, and low-cost characteristics, aptamer-based biosensors have successfully been developed for the detection of various mycotoxins with high sensitivity and selectivity compared with traditional instrumental methods and immunological approaches. In this article, we discuss and analyze the development of aptasensors for mycotoxins determination in food and agricultural products over the last 11 years and cover the literatures from the first report in 2008 until the present time. In addition, challenges and future trends for the selection of aptamers toward various mycotoxins and aptasensors for multi-mycotoxins analyses are summarized. Given the promising development and potential application of aptasensors, future research studies made will witness the great practicality of using aptamer-based biosensors within the field of food safety.Project of risk assessment on raw milk (GJFP2019026

Selection and characterization of DNA aptamers for estradiol and ethynylestradiol for aptasensor development

Small organic contaminants have been widely detected in the surface and ground waters of this nation. A sub-class of these contaminants called endocrine disrupting compounds (EDCs) are known to have adverse effects on aquatic and human health. Among the EDCs, natural hormone 17β-estradiol (E2) and synthetic hormone 17α-ethynylestradiol (EE) possess high estrogenic potency and hence are contaminants of interest. Conventional methods to detect these compounds are expensive, time consuming and need implementation by an expert. By contrast, antibody-based assays are relatively inexpensive and commercially available but suffer from poor selectivity. A promising alternative makes use of DNA aptamers as molecular recognition elements. In order to evaluate the potential of DNA aptamers and aptasensors to detect small organics in natural waters, the following objectives were pursued: (1) critically review DNA aptamers and aptasensors developed for small organic molecules and assess their use for monitoring environmentally relevant organics, (2) select and characterize DNA aptamers that bind to E2 and EE and, (3) study the effect of immobilization on the binding affinity of the selected E2 and EE aptamers. A review of ~80 aptamers and ~200 aptasensors for small organics was conducted to identify factors that affect binding affinity of the aptamer and limits of detection (LODs) of the aptasensor. Based on regression analyses, aptamer binding affinities are found to have a weak relationship with hydrophobicity of the target and length of the aptamer (p-values<0.05). Independent t-tests comparing aptasensor LODs suggest that the electrochemical platform is significantly more sensitive than colorimetric and fluorescence-based platforms. The inherent binding affinity of the aptamer was found to have a significant effect on the LOD of the aptasensor. While some fabricated aptasensors are sufficiently sensitive to detect contaminants at environmentally relevant concentrations, they are often associated with complex fabrication steps, and/or interference from structurally similar analogs. As a result, aptasensor commercialization faces many challenges including reusability, reproducibility and robustness. In vitro selections were conducted with different selection pressures to isolate sensitive and selective DNA aptamers for E2 and EE. An equilibrium-filtration assay was used to determine dissociation constants (Kd) of the aptamer towards its parent target and its analogues. The E2 aptamers, E2Apt1 and E2Apt2 were found to have Kd values of 0.6 µM. They bound to analogue estrone (E1) with a similar affinity but were at least 74-fold more selective over EE. The EE aptamers Kd values are 0.5-1 µM. While one EE aptamer (EEApt1) was 53-fold more selective for EE over E2 and E1, the second EE aptamer (EEApt2) bound to all three EDCs (E1, E2 and EE) with similar affinities. The aptamers maintained their binding affinities in natural waters samples (tap water and lake water). DMS probing of the structure of the DNA aptamer revealed that the binding regions were mostly located in the single-stranded loop regions of the aptamer. Aptasensors typically employ immobilized aptamers though the aptamers are selected and characterized while free or unattached in solution. The Kd values of immobilized selective aptamers were evaluated using magnetic microbeads surface for attachment. E2Apt1 immobilized at either end (5′ or 3′) and E2Apt2 immobilized at the 3′ end retain their binding affinity. The binding affinity is inversely correlated to the average linear distance of the binding pocket from the immobilized end. This result suggests that unwanted interactions between the aptamer and other moieties are more likely when the binding pocket is further away from the surface. Binding curve of E2Apt2 immobilized at the 5′ end indicates potential dimerization at high loadings of aptamer on the beads due to increased proximity between aptamer strands. EEApt1 loses its binding affinity upon immobilization potentially due to disruption in its tertiary structure upon attachment to the surface. Despite no loss in binding affinity upon immobilization, E2Apt1 (5′) shows no significant change in electrochemical current on binding to E2 when incorporated into an electrochemical sensor. This result implies an insufficient conformational change of the aptamer on binding to the target. Overall, this work identifies the first aptamers for EE and selective aptamers for E2, while also highlighting the issues with development of aptamers and their eventual incorporation into aptasensors to detect small organics. Two major concerns are (1) immobilizing aptamers in sensor platforms while selections of aptamers are conducted with free/unattached aptamers, resulting in loss of binding affinity and (2) insufficient conformational change of the aptamer on binding to small molecule targets, resulting in a lack of change in the sensor signal. The findings from this dissertation support additional research directions regarding employing free aptamers in sensors and/or conducting new selections for aptamers using a DNA pool that is attached to a surface

Jednostavna i ekonomična metoda detekcije saksitoksina praćenjem promjene struktura aptamera

A simple method to detect saxitoxin (STX), one of the main components of the paralytic shellfish poison from red tide, has been developed. By using a next generation dye for double-stranded DNA we were able to differentiate fluorescence from STX-binding aptamers when exposed to different concentrations of STX, suggesting a change in aptamer folding upon target binding. The developed method is extremely rapid, only requiring small sample volumes, with quantitative results in the concentration range of 15 ng/mL to 3 μg/mL of STX, with a detection limit of 7.5 ng/mL.Razvijena je jednostavna metoda detekcije saksitoksina, jednog od glavnih sastojaka paralitičkog toksina školjkaša prisutnih u moru preplavljenom dinoflagelatima (tzv. crvena plima). Upotrebom najnovije generacije fluorescentne boje za dvolančanu DNA bilo je moguće razlikovati fluorescenciju aptamera specifičnog za saksitoksin pri različitim koncentracijama, zbog čega smo pretpostavili da se struktura aptamera promijenila nakon vezanja na saksitoksin. Razvijena metoda je jako brza, za određivanje je potreban mali volumen uzorka, te se prisutnost saksitoksina može odrediti u rasponu koncentracija od 15 ng/mL do 3 µg/mL, s donjom granicom detekcije od 7,5 ng/mL

G-quadruplex DNA aptamers and their ligands: Structure, function and application

Highly specific and tight-binding nucleic acid aptamers have been selected against a variety of molecular targets for over 20 years. A significant proportion of these oligonucleotides display G-quadruplex structures, particularly for DNA aptamers, that enable molecular recognition of their ligands. G-quadruplex structures couple a common scaffold to varying loop motifs that act in target recognition. Here, we review DNA G-quadruplex aptamers and their ligands from a structural and functional perspective. We compare the diversity of DNA G-quadruplex aptamers selected against multiple ligand targets, and consider structure with a particular focus on dissecting the thrombin binding aptamer - thrombin interaction. Therapeutic and analytical applications of DNA G-quadruplex aptamers are also discussed. Understanding DNA G-quadruplex aptamers carries implications not only for therapeutics and diagnostics, but also in the natural biochemistry of guanine-rich nucleic acids. © 2012 Bentham Science Publishers.postprin

Rapid Detection of Mycotoxin Contamination

A compilation of 12 original research articles and a review on the development of instrumental and immunoanalytical methods for mycotoxins; on the enhancement of sample preparation and selection to improve method applicability; and on practical applications of analytical methods in laboratory fungal cultures, cereal and feed samples, surface water (as a novel matrix of mycotoxins as emerging surface water contaminants), and during mycotoxin decontamination by bacteria. Target analyte mycotoxins include aflatoxins, deoxynivalenol, diacetoxyscirpenol, fumonisins, fusarenone-X, HT-2 toxins, nivalenol, ochratoxins, sterigmatocystin, T-2 toxin, and zearalenone