Novel nandrolone aptamer for rapid colorimetric detection of anabolic steroids.

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result.

Low-Cost Platform for Multiplexed Electrochemical Melting Curve Analysis.

Detection and identification of single nucleotide polymorphisms (SNPs) have garnered increasing interest in the past decade, finding potential application in detection of antibiotic resistance, advanced forensic science, as well as clinical diagnostics and prognostics, moving toward the realization of personalized medicine. Many different techniques have been developed for genotyping SNPs, and ideally these techniques should be rapid, easy-to-use, cost-effective, flexible, scalable, easily automated, and requiring minimal end-user intervention. While high-resolution melting curve analysis has been widely used for the detection of SNPs, fluorescence detection does not meet many of the desired requirements, and electrochemical detection is an attractive alternative due to its high sensitivity, simplicity, cost-effectiveness, and compatibility with microfabrication. Herein, we describe the multiplexed electrochemical melting curve analysis of duplex surfaces tethered to electrodes of an array. In this approach, thiolated probes designed to hybridize to a DNA sequence containing the SNP to be interrogated are immobilized on gold electrodes. Asymmetric PCR using a ferrocene-labeled forward primer is used to generate this single-stranded redox-labeled PCR amplicon. Following hybridization with the probe immobilized on the electrode surface, the electrode array is exposed to a controlled ramping of temperature, with concomitant constant washing of the electrode array surface while simultaneously carrying out voltammetric measurements. The optimum position of the site complementary to the SNP site in the immobilized probe to achieve maximum differentiation in melting temperature between wild-type and single base mismatch, thus facilitating allelic discrimination, was determined and applied to the detection of a cardiomyopathy associated SNP.

A biophotoelectrode based on boronic acid-modified Chlorella vulgaris cells integrated within a redox polymer.

Green microalgae are gaining attention in the renewable energy field due to their ability to convert light into energy in biophotovoltaic (BPV) cells. The poor exogenous electron transfer kinetics of such microorganisms requires the use of redox mediators to improve the performance of related biodevices. Redox polymers are advantageous in the development of subcellular-based BPV devices by providing an improved electron transfer while simultaneously serving as immobilization matrix. However, these surface-confined redox mediators have been rarely used in microorganism-based BPVs. Since electron transfer relies on the proximity between cells and the redox centres at the polymer matrix, the development of molecularly tailored surfaces is of great significance to fabricate more efficient BPV cells. We propose a bioanode integrating Chlorella vulgaris embedded in an Os complex-modified redox polymer. Chlorella vulgaris cells are functionalized with 3-aminophenylboronic acid that exhibits high affinity to saccharides in the cell wall as a basis for an improved integration with the redox polymer. Maximum photocurrents of (5 ± 1) µA cm-2 are achieved. The developed bioanode is further coupled to a bilirubin oxidase-based biocathode for a proof-of-concept BPV cell. The obtained results encourage the optimization of electron-transfer pathways toward the development of advanced microalgae-based biophotovoltaic devices.

Aptasensors for mycotoxin detection: A review.

Mycotoxins are toxic compounds produced by fungi, which represent a risk to the food and feed supply chain, having an impact on health and economies. A high percentage of feed samples have been reported to be contaminated with more than one type of mycotoxin. Systematic, cost-effective and simple tools for testing are critical to achieve a rapid and accurate screening of food and feed quality. In this review, we describe the various aptamers that have been selected against mycotoxins and their incorporation into optical and electrochemical aptasensors, outlining the strategies exploited, highlighting the advantages and disadvantages of each approach. The review also discusses the different materials used and the immobilization methods employed, with the aim of achieving the highest sensitivity and selectivity.

Aptamer Sandwich Assay for the Detection of SARS-CoV-2 Spike Protein Antigen.

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) emerged at the end of 2019, resulting in the ongoing COVID-19 pandemic. The high transmissibility of the virus and the substantial number of asymptomatic individuals have led to an exponential rise in infections worldwide, urgently requiring global containment strategies. Reverse transcription-polymerase chain reaction is the gold standard for the detection of SARS-CoV-2 infections. Antigen tests, targeting the spike (S) or nucleocapsid (N) viral proteins, are considered as complementary tools. Despite their shortcomings in terms of sensitivity and specificity, antigen tests could be deployed for the detection of potentially contagious individuals with high viral loads. In this work, we sought to develop a sandwich aptamer-based assay for the detection of the S protein of SARS-CoV-2. A detailed study on the binding properties of aptamers to the receptor-binding domain of the S protein in search of aptamer pairs forming a sandwich is presented. Screening of aptamer pairs and optimization of assay conditions led to the development of a laboratory-based sandwich assay able to detect 21 ng/mL (270 pM) of the protein with negligible cross-reactivity with the other known human coronaviruses. The detection of 375 pg of the protein in viral transport medium demonstrates the compatibility of the assay with clinical specimens. Finally, successful detection of the S antigen in nasopharyngeal swab samples collected from suspected patients further establishes the suitability of the assay for screening purposes as a complementary tool to assist in the control of the pandemic.

Hybrid Antibody-Aptamer Assay for Detection of Tetrodotoxin in Pufferfish.

The marine toxin tetrodotoxin (TTX) poses a great risk to public health safety due to its severe paralytic effects after ingestion. Seafood poisoning caused by the consumption of contaminated marine species like pufferfish due to its expansion to nonendemic areas has increased the need for fast and reliable detection of the toxin to effectively implement prevention strategies. Liquid chromatography-mass spectrometry is considered the most accurate method, although competitive immunoassays have also been reported. In this work, we sought to develop an aptamer-based assay for the rapid, sensitive, and cost-effective detection of TTX in pufferfish. Using capture-SELEX combined with next-generation sequencing, aptamers were identified, and their binding properties were evaluated. Finally, a highly sensitive and user-friendly hybrid antibody-aptamer sandwich assay was developed with superior performance compared to several assays reported in the literature and commercial immunoassay kits. The assay was successfully applied to the quantification of TTX in pufferfish extracts, and the results obtained correlated very well with a competitive magnetic bead-based immunoassay performed in parallel for comparison. This is one of the very few works reported in the literature of such hybrid assays for small-molecule analytes whose compatibility with field samples is also demonstrated.

Gold nanoparticle aptamer assay for the determination of histamine in foodstuffs.

The development of a gold nanoparticle aptamer assay is persued for rapid and sensitive determination of histamine in foodstuffs, which could be deployed for on-site use. The assay is based on a histamine-specific aptamer and gold nanoparticles and the salt-induced aggregation of the particles in the presence of histamine indicated by the color change from red to blue. Gold nanoparticle size, salt type, and concentration as well as aptamer concentration were optimized, and using optimum conditions, a limit of detection of 8 nM (~ 0.05 mg/kg) was obtained. Finally, the aptamer AuNP assay was applied to the determination of histamine in quality control fish samples. The histamine levels of these samples had previously been determined using HPLC and commercial ELISA kits by numerous independent laboratories and a good correlation was obtained. The developed AuNP assay is rapid, sensitive, and reproducible. Graphical abstract.

Yersinia pestis detection using biotinylated dNTPs for signal enhancement in lateral flow assays.

Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications.

One-Pot SELEX: Identification of Specific Aptamers against Diverse Steroid Targets in One Selection.

Aptamers are well-established biorecognition molecules used in a wide variety of applications for the detection of their respective targets. However, individual SELEX processes typically performed for the identification of aptamers for each target can be quite time-consuming, labor-intensive, and costly. An alternative strategy is proposed herein for the simultaneous identification of different aptamers binding distinct but structurally similar targets in one single selection. This one-pot SELEX approach, using the steroids estradiol, progesterone, and testosterone as model targets, was achieved by combining the benefits of counter-SELEX with the power of next-generation sequencing and bioinformatics analysis. The pools from the last stage of the selection were compared in order to discover sequences with preferential abundance in only one of the pools. This led to the identification of aptamer candidates with potential specificity to a single steroid target. Binding studies demonstrated the high affinity of each selected aptamer for its respective target, and low nanomolar range dissociation constants calculated were similar to those previously reported for steroid-binding aptamers selected using traditional SELEX approaches. Finally, the selected aptamers were exploited in microtiter plate assays, achieving nanomolar limits of detection, while the specificity of these aptamers was also demonstrated. Overall, the one-pot SELEX strategy led to the discovery of aptamers for three different steroid targets in one single selection without compromising their affinity or specificity, demonstrating the power of this approach of aptamer discovery for the simultaneous selection of aptamers against multiple targets.

High Affinity Aptamer for the Detection of the Biogenic Amine Histamine.

The importance of histamine in various physiological functions and its involvement in allergenic responses make this small molecule one of the most studied biogenic amines. Even though a variety of chromatography-based methods have been described for its analytical determination, the disadvantages they present in terms of cost, analysis time, and low portability limit their suitability for in situ routine testing. In this work, we sought to identify histamine-binding aptamers that could then be exploited for the development of rapid, facile, and sensitive assays for histamine detection suitable for point-of-need analysis. A classic SELEX process was designed employing magnetic beads for target immobilization and the selection was completed after ten rounds. Following Next Generation Sequencing of the last selection rounds from both positive and counter selection magnetic beads, several sequences were identified and initially screened using an apta-PCR affinity assay (APAA). Structural and functional characterization of the candidates resulted in the identification of the H2 aptamer. The high binding affinity of the H2 aptamer to histamine was validated using four independent assays ( KD of 3-34 nM). Finally, the H2 aptamer was used for the development of a magnetic beads-based competitive assay for the detection of histamine in both buffer and synthetic urine, achieving very low limits of detection of 18 pM and 76 pM, respectively, while no matrix effects were observed. These results highlight the suitability of the strategy followed for identifying small molecule-binding aptamers and the compatibility of the selected H2 aptamer with the analysis of biological samples, thus facilitating the development of point-of-care devices for routine testing. Ongoing work is focused on extending the application of the H2 aptamer to the detection of spoilage in meat, fish, and beverages, as well as evaluating the affinity of truncated forms of the aptamer.

Duplex PCR-ELONA for the detection of pork adulteration in meat products.

In this work, a duplex PCR-Enzyme Linked Oligonucleotide Assay (ELONA) is reported for the sensitive and reliable detection of pork adulteration in beef and chicken products, two of the most widely consumed meat types in the world. The strategy relies on the use of species-specific tailed primers for duplex amplification and simple dilution of the PCR reactions for direct colorimetric detection via hybridization, eliminating the need for any other post-amplification steps. A high sensitivity was achieved, with as low as 71-188 pg of genomic DNA able to be detected using mixtures of control DNA from each species. The strategy was validated using DNA add-mixtures as well as DNA extracted from raw meat mixtures and 0.5-1% w/w pork could be easily detected when mixed with beef or chicken. The proposed approach is simple, sensitive and cost-effective compared to equivalent commercial kits suitable for detecting adulterant pork levels in meat products.

Aptamer Selection against a Trichomonas vaginalis Adhesion Protein for Diagnostic Applications.

Trichomoniasis, caused by Trichomonas vaginalis, is the leading nonviral sexually transmitted infection worldwide. We report the selection of a DNA aptamer against a T. vaginalis adhesion protein, AP65, using a microtiter plate-based in vitro combinatorial chemistry process termed systematic evolution of ligands by exponential enrichment. The enriched library pool was sequenced by next-generation sequencing, and several aptamer candidates with high affinity and specificity were identified. The aptamer with the highest affinity and specificity had a KD in the low nanomolar range, as confirmed by three different techniques: surface plasmon resonance, enzyme-linked aptamer assay, and biolayer interferometry. The selected aptamer was demonstrated to have a high specificity to the AP65 protein and to T. vaginalis cells with no cross-reactivity to other enteric and urogenital microorganisms. Current work is focused on the development of inexpensive and easy-to-use aptamer-based diagnostic assays for the reliable and rapid detection of T. vaginalis in vaginal swabs.

Aptatope mapping of the binding site of a progesterone aptamer on the steroid ring structure.

In this work we report the mapping of the binding site of the only progesterone aptamer published to date, in an approach referred to as aptatope mapping. By linking the binding data obtained from microscale thermophoresis analysis to the structural differences on the ring structure of a range of steroids, we elucidated the moieties involved in aptamer-progesterone binding. This approach can be further exploited for the characterization of aptamer specificity and ultimately facilitate the development of aptamer-based assays depending on the desired specificity.

The characterization and validation of 17ß-estradiol binding aptamers.

The rapid and sensitive detection of small molecules is garnering increasing importance, and aptamers show great promise in replacing expensive, elaborate detection platforms exploiting chromatographic separation or antibody-based assays. The characterization of aptamer interaction with small molecule targets is not facile, and there is a mature need for a rapid, high-throughput technique for the analysis of aptamer-small molecule kinetics and affinity. In this work we present methodologies for the evaluation of aptamer-small molecule interactions, using the aptamers reported against the steroid 17ß-estradiol as a model system. Microscale thermophoresis, apta-PCR affinity assay and surface plasmon resonance were explored to evaluate the reported aptamers' binding properties in terms of affinity and specificity, and were demonstrated to be successfully applied to the analysis of aptamer-small molecule interactions.

Ultrasensitive and rapid detection of ß-conglutin combining aptamers and isothermal recombinase polymerase amplification.

Lupin is increasingly being used in a variety of food products due to its nutritional, functional and nutraceutical properties. However, several examples of severe and even fatal food-associated anaphylaxis due to lupin inhalation or ingestion have been reported, resulting in the lupin subunit ß-conglutin, being defined as the Lup an 1 allergen by the International Union of Immunological Societies (IUIS) in 2008. Here, we report an innovative method termed aptamer-recombinase polymerase amplification (Apta-RPA) exploiting the affinity and specificity of a DNA aptamer selected against the anaphylactic ß-conglutin allergen termed ß-conglutin binding aptamer II (ß-CBA II), facilitating ultrasensitive detection via isothermal amplification. Combining magnetic beads as the solid phase with Apta-RPA detection, the total assay time was reduced from 210 min to just 25 min, with a limit of detection of 3.5 × 10-11 M, demonstrating a rapid and ultrasensitive generic methodology that can be used with any aptamer. Future work will focus on further simplification of the assay to a lateral flow format. Graphical Abstract Schematic representation of the rapid and novel bead-based Apta-RPA assay.

The Thermodynamic and Kinetic Properties of 2-Hydroxypyridine/2-Pyridone Tautomerization: A Theoretical and Computational Revisit.

The gas-phase thermal tautomerization reaction between 2-hydroxypyridine (2-HPY) and 2-pyridone (2-PY) was investigated by applying 6-311++G** and aug-cc-pvdz basis sets incorporated into some density functional theory (DFT) and coupled cluster with singles and doubles (CCSD) methods. The geometrical structures, dipole moments, HOMO-LUMO energy gaps, total hyperpolarizability, kinetics and thermodynamics functions were monitored against the effects of the corrections imposed on these functionals. The small experimental energy difference between the two tautomers of 3.23 kJ/mol; was a real test of the accuracy of the applied levels of theory. M062X and CCSD methods predicted the preference of 2-HPY over 2-PY by 5-9 kJ/mol; while B3LYP functional favoured 2-PY by 1-3 kJ/mol. The CAM-B3LYP and ωB97XD functionals yielded mixed results depending on the basis set used. The source of preference of 2-HPY is the minimal steric hindrance and electrostatic repulsion that subdued the huge hyperconjugation in 2-PY. A 1,3-proton shift intramolecular gas-phase tautomerization yielded a high average activation of 137.152 kJ/mol; while the intermolecular mixed dimer interconversion gave an average barrier height of 30.844 kJ/mol. These findings are boosted by a natural bond orbital (NBO) technique. The low total hyperpolarizabilities of both tautomers mark out their poor nonlinear optical (NLO) behaviour. The enhancement of the total hyperpolarizability of 2-HPY over that of 2-PY is interpreted by the bond length alternation.

UV excitations of halons.

In the present study, we examined the UV excitations of a newly introduced molecular set, Halons-9, composed of nine gaseous halon molecules. The performance of the density functional-based multi-reference configuration interaction method (DFT/MRCI) and time-dependent density functional theory with CAM-B3LYP functional (TD-CAM-B3LYP) in the computation of singlet and triplet excited states of this set was evaluated against coupled-cluster with singles and doubles (CCSD). Excited states up to the corresponding ionization limits, including both localized and delocalized excitations, have been benchmarked. TD-CAM-B3LYP significantly underestimates excitation energies of the higher mixed valence-Rydberg and Rydberg states, with computed mean absolute deviations from the equation of motion (EOM)-CCSD results 1.06 and 0.76 eV, respectively. DFT/MRCI gives a significantly better description of higher excited states, albeit still poor, compared to the TD-CAM-B3LYP. The mean absolute deviations of mixed valence-Rydberg and Rydberg states from the reference EOM-CCSD values are 0.66 and 0.47 eV, respectively. The performance of DFT/MRCI for description of strongly correlated states with valence-Rydberg mixing is still not satisfactory enough. On the other hand, oscillator strengths of most of singlet states obtained with both methods are close to the EOM-CCSD values. The largest deviations, occurring in the case of several high-lying multiconfigurational states, are of an order of magnitude.

Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay.

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10-11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need.

Aptamer Lateral Flow Assays for Ultrasensitive Detection of ß-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers.

In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which ß-conglutin immobilized on the test line of a nitrocellulose membrane and ß-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the ß-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized ß-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

Tungsten Doped TiO2 with Enhanced Photocatalytic and Optoelectrical Properties via Aerosol Assisted Chemical Vapor Deposition.

Tungsten doped titanium dioxide films with both transparent conducting oxide (TCO) and photocatalytic properties were produced via aerosol-assisted chemical vapor deposition of titanium ethoxide and dopant concentrations of tungsten ethoxide at 500 °C from a toluene solution. The films were anatase TiO2, with good n-type electrical conductivities as determined via Hall effect measurements. The film doped with 2.25 at.% W showed the lowest resistivity at 0.034 Ω.cm and respectable charge carrier mobility (14.9 cm(3)/V.s) and concentration (×10(19) cm(-3)). XPS indicated the presence of both W(6+) and W(4+) in the TiO2 matrix, with the substitutional doping of W(4+) inducing an expansion of the anatase unit cell as determined by XRD. The films also showed good photocatalytic activity under UV-light illumination, with degradation of resazurin redox dye at a higher rate than with undoped TiO2.