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Because accidents, disease and aging compromise the structural and physiological functions of bones, the development of an in vivo bone imaging test is critical to identify, detect and diagnose bone related development and dysfunctions. Recent advances in fluorescence instrumentation offer a new alternative for traditional bone imaging methods. However, the development of new in vivo bone imaging fluorescence materials has significantly lagged behind. Here we show that carbon dot nanoparticles (C-dots) with low quantum yield (“dark”) bind to calcified bone structures of live zebrafish larvae with high affinity and selectivity. Binding resulted in a strong enhancement of luminescence that was not observed in other tissues, including non-calcified endochondral elements. Retention of C-dots by bones was very stable, long lasting, and with no detectable toxicity. Furthermore, we found C-dots to be a suitable carrier to deliver fluorescein to bones. These observations support a novel and revolutionary use of C-dots as highly specific bioagents for bone imaging and diagnosis, and as bone-specific drug delivery vehicles. [ABSTRACT FROM AUTHOR]

Two novel and low cost liquid-liquid extraction methods for the separation of bismuth(III) at trace level from aqueous medium have been developed. The two methods were based upon the formation of yellow colored ternary complex ion associates of tetraiodobismuth(III) complex anion, BiI4 - with the ion-pairing reagent 2,3,5-tetraphenyltetrazoliumchloride (Tz+.Cl-) and 1, 10 phenanthroline (Phen) in sulfuric acid medium. The effect of various parameters e.g. pH, organic solvent, shaking time, etc. on the preconcentration of bismuth(III) from the aqueous media by the reagent was investigated. The developed colored complex ion associates [Tz+.BiI4 -] and [Phen+.BiI4 -] were extracted quantitatively into acetone-chloroform (1:1v/v) and methylisobutylketone (MIBK), respectively. The compositions of the formed complex ion associates [Tz+.BiI4 -] and [Phen+.BiI4 -] were determined by the Job's method at 500 and 490 nm, respectively. The plots of bismuth(III) concentration (0-17 μg mL-1) versus absorbance of the associates at 500 and 490 nm were linear with good correlation coefficient (R²=0.998). The developed method of the ion associate [Tz+.BiI4 -] two methods was applied successfully for the analysis of bismuth in water. [ABSTRACT FROM AUTHOR]

The chromogenic reagent 4,4′-dichloro(3-mercapto-1,5-diphenylformazan), Cl 2 H 2 D Z , forms a yellow–red-coloured complex with selenium(IV). The produced complex species was extracted quantitatively into n -hexane, and its absorbance was measured at 416  nm. The chemical composition of the extracted selenium(IV)-Cl 2 H 2 D Z chelate and the molar absorptivity at 416  nm were found to be [SeO (Cl 2 HD Z ) 2 ] and 9  ×  10 4   L  mol -1   cm -1 , respectively. The values of the extraction constants ( K D , K ex , β ) enable a convenient application of the proposed system for the liquid–liquid extraction procedure and sequential spectrophotometric determination of traces of inorganic selenium(IV) and/or selenium(VI) after reduction of the later to selenium(IV) with HCl (6  M). Beer's law and Ringbom's plots were obeyed in the concentration range 0.01–20 and 0.5–19  µg  mL -1 of selenium(IV), respectively, with a relative standard deviation of 2.2%. The proposed method has been successfully applied to the determination of selenium(IV) or (VI) and total inorganic selenium(IV) and (VI) in tap and freshwater samples. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The use of lateral flow assays exploiting antibodies is well established in different fields due to their advantages, which include low cost, ease of production and rapid response, with the only required end-user intervention being sample addition. In recent years, aptamer-based lateral flow assays are garnering increasing interest offering a highly cost-effective and more flexible alternative to antibodies. In this review, an overview of the aptamer-based lateral flow assays developed to date is provided, highlighting the advantages of using aptamers and their ability to be incorporated into formats not possible with antibodies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Image 1 • Ultrasensitive molecular detection of Yersinia pestis using biotinylated dNTPs and tailed primer is demonstrated. • Optimisation of ratio of biotinylated dNTPs: natural dNTPs is carried out using PCR and ELONA. • Optimised parameters were exploited using RPA. • Lateral flow assays for detection of single tailed amplicon and biotinylated dNTPs with SA-AuNPs was developed. • Real samples were tested. [ABSTRACT FROM AUTHOR]

Highlights • Duplex PCR amplification of pork in beef and chicken products. • Tailed primers allow the generation of duplex flanked by single stranded tails. • Enzyme-linked oligonucleotide assay for direct detection via hybridisation of PCR products. • Sensitivity of 71–188 pg of genomic DNA from each species was achieved. • The presence of 0.5–1% pork in binary DNA add-mixtures and in raw meat mixtures could be detected. Abstract 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. [ABSTRACT FROM AUTHOR]

Cholera and milder diarrheal disease are caused by Vibrio cholerae and enterotoxigenic Escherichia coli and are still a prominent public health concern. Evaluation of suspicious isolates is essential for the rapid containment of acute diarrhea outbreaks or prevention of epidemic cholera. Existing detection techniques require expensive equipment, trained personnel and are time-consuming. Antibody-based methods are also available, but cost and stability issues can limit their applications for point-of-care testing. This study focused on the selection of single stranded DNA aptamers as simpler, more stable and more cost-effective alternatives to antibodies for the co-detection of AB 5 toxins secreted by enterobacteria causing acute diarrheal infections. Cholera toxin and Escherichia coli heat-labile enterotoxin, the key toxigenicity biomarkers of these bacteria, were immobilized on magnetic beads and were used in a SELEX-based selection strategy. This led to the enrichment of sequences with a high % GC content and a dominant G-rich motif as revealed by Next Generation Sequencing. Enriched sequences were confirmed to fold into G-quadruplex structures and the binding of one of the most abundant candidates to the two enterotoxins was confirmed. Ongoing work is focused on the development of monitoring tools for potential environmental surveillance of epidemic cholera and milder diarrheal disease. [Display omitted] • Vibrio cholerae toxin and Escherichia coli heat-labile enterotoxin were targeted for aptamer development. • Magnetic-bead SELEX was performed and combined with Next Generation Sequencing for sequence analysis. • Enrichment in common enterotoxin-binding sequences containing a dominant G-rich motif was observed. • The folding of G-rich enriched sequences into G-quadruplex structures was confirmed. • A G-quadruplex aptamer with good binding affinity for both AB 5 enterotoxins was identified. [ABSTRACT FROM AUTHOR]

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 17β-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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Selection of novel aptamers binding to nandrolone by sepharose-SELEX. • High binding affinity of the selected aptamers demonstrated with different assays. • Development of a colorimetric gold nanoparticles-aptamer assay for nandrolone. • No cross-reactivity of the assay with endogenous steroids. • Use for screening suspicious samples for presence of nandrolone and its metabolites. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Review of aptasensors for the detection of mycotoxins. • Detection of Patulin, T-2 toxin, Aflatoxin B1, Aflatoxin B2, Zearalenone, Fumonisin B1, Ochratoxin. • Examples of optical and electrochemical aptasensors as well as lateral flow assays. • Presentation of different materials and immobilization approaches for aptasensor development. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

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 ( K D 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.

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 K D 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.