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The cultivation of genetically modified organisms (GMO) continues to expand worldwide. Still, many consumers express concerns about the use of GMO in food or feed, and many countries have legislated on labelling systems to indicate the presence of GMO in commercial products. To deal with the increased number of GMO events and to address related regulations, alternative detection methods for GMO inspection are required. In this work, a genosensor based on Surface Plasmon Resonance under continuous flow was developed for the detection and quantification of a genetically modified soybean (event GTS 40-3-2). In a single chip, the simultaneous detection of the event-specific and the taxon-specific samples were achieved, whose detection limits were 20 pM and 16 pM, respectively. The reproducibility was 1.4%, which supports the use of the chip as a reliable and cost-effective alternative to other DNA-based techniques. The results indicate that the proposed method is a versatile tool for GMO quantification in food and feed samples. [ABSTRACT FROM AUTHOR]

The main goal of food safety assessment is to provide reliable information on the identity and composition of food and reduce the presence of harmful components. Nowadays, there are many countries where rather than the presence of pathogens, common public concerns are focused on the presence of hidden allergens, fraudulent practices, and genetic modifications in food. Accordingly, food regulations attempt to offer a high level of protection and to guarantee transparent information to the consumers. The availability of analytical methods is essential to comply these requirements. Protein-based strategies are usually employed for this purpose, but present some limitations. Because DNA is a more stable molecule, present in most tissues, and can be amplified, there has been an increasing interest in developing DNA-based approaches (polymerase chain reaction, microarrays, and genosensors). In this regard, electrochemical genosensors may play a major role in fulfilling the needs of food industry, such as reliable, portable, and affordable devices. This work reviews the achievements of this technology applied to allergen detection, species identification, and genetically modified organisms testing. We summarized the legislative framework, current design strategies in sensor development, their analytical characteristics, and future prospects. [ABSTRACT FROM PUBLISHER]

Despite having been underappreciated in favor of their protein-coding counterparts for a long time, long noncoding RNAs (lncRNAs) have emerged as functional molecules, which defy the central dogma of molecular biology, with clear implications in cancer. Altered expression levels of some of these large transcripts in human body fluids have been related to different cancer conditions that turns them into potential noninvasive cancer biomarkers. In this review, a brief discussion about the importance and current challenges in the determination of lncRNAs associated to cancer is provided. Different electrochemical nucleic acid-based strategies for lncRNAs detection are critically described. Future perspectives and remaining challenges for the practical implementation of these methodologies in clinical medicine are also discussed. [ABSTRACT FROM AUTHOR]

The detection of bacterial pathogens may be accomplished through their genome material. With that aim, current methods usually rely on polymerase chain reaction (PCR) amplifications, which are time consuming and require specialist equipment. Here, we optimized an on‐chip isothermal recombinase polymerase amplification (RPA) with chronoamperometric detection, which could be run with simple equipment. A short thiol‐modified oligonucleotide, complementary to a fragment of bipA gene of Salmonella spp. is chemisorbed onto the gold chip surface together with p‐aminothiophenol as a blocking agent. Remarkably, we demonstrate that, in the presence of Salmonella genome, this oligonucleotide, acting as a primer in RPA, is directly elongated on the surface, copying the genome and giving rise to an on‐surface enzyme‐tagged dsDNA. After 30 min of isothermal amplification, the chronoamperometric signal, related to one of the products of the enzyme reaction, increased with the amount of Salmonella genome, which allowed the detection of 105 copies. The results of this study may be of general utility in the design of sensors for detecting other bacteria. Under the influence: The mechanism for the selective and isothermal amplification with electrochemical detection of Salmonella genome on gold chips is investigated. The recombinase‐polymerase reaction takes place directly on the surface, elongating the anchored primer, which enables the detection of 105 genome units. [ABSTRACT FROM AUTHOR]

Abstract: Herein, we show the feasibility of using deep eutectic solvents as a faster way of selecting aptamers targeting poorly water‐soluble species. This unexplored concept is illustrated for gluten proteins. In this way, aptamer‐based gluten detection can be performed directly in the extraction media with improved detectability. We envision deep implications for applications not only in food safety control but also in biomedicine. [ABSTRACT FROM AUTHOR]

The InCl3-catalyzed reaction of ferrocene with orthohydroxybenzyl alcohols is reported and represents a convenient route for the synthesis of ferrocenyl phenols. This carbon–carbon bond forming process is believed to proceed through an ortho-quinone methide intermediate that can be interceptedby ferrocene through a Friedel–Crafts-type process. Preliminary cytotoxic screening carried out on several cancer cell lines revealed that some of the compounds exhibit moderate cytotoxicity [ABSTRACT FROM AUTHOR]

The design of screening methods for the detection of genetically modified organisms (GMOs) in food would improve the efficiency in their control. We report here a PCR amplification method combined with a sequence-specific electrochemical genosensor for the quantification of a DNA sequence characteristic of the 35S promoter derived from the cauliflower mosaic virus (CaMV). Specifically, we employ a genosensor constructed by chemisorption of a thiolated capture probe and p-aminothiophenol gold surfaces to entrap on the sensing layer the unpurified PCR amplicons, together with a signaling probe labeled with fluorescein. The proposed test allows for the determination of a transgene copy number in both hemizygous (maize MON810 trait) and homozygous (soybean GTS40-3-2) transformed plants, and exhibits a limit of quantification of at least 0.25% for both kinds of GMO lines. [ABSTRACT FROM AUTHOR]

Celiac disease is a lifelong autoimmune disorder triggered by foods containing gluten, the storage protein in wheat, rye, and barley. The rapidly escalating number of patients diagnosed with this disease poses a great challenge to both food industry and authorities to guarantee food safety for all. Therefore, intensive efforts are being made to establish minimal disease-eliciting doses of gluten and consequently to improve gluten-free labeling. These efforts depend to a high degree on the availability of methods capable of detecting the protein in food samples at levels as low as possible. Current analytical approaches rely on the use of antibodies as selective recognition elements. With limited sensitivity, these methods exhibit some deficiencies that compromise the accuracy of the obtained results. Aptamers provide an ideal alternative for designing biosensors for fast and selective measurement of gluten in foods. This article highlights the challenges in gluten detection, the current status of the use of aptamers for solving this problem, and what remains to be done to move these systems into commercial applications. [ABSTRACT FROM AUTHOR]

Current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) with a minimum content of 0.9% would benefit from the availability of reliable and rapid methods to detect and quantify DNA sequences specific for GMOs. Different genosensors have been developed to this aim, mainly intended for GMO screening. A remaining challenge, however, is the development of genosensing platforms for GMO quantification, which should be expressed as the number of event-specific DNA sequences per taxon-specific sequences. Here we report a simple and sensitive multiplexed electrochemical approach for the quantification of Roundup-Ready Soybean (RRS). Two DNA sequences, taxon (lectin) and event-specific (RR), are targeted via hybridization onto magnetic beads. Both sequences are simultaneously detected by performing the immobilization, hybridization and labeling steps in a single tube and parallel electrochemical readout. Hybridization is performed in a sandwich format using signaling probes labeled with fluorescein isothiocyanate (FITC) or digoxigenin (Dig), followed by dual enzymatic labeling using Fab fragments of anti-Dig and anti-FITC conjugated to peroxidase or alkaline phosphatase, respectively. Electrochemical measurement of the enzyme activity is finally performed on screen-printed carbon electrodes. The assay gave a linear range of 2–250 pM for both targets, with LOD values of 650 fM (160 amol) and 190 fM (50 amol) for the event-specific and the taxon-specific targets, respectively. Results indicate that the method could be applied for GMO quantification below the European labeling threshold level (0.9%), offering a general approach for the rapid quantification of specific GMO events in foods. [ABSTRACT FROM AUTHOR]

In recent years, several devices have been developed for the direct measurement of hydrogen peroxide (H 2 O 2 ), a key compound in biological processes and an important chemical reagent in industrial applications. Classical enzymatic biosensors for H 2 O 2 have been recently outclassed by electrochemical sensors that take advantage of material properties in the nano range. Electrodes with metal nanoparticles (NPs) such as Pt, Au, Pd and Ag have been widely used, often in combination with organic and inorganic molecules to improve the sensing capabilities. In this review, we present an overview of nanomaterials, molecules, polymers, and transduction methods used in the optimization of electrochemical sensors for H 2 O 2 sensing. The different devices are compared on the basis of the sensitivity values, the limit of detection (LOD) and the linear range of application reported in the literature. The review aims to provide an overview of the advantages associated with different nanostructures to assess which one best suits a target application. [ABSTRACT FROM AUTHOR]

A personal glucose meter (PGM)-based method for quantitative detection of a urinary nucleic acid biomarker in prostate cancer screening, the so-called PCA3, is reported herein. A sandwich-type genoassay is conducted on magnetic beads to collect the target from the sample by specific hybridization, making the assay appropriate for PCA3 detection in biological fluids. The success of the method hinges on the use of alkaline phosphatase (ALP) to link the amount of nucleic acid biomarker to the generation of glucose. In particular, specifically attached ALP molecules hydrolyze D-glucose-1-phosphate into D-glucose, thus enabling the amplification of the recorded signal on the personal glucose meter. The developed genoassay exhibits good sensitivity (3.3 ± 0.2 mg glucose dL−1 pM−1) for PCA3, with a dynamic range of 5 to 100 pM and a quantification limit of 5 pM. Likewise, it facilitates point-of-care testing of nucleic acid biomarkers by using off-the-shelf PGM instead of complex instrumentation involved in traditional laboratory-based tests. [ABSTRACT FROM AUTHOR]

Affinity characterization is essential to develop reliable aptamers for tumor biomarker detection. For alpha-fetoprotein (AFP), a biomarker of hepatocellular carcinoma (HCC), two DNA aptamers were described with very different affinity. In this work, we estimate the dissociation constant of both of them by means of a direct assay on magnetic beads modified with AFP and electrochemical detection on carbon screen-printed electrodes (SPCE). Unlike previous works, both aptamers showed similar dissociation constant (Kd) values, in the subµM range. In order to improve the performance of these aptamers, we proposed the isothermal amplification of the aptamers by both terminal deoxynucleotidyl transferase (TdT) and rolling circle amplification (RCA). Both DNA amplifications improved the sensitivity and also the apparent binding constants from 713 nM to 189 nM for the short aptamer and from 526 nM to 32 nM for the long aptamer. This improvement depends on the true affinity of the binding pair, which ultimately limits the analytical usefulness. [ABSTRACT FROM AUTHOR]

The reaction of para-hydroxybenzyl alcohols with ferrocene in the presence of a catalytic amount of InCl3 provided ferrocenyl phenol derivatives, an interesting class of organometallic compounds with potential applications in medicinal chemistry. This transformation exhibited a reasonable substrate scope delivering the desired products in synthetically useful yields. Evidence of involvement of a para-quinone methide intermediate in this coupling process was also provided. Preliminary biological evaluation demonstrated that some of the ferrocene derivatives available by this methodology exhibit significant cytotoxicity against several cancer cell lines with IC50 values within the range of 1.07–4.89 μM. [ABSTRACT FROM AUTHOR]

Real-time PCR is the gold standard technique for the unambiguous detection of sequence-specific fragments of pathogen DNA in many fields but it does not always fulfill the extremely low detectability legislation demands. Herein we demonstrate that rationally designed genosensors can rival or even supersede it when combined with previous amplification strategies even isothermal ones. Among the latter, helicase-dependent amplification (HDA) is a simple and powerful DNA amplification using helicase instead of temperature cycles to unwind DNA; it follows an amplification scheme very similar to PCR, which could facilitate its widespread adoption for point-of-need applications. Unfortunately, the primer design for HDA is more restrictive than for PCR. We show that it is possible to adapt PCR primers to isothermal amplification of a DNA sequence specific for Salmonella . We design an electrochemical DNA sensor, based on a sandwich hybridization assay on the surface of indium-tin oxide electrodes, which allows monitoring both PCR and HDA amplicons. The resulting sequence-specific detection strategy not only increased the selectivity of the detection but also its sensitivity, improving real-time PCR in two-fold and a hundred-fold when combined with HDA and PCR, respectively, achieving the single molecule detection. [ABSTRACT FROM AUTHOR]

Reliable detection of decreasing amounts of gluten in food is the only way of ensuring the safety of all coeliac patients. Results obtained with the method of choice, immunochemical assays, are not entirely comparable and many of them are sandwich assays that cannot recognize hydrolyzed proteins. In this work, we propose a competitive electrochemical sensor based on a recently described aptamer targeting the gliadin immunodominant peptide 33-mer that triggers the coeliac disease. The sensing layer is built on the surface of a screen-printed carbon electrode (SPCE) by adsorption of streptavidin and subsequent peptide immobilization. A competition between the peptide and gluten proteins from samples for a defined concentration of biotinylated Gli 4 aptamer is established. The aptamer bound to the peptide on the surface is finally measured after enzyme labelling and chronoamperometric detection of an enzymatically obtained electrochemically active product. This method is able to detect as low as 0.113 μg L −1 of gliadin, which corresponds to 380 μg kg −1 of gluten in food, taking all dilutions and conversion factors into consideration, with a reproducibility lower than 11%. The aptasensor was applied to food samples with gluten contents above and below the legislated threshold for gluten-free labelling in the EU, obtaining good agreement with the official R5 immunochemical method. [ABSTRACT FROM AUTHOR]

A highly sensitive and robust method for the quantification of specific DNA sequences based on coupling asymmetric helicase-dependent DNA amplification to electrochemical detection is described. This method relies on the entrapment of the amplified ssDNA sequences on magnetic beads followed by a post-amplification hybridization assay to provide an added degree of specificity. As a proof-of-concept a 84-bases long sequence specific of Mycobacterium tuberculosis is amplified at 65 °C, providing 3×10 6 amplification after 90 min. Using this system 0.5 aM, corresponding to 15 copies of the target gene in 50 µL of sample, can be successfully detected and reliably quantified under isothermal conditions in less than 4 h. The assay has been applied to the detection of M. tuberculosis in sputum, pleural fluid and urine samples. Besides this application, the proposed assays is a powerful and general tool for molecular diagnostic that can be applied to the detection of other specific DNA sequences, taking full advantage of the plethora of genomic information now available. [ABSTRACT FROM AUTHOR]

Prostate specific antigen (PSA) is the common biomarker for prostate cancer (PCa). However, its lack of specificity to differentiate PCa from benign prostate disorders stimulates the search for alternative cancer biomarkers to improve the clinical management of the patients. Different studies have described changes in the core-fucosylation level of PSA between PCa patients and healthy controls. To exploit these findings, we have adapted an impedimetric aptamer-based sensor to the dual recognition of PSA. Two different aptamers, PSAG-1 and anti-PSA, are immobilized onto two adjacent nanostructured gold electrodes. The direct binding from diluted serum samples of specific glycosylated-PSA to the first sensor and total PSA to the second one leads to changes in the charge transfer resistance, which correlate to the amount of glycosylated and total PSA in the sample. The sensors are able to measure PSA in serum with a dynamic range between 0.26 and 62.5 ng/mL (PSAG-1) and from 0.64 to 62.5 ng/mL (anti-PSA), with a reproducibility of 5.4 %. The final output of the proposed platform is the ratio between PSAG-1 reactive PSA and total PSA, defined as the glycan score. The glycan score was tested in serum samples from patients with different pathologies, showing excellent correlation between the measured score and the known diagnosis of the patients. Hence this dual aptamer-based impedimetric biosensor could be used as a minimally invasive method for the diagnosis of prostate cancer. Image 1 • A dual impedimetric aptamer-based platform directly deployed in diluted serum samples. • Combination of two aptamers recognizing different regions of PSA. • The platform provides as output a glycan PSA score. • The glycan score discriminates prostate cancer from other benign diseases. [ABSTRACT FROM AUTHOR]

The tunability of SELEX procedure is an essential feature to supply bioaffinity receptors (aptamers) almost on demand for analytical and therapeutic purposes. This longstanding ambition is, however, not straightforward. Non-invasive cancer diagnosis, so called liquid biopsy, requires collection of body fluids with minimal or no sample pretreatment. In those raw matrices, aptamers must recognize minute amounts of biomarkers that are not unique entities but large sets of variants evolving with the disease stage. The susceptibility of aptasensors to assay conditions has driven the selection of aptamers to natural environments to ensure their optimum performance in clinical samples. We present herein a compilation of the SELEX procedures in natural milieus. By revising the electrochemical aptasensors applied to clinical samples for cancer diagnosis and tracing back to the original SELEX we analyze whether aptamers raised using these SELEX strategies are being incorporated to the diagnostic devices and how aptasensors are finding their way to a market dominated by antibody-based assays. Image 1 • Electrochemical aptasensors tested in clinical samples for cancer biomarkers. • Critical revision of aptasensors to detect CTCs, exosomes and proteins. • Relevance of natural environment during SELEX on aptamer performance. • Prospects of in-vitro diagnostic devices based on electrochemical aptasensors. [ABSTRACT FROM AUTHOR]

Highlights • Simultaneous electrochemical detection of two Roundup Ready™ soybean events is feasible. • Core@shell Fe 3 O 4 @Au MNPs proved to be a suitable nanosupport. • Detection limits at the subnM level were achieved. • Methodology specific for these events with no cross-reactivity. Abstract Development of expeditious analytical methods for the detection of genetically modified organisms (GMOs) is increasingly necessary, not only to verify compliance with labelling, but also to help industry to efficiently control the reception of raw materials. On the basis of this, a disposable electrochemical magnetogenoassay is proposed for simultaneous detection of two Roundup Ready (RR) soybean lines GTS 40-3-2 and MON89788, using gold-coated magnetic nanoparticles (Fe 3 O 4 @Au) as nanosupport. To perform this magnetogenoassay, a sandwich-type hybridization assay was used with different enzymatic labelling systems (fluorescein isothiocyanate and digoxigenin) and dual screen-printed carbon electrodes (SPdCEs), which allowed the simultaneous readout of each target. A linear relationship ranging from 0.1 to 2.5 nM and from 0.1 to 1.0 nM was achieved for GTS 40-3-2 and MON89788 events, respectively, and both assays showed a similar detection limit of about 0.1 nM. Furthermore, a good performance in terms of precision and selectivity was achieved. The proposed approach is a step forward for event-specific multiplex detection. [ABSTRACT FROM AUTHOR]

Simple, cost-effective and reliable tools for the quantification of genetically modified organisms (GMO) in food and feed are highly demanded to enforce labelling legislation in the EU. Herein, we report a novel method for quantitative analysis of genetically modified soybean with the event GTS-40-3-2, also known as Roundup Ready (RR) soybean, using magnetoassays with electrochemical detection, coupled to DNA amplification by end-point polymerase chain reaction (PCR). For the proposed work, two DNA sequences were targeted via hybridisation onto magnetic beads, one specific for the transgenic event and the other for the taxon or species-specific lectin gene. Enzymatic labelling was performed to obtain an electrochemically active product measured by chronoamperometry. By optimising the number of PCR cycles, among other parameters, two magnetoassays coupled to PCR were successfully accomplished and linearity was obtained in the ranges of 53–4425 and 1093–88,496 DNA copies for the event-specific and lectin sequences, respectively. The proposed method provides accurate and precise RR soybean quantitative results, being effectively compared to those obtained by real-time PCR, as the reference method. These findings confirm the suitability of the method as an alternative tool for GMO quantification. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) plays an essential role in tumor progression and invasion through its continuous remodeling. The growth of most carcinomas is associated with an excessive collagen deposition that provides the proper environment for tumor development and chemoresistance. The α1 chain of a minor human collagen, type XI, is overexpressed in some tumor stroma, but not found in normal stroma. To test the clinical utility of this collagen as a cancer biomarker, specific receptors are needed. Available antibodies do not show enough selectivity or are directed toward the propeptide region that is cleaved when the protein is released to the ECM. Here we show the selection of an aptamer for the specific C-telopeptide region using a 16-mer peptide as the target for the SELEX. The aptamer selected with a K d of ∼25 nM was able to capture the collagen XI from cell lysates. It was also used for target detection in a mixed antibody-aptamer sandwich assay showing it can be useful for diagnostic purposes in biological fluids. [Display omitted] • Extracellular matrix components are an understudied source of cancer biomarkers. • Detection of collagen XI, overexpressed in some cancers, needs suitable probes. • A 16mer peptide was the target for the first aptamer for α chain of human collagen XI. • Mass spectrometry verifies collagen XI as the target of D1 aptamer from cell lysates. • Antibody-aptamer sandwich assay detects colXIα1 in cell lysates. [ABSTRACT FROM AUTHOR]

Enzyme-linked immunosorbent assays are currently the most popular methods to quantify gluten in foods. Unfortunately, the antibodies used as specific receptors in such methods are not compatible with the usual solvents for the extraction of gluten proteins. In consequence, commercial tests require a high dilution of the sample after the extraction, increasing the limit of quantification and decreasing convenience. In this work, we have rationally truncated an aptamer capable of recognizing gliadin in a deep eutectic solvent (DES). The truncated aptamer is a 19-nucleotides-long DNA that minimizes self-hybridization, allowing the development of an electrochemical sandwich-based sensor for the quantification of gluten in the DES ethaline. The sensor incorporates two identical biotin-labeled truncated aptamers, one of which is immobilized on a carbon screen-printed electrode and the other reports the binding of gliadin after incubation in streptavidin-peroxidase. This sensor can detect gliadin in DES, with a dynamic range between 1 and 100 μg/L and an intra-assay coefficient of variation of 11%. This analytical performance allows the quantification of 20 μg of gluten/kg of food when 1 g of food is extracted with 10 mL of ethaline. We demonstrate the ability of this method to achieve the measurement of gluten in food samples, after the extraction with pure ethaline. The assay is useful for the analysis of residual gluten levels in foods, thus facilitating the evaluation of any potential health risk associated with the consumption of such food by people with celiac disease or other gluten-related disorders. Image 1 • First aptamer-based sensor directly deployed in a deep-eutectic solvent. • A truncated aptamer is designed to be applied in a sandwich format. • Electrochemical sensor exhibits a limit of quantification of 20 μg gluten/kg sample. • Detection of residual gluten levels in food for highly-sensitive celiac patients. [ABSTRACT FROM AUTHOR]