Search

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]

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]

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]

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]

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]

Latest technological advancement has tremendously expanded the knowledge on the composition of body fluids and the cancer-associated changes, which has fueled the replacement of invasive biopsies with liquid biopsies by using appropriate specific receptors. DNA emerges as a versatile analytical reagent in electrochemical devices for hybridization-based or aptamer-based recognition of all kind of biomarkers. In this mini review, we briefly introduce the current affordable targets (tumor-derived nucleic acids, circulating tumor cells and exosomes) in body fluids, and then we provide an overview of selected electrochemical methods already applied in clinical samples by dividing them into three large categories according to sample type: red (blood), yellow (urine), and white (saliva and sweat) diagnostics. This review focuses on the hurdles of the complex matrices rather than a comprehensive and detailed revision of the format schemes of DNA-based electrochemical sensing. This diverse perspective compiles some challenges that are often forgotten and critically underlines real sample analysis or clinical validation assays. Finally, the needs and trends to reach the market are briefly outlined., (Copyright © 2020 Miranda-Castro, Palchetti and de-los-Santos-Álvarez.)