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In the present work, the development of a genosensor for the event-specific detection of MON810 transgenic maize is proposed. Taking advantage of nanostructuration, a cost-effective three dimensional electrode was fabricated and a ternary monolayer containing a dithiol, a monothiol and the thiolated capture probe was optimized to minimize the unspecific signals. A sandwich format assay was selected as a way of precluding inefficient hybridization associated with stable secondary target structures. A comparison between the analytical performance of the Au nanostructured electrodes and commercially available screen-printed electrodes highlighted the superior performance of the nanostructured ones. Finally, the genosensor was effectively applied to detect the transgenic sequence in real samples, showing its potential for future quantitative analysis. [ABSTRACT FROM AUTHOR]

Abstract: Electrochemical genosensors are becoming cost-effective miniaturizable alternatives to real-time PCR (RT-PCR) methods for the detection of sequence-specific DNA fragments. We report on the rapid detection of PCR amplicons without the need of purification or strand separation. A challenging target sequence for both PCR amplification and electrochemical detection allowed us to address some difficulties associated to hybridization on electrode surfaces. The target was a highly specific oligonucleotide sequence of wheat encoding the most immunogenic peptide of gliadin that triggers the immune response of celiac disease (CD), the 33-mer. With a sandwich assay format and a rational design of the capture and tagged-signaling probes the problems posed by the strong secondary structure of the target and complementary probes were alleviated. Using a binary self-assembled monolayer and enzymatic amplification, a limit of detection of 0.3nM was obtained. The genosensor did not respond to other gluten-containing cereals such as rye and barley. Coupling to PCR to analyze wheat flour samples required tailoring both the capture and signaling probes. This is the first time that deleterious steric hindrance from long single-stranded regions adjacent to the electrode surface is reported for relatively short amplicons (less than 200bp). The importance of the location of the recognition site within the DNA sequence is discussed. Since the selected gene fragment contains several repetitions of short sequences, a careful optimization of the PCR conditions had to be performed to circumvent the amplification of non-specific fragments from wheat flour. [Copyright &y& Elsevier]

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]

A magnetic microsphere-based hybridization assay for the electrochemical detection of pathogen-specific DNA sequences is reported. Enzyme-linked sandwich hybridization assays with different tags (biotin, fluorescein or digoxigenin) and enzyme conjugates (streptavidin-alkaline phosphatase, streptavidin-peroxidase, antifluorescein-peroxidase or antidigoxigenin-peroxidase) are comparatively evaluated. Sensitivity and limit of detection depend on both the choice of the enzyme and the tag. The highest sensitivity and lowest limit of detection were obtained when antibody Fab fragments conjugated to peroxidase were used as reporter molecule. The digoxigenin system yields the lowest detection limit, mainly due to producing much lower background signals. [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]

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]

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]