Electrochemical Investigation of Oligonucleotide-DNA Hybridization on Poly(4-Methoxyphenethylamine)

This work describes the immobilization of purine and pyrimidine bases and immobilization/hybridization of synthetic oligonucleotides on graphite electrodes modified with poly(4-methoxyphenethylamine) produced in acid medium. The immobilization of adenine, guanine, cytosine and thymine on these modified electrodes was efficient, producing characteristic peaks. Another relevant observation is that, according to the literature, pyrimidine bases, cytosine and thymine are more difficult to detect. However, when immobilized onto the poly(4-methoxyphenethylamine), a significant increase in the magnitude of the current was obtained. The observation of the hybridization between the poly(GA) probe and its complementary, poly(CT) target, was possible by monitoring the guanosine and adenosine peaks or through methylene blue indicator, using differential pulse voltammetry. Hybridization results in a decrease of the peak current of guanosine and adenosine or the signal of methylene blue accumulated on the modified electrode surface. The hybridization with the complementary target was also investigated by electrochemical impedance spectroscopy. The results showed a significant modification in the Nyquist plot, after addition of the complementary target, with increase of the charge transference resistance

Label-free and reagentless electrochemical genosensor based on graphene acid for meat adulteration detection

Altres ajuts: CERCA Programme/Generalitat de CatalunyaWith the increased demand for beef in emerging markets, the development of quality-control diagnostics that are fast, cheap and easy to handle is essential. Especially where beef must be free from pork residues, due to religious, cultural or allergic reasons, the availability of such diagnostic tools is crucial. In this work, we report a label-free impedimetric genosensor for the sensitive detection of pork residues in meat, by leveraging the biosensing capabilities of graphene acid - a densely and selectively functionalized graphene derivative. A single stranded DNA probe, specific for the pork mitochondrial genome, was immobilized onto carbon screen-printed electrodes modified with graphene acid. It was demonstrated that graphene acid improved the charge transport properties of the electrode, following a simple and rapid electrode modification and detection protocol. Using non-faradaic electrochemical impedance spectroscopy, which does not require any electrochemical indicators or redox pairs, the detection of pork residues in beef was achieved in less than 45 min (including sample preparation), with a limit of detection of 9% w/w pork content in beef samples. Importantly, the sample did not need to be purified or amplified, and the biosensor retained its performance properties unchanged for at least 4 weeks. This set of features places the present pork DNA sensor among the most attractive for further development and commercialization. Furthermore, it paves the way for the development of sensitive and selective point-of-need sensing devices for label-free, fast, simple and reliable monitoring of meat purity

Label-free and reagentless electrochemical genosensor based on graphene acid for meat adulteration detection

With the increased demand for beef in emerging markets, the development of quality-control diagnostics that are fast, cheap and easy to handle is essential. Especially where beef must be free from pork residues, due to religious, cultural or allergic reasons, the availability of such diagnostic tools is crucial. In this work, we report a label-free impedimetric genosensor for the sensitive detection of pork residues in meat, by leveraging the biosensing capabilities of graphene acid - a densely and selectively functionalized graphene derivative. A single stranded DNA probe, specific for the pork mitochondrial genome, was immobilized onto carbon screen-printed electrodes modified with graphene acid. It was demonstrated that graphene acid improved the charge transport properties of the electrode, following a simple and rapid electrode modification and detection protocol. Using non-faradaic electrochemical impedance spectroscopy, which does not require any electrochemical indicators or redox pairs, the detection of pork residues in beef was achieved in less than 45 min (including sample preparation), with a limit of detection of 9% w/w pork content in beef samples. Importantly, the sample did not need to be purified or amplified, and the biosensor retained its performance properties unchanged for at least 4 weeks. This set of features places the present pork DNA sensor among the most attractive for further development and commercialization. Furthermore, it paves the way for the development of sensitive and selective point-of-need sensing devices for label-free, fast, simple and reliable monitoring of meat purity.We acknowledge funding from the European Union Horizon2020 Programme under Grant No. 881603 (Graphene Flagship Core 3). This article reflects only the author's view, and the European Commission is not responsible for any use that may be made of the information it contains. ICN2 is funded by the CERCA programme, Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa Centres of Excellence programme, funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706). J. M. R. Flauzino is grateful for the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior scholarship (CAPES-PRINT, Brazil, Grant number: 88887.371591/2019–00). D. Panáček acknowledges the Internal Student Grant Agency of the Palacký University in Olomouc, (IGA_PrF_2021_031). A. G. Brito-Madurro acknowledges the funding from Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (310782/2018–0). J. M. Madurro acknowledges the funding from Fundação de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG (CEX-APQ-02902-17) and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (311737/2018–8). A. Bakandritsos acknowledges the funding from the Czech Science Foundation, (project GA CR – EXPRO, 19–27454X). M. Otyepka acknowledges the ERC grand 2D-CHEM (No 683024 from H202). The work was supported also by the ERDF/ESF project “Nano4Future” (No.CZ.02.1.01/0.0/0.0/16_019/0000754)

A single amino acid substitution in one of the lipases of Aspergillus nidulans confers resistance to the antimycotic drug undecanoic acid

A plausible approach to evaluate the inhibitory action of antifungals is through the investigation of the fungal resistance to these drugs. We describe here the molecular cloning and initial characterization of the A. nidulans lipA gene, where mutation (lipA1) conferred resistance to undecanoic acid, the most fungitoxic fatty acid in the C(7:0)-C(18:0) series. The lipA gene codes for a putative lipase with the sequence consensus GVSIS and WIFGGG as the catalytic signature. Comparison of the wild-type and LIP1 mutant strain nucleotide sequences showed a G -> A change in lipA1 allele, which results in a Glu(214) -> Lys substitution in LipA protein. This ionic charge change in a conserved LipA region, next to its catalytic site, may have altered the catalytic properties of this enzyme resulting in resistance to undecanoic acid.FAPESPCNPqCAPESFAEP

Functional Epitope Core Motif of the Anaplasma marginale Major Surface Protein 1a and Its Incorporation onto Bioelectrodes for Antibody Detection

Anaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale, causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequence SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences ’ variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized: one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96 % and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated b

Antibody detection by ELISA.

<p>Detection of immunoglobulin G antibodies anti-<i>Anaplasma marginale</i> MSP1a in serum samples from infected and non-infected bovine with a definitive diagnosis of anaplasmosis (n = 24), and apparently healthy individuals (n = 52) by enzyme-linked immunosorbent assay using the Am1 (A) and Am2 (B), and the respectively ROC curve.</p

Impedance response of graphite electrode.

<p>Nyquist diagrams of a the polymeric film poly(3-HPA) (-□-), poly(3-HPA)/Am1 (-Δ-), poly(3-HPA)/Am1:IgG+ (-☆-), and poly(3-HPA)/Am1:IgG- (-○-) obtained in aqueous solution containing K₃Fe(CN)₆/K₄Fe(CN)₆ (5 mmol.L⁻¹) and KCl (0.1 mol.L⁻¹), recorded from an applied potential of +0.24 V, amplitude of 10 mV, and frequency range from 100 KHz to 10 Hz. The continuous lines represent the fitting curve to the equivalent circuit. Inset: amplification of high frequencies region.</p

AFM topographical images of modified graphite electrode with poly(3-HPA).

<p>(A) Without biomolecules; (B) polymeric film after immobilization of the synthetic peptide Am1, and (C) agglutination process (Am1:IgG+).</p