Ana G. Brito-Madurro

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.

Detection of a specific biomarker for Epstein-Barr virus using a polymer-based genosensor.

This paper describes methodology for direct and indirect detections of a specific oligonucleotide for Epstein-Barr virus (EBV) using electrochemical techniques. The sequence of oligonucleotide probe (EBV1) revealed a high sequence identity (100%) with the EBV genome. For the development of the genosensor, EBV1 was grafted to the platform sensitized with poly(4-aminothiophenol). After that, the hybridization reaction was carried out with the complementary target (EBV2) on the modified electrode surface using ethidium bromide as DNA intercalator. The oxidation peak currents of ethidium bromide increased linearly with the values of the concentration of the complementary sequences in the range from 3.78 to 756 μmol·L−1. In nonstringent experimental conditions, this genosensor can detect 17.32 nmol·L−1 (three independent experiments) of oligonucleotide target, discriminating between complementary and non-complementary oligonucleotides, as well as differentiating one-base mismatch, as required for detection of genetic diseases caused by point mutations. The biosensor also displayed high specificity to the EBV target with elimination of interference from mix (alanine, glucose, uric acid, ascorbic acid, bovine serum albumin (BSA), glutamate and glycine) and good stability (120 days). In addition, it was possible to observe differences between hybridized and non-hybridized surfaces through atomic force microscopy.

Electropolymerization of hydroxyphenylacetic acid isomers and the development of a bioelectrode for the diagnosis of bacterial meningitis

In this work, three polymeric films derived from hydroxyphenylacetic acid isomers were electropolymerized onto the surfaces of graphite electrodes through cyclic voltammetry. Analysis of electrochemical behaviors and electrical properties of the three polymeric films were investigated and their probably structures were explored by molecular modeling. The best functionalization strategy for the incorporation and retention of adenine and guanine nitrogenous bases was the poly(3-hydroxyphenylacetic acid). It was applied for the immobilization of a nucleotide probe specific to DNA of the bacteria Neisseria meningitidis operated in amperometric and impedimetric indirect modes. The simple protocol of electrochemical detection of DNA by the bioelectrode, developed in this work, could be further enhanced and applied in a low-cost and pain-less platform to diagnose human meningitis infection.

A Biosensor Using Poly(4-Aminophenol)/acetylcholinesterase modified graphite electrode for the detection of dichlorvos

The properties of poly(4-aminophenol) modified graphite electrode as material for the immobilization of acetylcholinesterase were investigated by the Cyclic Voltammetry, Electrochemical Impedance Spectroscopy and Atomic Force Microscopy. The polymer was deposited on graphite electrode surface by the oxidation of 4-aminophenol and then acetylcholinesterase was immobilized on the surface of the electrode. The biosensor coupled in the continuous flow system was employed for the detection of dichlorvos. The detection and quantification limits were 0.8 and 2.4 μmol L-1 dichlorvos, respectively. Graphite electrodes modified with the poly(4-aminophenol) showed to be an efficient and promising material for immobilization of acetylcholinesterase enzyme. The proposed method requires simple parts which are easy to build, involves only one biosensor and the potentiometric detection is simple.

Development of direct assays for Toxoplasma gondii and its use in genomic DNA sample

Graphical abstract Figure. No Caption available. HighlightsA new biosensor to toxoplasmosis was developed based on modified electrode.The biosensor was able of detect until 100 ng mL−1 of the T. gondii genomic DNA.The genosensor showed high selectivity, discriminating non‐specific targets;.Optical assays showed significant change in the absorbance peak in presence of Toxoplasma gondii genomic DNA. Abstract This work describes an approach for the selection and detection of specific DNA probes related to Toxoplasma gondii, a protozoan parasite responsible for toxoplasmosis. The detection system was developed on graphite carbon electrode modified with poly(3‐hydroxybenzoic acid) sensitized with ToxG1 probe. The hybridization of the specific genomic DNA related to T. gondii showed good response by direct detection of guanine residue oxidation using differential pulse voltammetry (DPV). The biosensor was able to distinguish both the complementary and non‐complementary targets and detect up to 100 ng &mgr;L−1 of the T. gondii genomic DNA. The hybridization (ToxG1: T. gondii genomic DNA) was confirmed by optical measurement. Optical assays using gold nanoparticles:ToxG1 probe showed a significant change in the absorbance peak in the presence of the T. gondii genomic DNA according to the electrochemical results. This novel biosensor shows potential as electrochemical transducer and was successfully applied in the biological sample.

Use of gold nanoparticles on graphite electrodes functionalized with poly (4-aminophenol) in the development of a bioelectrode for hepatitis B

Background Hepatitis B is an infectious disease of the liver, highly prevalent in society, caused by hepadnavirus (HBV), which affects approximately 350 million individuals worldwide [1]. It is estimated that the number of deaths from the disease is in the range of 2000 to 4000 per year, mainly by hepatic cirrhosis and hepatocellular carcinoma [2]. Due to the fact that in adults in most cases the initial infection shows no symptoms, the diagnosis is difficult and late. The development of biosensors for disease diagnosis has been a much valued currently, and in this area there are already several innovative research. Among the various detection techniques used in these sensors, due to the low cost and high sensitivity, electrochemical detection has been widely used [3]. Some polymers have high applicability in this area, because through chemical affinity can immobilize the molecule that will bind to its target. Studies realized by our group [4] indicates that the poly(4aminophenol) electrodeposited in acid medium is interesting for the immobilization of oligonucleotides. Polymer embedded with metal nanoparticles provided the suitable microenvironment for biomolecules, since improved the electron transfer with the electrode surfaces, resultant in enhanced sensing performance [5]. In this work, specific graphite electrode modified with poly(4-aminophenpol)/gold nanoparticles was sensibilized with specific oligonucleotide for the detection of hepatitis B virus and compared to the bioelectrode in the absence of the gold nanoparticles. Methods The monomer solution was prepared in 0.5 mol.l HClO4 and the electropolymerization was carried out by cyclic voltammetry in a three compartment cell using a CH Instruments potentiostat model 420A. Gold nanoparticles (1:3 solute/solvent) was added onto graphite electrode containing poly(4-aminophenol). Then, the solution containing the HBV oligonucleotide (0,414 mg.mL) was added onto poly(4-aminophenol) with gold nanoparticles. Other experiments were conducted in the absence of the gold nanoparticles. The detection of the immobilization of oligonucleotides was carried out by differential pulse voltammetry in a electrochemical cell of one compartment. The oxidation peak of guanosine present in the oligonucleotide was monitored in the potential of +0.9 V (vs. Ag/AgCl).

Application of nanomaterials for the electrical and optical detection of the hepatitis B virus

This work describes different approaches for the detection of hepatitis B virus (HBV) genomic DNA, using electrochemical and optical techniques. The platforms consisted of a single-stranded DNA probe (HEPB1S), specific to HBV, grafted on a gold electrode modified with reduced graphene oxide or gold nanoparticles. Differential pulse voltammetry analysis indicates that the addition of HBV genomic DNA caused an increase of about 1.4 times in the current peak value, when compared to the negative control. It was observed a linear dependence with the log HBV-genomic DNA concentration and the electrochemical biosensor detected until 7.65 pg μL-1 of the target. Electrochemical impedance spectroscopy measurements showed an increase of about 2 times in the charge transfer resistance, after the addition of HBV genomic DNA. Assays using colloidal suspension of gold nanoparticles showed a shift of the peak wavelength, linearly proportional to the HBV-genomic DNA concentration, with a detection limit of 0.15 ng μL-1. The applicability of the gold nanoparticles for clinical samples was tested with success in the blood plasma. All the approaches used in this work were effective in detecting genomic DNA or blood plasma in positive samples for HBV.

Frontiers of biology in human diseases: strategies for biomolecule's discovery, nanobiotechnologies and biophotonics.

The current frontiers in biological sciences demand an interface among disciplines of biology, chemistry, and physics to achieve new paradigms in applied nanobiotechnologies to health. An extraordinary amount of information on genomes, transcriptomes, proteomes, metabolomes, miRNAs, lncRNAs, protein editing, post-translational modifications, exosomes, molecular interactions, cell signaling, structural biology, immunology, cellular receptors, interactomes, and bioinformatics are opening new possibilities for biological manipulations aiming improved diagnostics and therapeutics. New rationale is required to use available technologies that intersect among imaging, electrochemistry, biophotonics, nanotechnologies and combinatorial molecules. A massive, diverse and broad knowledge on multidisciplinary aspects have been provided in the last few years, and the challenge is to combine recent technologies and information to identify revolutionary platforms for the progression of life sciences. This brief review will discuss examples of epitope-based and combinatorial antibody and nucleic acid (aptamers) selection technologies in association with nanobiotechnologies and their multiple applications in biomedical sciences.

Electrochemical evaluation of anti-CRP/CRP interaction for the molecular diagnosis of the cardiovascular risk

Background The C-reactive protein (CRP or PTX1) is an acute phase protein, expressed by hepatocytes, that is regarded as a clinical marker for infection and has been increasingly used as a risk indicator in chronic inflammatory diseases [1]. Atherosclerosis is an inflammatory disease that remains a major cause of morbidity and mortality. Its progression is associated with the accumulation of lipoproteins in the endothelium, establishing plaques whose rupture may result in the formation of a thrombus, arising several cardiovascular complications [2]. Studies show there is a correlation between the risk of cardiac events (such as myocardial infarction) and increasing levels of CRP [3]. Currently, there are several methods to detect CRP, including immunoassays and agglutination. However, these methods are not sensitive enough, time-consuming or cost-ineffective [4]. Therefore, new diagnosis methods are being developed. Electrochemical biosensors are small devices that combine the selectivity of biochemical molecular recognition with the sensitivity of electrode transducers, being remarkable for their sspecificity, speed, portability and low cost [5]. In this work, a graphite electrode surface is modified with poly (3-aminotiophenol) and a specific antibody (anti-CRP), aiming the detection of its antigen (CRP), through electrochemical methods.