Rosa F. Dutra

Smart plastic antibody material (SPAM) tailored on disposable screen printed electrodes for protein recognition: Application to myoglobin detection

This work introduces two major changes to the conventional protocol for designing plastic antibodies: (i) the imprinted sites were created with charged monomers while the surrounding environment was tailored using neutral material; and (ii) the protein was removed from its imprinted site by means of a protease, aiming at preserving the polymeric network of the plastic antibody. To our knowledge, these approaches were never presented before and the resulting material was named here as smart plastic antibody material (SPAM). As proof of concept, SPAM was tailored on top of disposable gold-screen printed electrodes (Au-SPE), following a bottom-up approach, for targeting myoglobin (Myo) in a point-of-care context. The existence of imprinted sites was checked by comparing a SPAM modified surface to a negative control, consisting of similar material where the template was omitted from the procedure and called non-imprinted materials (NIMs). All stages of the creation of the SPAM and NIM on the Au layer were followed by both electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). AFM imaging was also performed to characterize the topography of the surface. There are two major reasons supporting the fact that plastic antibodies were effectively designed by the above approach: (i) they were visualized for the first time by AFM, being present only in the SPAM network; and (ii) only the SPAM material was able to rebind to the target protein and produce a linear electrical response against EIS and square wave voltammetry (SWV) assays, with NIMs showing a similar-to-random behavior. The SPAM/Au-SPE devices displayed linear responses to Myo in EIS and SWV assays down to 3.5 μg/mL and 0.58 μg/mL, respectively, with detection limits of 1.5 and 0.28 μg/mL. SPAM materials also showed negligible interference from troponin T (TnT), bovine serum albumin (BSA) and urea under SWV assays, showing promising results for point-of-care applications when applied to spiked biological fluids.

Occurrence of natural vertical transmission of dengue-2 and dengue-3 viruses in Aedes aegypti and Aedes albopictus in Fortaleza, Ceará, Brazil.

Background Aedes aegypti and Aedes albopictus perform an important role in the transmission of the dengue virus to human populations, particularly in the tropical and subtropical regions of the world. Despite a lack of understanding in relation to the maintenance of the dengue virus in nature during interepidemic periods, the vertical transmission of the dengue virus in populations of A. aegypti and A. albopictus appears to be of significance in relation to the urban scenario of Fortaleza. Methods From March 2007 to July 2009 collections of larvae and pupae of Aedes spp were carried out in 40 neighborhoods of Fortaleza. The collections yielded 3,417 (91%) A. aegypti mosquitoes and 336 (9%) A. albopictus mosquitoes. Only pools containing females, randomly chosen, were submitted to the following tests indirect immunofluorescence (virus isolation), RT-PCR/nested-PCR and nucleotide sequencing at the C-prM junction of the dengue virus genome. Results The tests on pool 34 (35 A. albopictus mosquitoes) revealed with presence of DENV-3, pool 35 (50 A. aegypti mosquitoes) was found to be infected with DENV-2, while pool 49 (41 A. albopictus mosquitoes) revealed the simultaneous presence of DENV-2 and DENV-3. Based on the results obtained, there was a minimum infection rate of 0.5 for A. aegypti and 9.4 for A. albopictus. The fragments of 192 bp and 152 bp related to DENV-3, obtained from pools 34 and 49, was registered in GenBank with the access codes HM130699 and JF261696, respectively. Conclusions This study recorded the first natural evidence of the vertical transmission of the dengue virus in populations of A. aegypti and A. albopictus collected in Fortaleza, Ceará State, Brazil, opening a discuss on the epidemiological significance of this mechanism of viral transmission in the local scenario, particularly with respect to the maintenance of these viruses in nature during interepidemic periods.

Myoglobin-biomimetic electroactive materials made by surface molecular imprinting on silica beads and their use as ionophores in polymeric membranes for potentiometric transduction

Myoglobin (Mb) is among the cardiac biomarkers playing a major role in urgent diagnosis of cardiovascular diseases. Its monitoring in point-of-care is therefore fundamental. Pursuing this goal, a novel biomimetic ionophore for the potentiometric transduction of Mb is presented. It was synthesized by surface molecular imprinting (SMI) with the purpose of developing highly efficient sensor layers for near-stereochemical recognition of Mb. The template (Mb) was imprinted on a silane surface that was covalently attached to silica beads by means of self-assembled monolayers. First the silica was modified with an external layer of aldehyde groups. Then, Mb was attached by reaction with its amine groups (on the external surface) and subsequent formation of imine bonds. The vacant places surrounding Mb were filled by polymerization of the silane monomers 3-aminopropyltrimethoxysilane (APTMS) and propyltrimethoxysilane (PTMS). Finally, the template was removed by imine cleavage after treatment with oxalic acid. The results materials were finely dispersed in plasticized PVC selective membranes and used as ionophores in potentiometric transduction. The best analytical features were found in HEPES buffer of pH 4. Under this condition, the limits of detection were of 1.3 × 10(-6)mol/L for a linear response after 8.0 × 10(-7) mol/L with an anionic slope of -65.9 mV/decade. The imprinting effect was tested by preparing non-imprinted (NI) particles and employing these materials as ionophores. The resulting membranes showed no ability to detect Mb. Good selectivity was observed towards creatinine, sacarose, fructose, galactose, sodium glutamate, and alanine. The analytical application was conducted successfully and showed accurate and precise results.

Disposable immunosensor for human cardiac troponin T based on streptavidin-microsphere modified screen-printed electrode

Screen-printed electrodes (SPE) have been widely used in the design of disposable sensors bringing advances in the use of electrochemical immunosensors for in field-clinical analysis. In this work, streptavidin polystyrene microspheres were incorporated to the electrode surface of SPEs in order to increase the analytical response of the cardiac troponin T (cTnT), a specific biomarker for the acute myocardial infarction diagnosis. The precise calculation of the stoichiometric streptavidin-biotin ratio [1:4] allowed the increase of sensitivity and stability of the immunosensor response to the cTnT analyte. The surface of the immunosensor was characterized by scanning electron microscopy and cyclic voltammetry. It was observed that the use of streptavidin microspheres significantly increased the analytical sensitivity of the electrode in 8.5 times, showing a curve with a linear response range between 0.1 and 10 ngmL(-1) of cTnT and a detection limit of 0.2 ngmL(-1). The proposed SPE showed ease preparation and high sensitivity allowing the detection of cTnT in the range of clinical levels. The new device coupled with a portable electrochemical analyzer shows great promise for point-of-care quantitative testing of necrosis cardiac proteins.

A sensor tip based on carbon nanotube-ink printed electrode for the dengue virus NS1 protein.

An immunosensor for the non-structural protein 1 (NS1) of the dengue virus based on carbon nanotube-screen printed electrodes (CNT-SPE) was successfully developed. A homogeneous mixture containing carboxylated carbon nanotubes was dispersed in carbon ink to prepare a screen printed working electrode. Anti-NS1 antibodies were covalently linked to CNT-SPE by an ethylenediamine film strategy. Amperometrical responses were generated at -0.5 V vs. Ag/AgCl by hydrogen peroxide reaction with peroxidase (HRP) conjugated to the anti-NS1. An excellent detection limit (in the order of 12 ng mL(-1)) and a sensitivity of 85.59 μA mM(-1)cm(-2) were achieved permitting dengue diagnostic according to the clinical range required. The matrix effect, as well as the performance of the assays, was successfully evaluated using spiked blood serum sample obtaining excellent recovery values in the results. Carbon nanotubes incorporated to the carbon ink improved the reproducibility and sensitivity of the CNT-SPE immunosensor. This point-of-care approach represents a great potential value for use in epidemic situations and can facilitate the early screening of patients in acute phase of dengue virus.

Potential of a simplified measurement scheme and device structure for a low cost label-free point-of-care capacitive biosensor

A simplified measurement scheme and device structure aiming at developing a low cost, label-free, point-of-care capacitive biosensor were investigated. The detection principle is the increase of low frequency capacitance between two planar Al electrodes observed after antibody-antigen interaction. The electrodes, deposited on oxidized Si wafers, were covered with an antibody layer, with and without using self-assembled thiol monolayer. Immunoglobulin G (IgG) and cardiac troponin T (TnT) were used as analytes to asses this proposal. The device was able to detect successfully TnT levels in the range 0.07 to 6.83ng/mL in human serum from patients with cardiac diseases and in the range 0.01ng/mL to 5ng/mL for TnT in phosphate buffer saline. An equivalent circuit model able to reproduce the general behavior of experimental capacitance versus frequency curves was presented. The investigated features that have potential to reduce costs and simplify measurements were: use of single, low frequency (1kHz) measurement signal, within the range of low cost portable capacitance meters; employment of a lower cost electrode material, aluminum, instead of gold electrodes; and use of simple and miniaturized planar two-electrodes arrangement, thus making a portable system for point-of-care applications.

An ultrasensitive human cardiac troponin T graphene screen-printed electrode based on electropolymerized-molecularly imprinted conducting polymer

A nano-molecularly imprinted polymer (N-MIP) assembled on a screen-printed electrode for the cardiac troponin T (cTnT) was developed. The biomimetic surface was obtained by a co-polymer matrix assembled on the reduced graphene oxide (RGO) electrode surface. The cTnT active sites were engineered using pyrrole and carboxylated pyrrole that was one-step electropolymerized jointly with cTnT by cyclic voltammetry. The stepwise preparation of the biomimetic surface was characterized by cyclic and differential pulse voltammetries using the ferrocyanide/ferricyanide as redox probe. Structural and morphological characterization was also performed. The optimal relation of pyrrole and pyrrole-3-acid carboxylic to perform the cTnT biomimetic nanosurface was obtained at 1:5 ratio. The analytical performance of cTnT N-MIP performed by differential pulse voltammetry showed a linear range from 0.01 to 0.1 ngmL(-1) (r=0.995, p«0.01), with a very low limit of detection (0.006 ngmL(-1)). The synergic effect of conductive polymer and graphene forming 3D structures of reactive sites resulted in a N-MIP with excellent affinity to cTnT binding (KD=7.3 10(-13) molL(-1)). The N-MIP proposed is based on a simple method of antibody obtaining with a large potential for point-of-care testing applications.

Salmonella spp., importante agente patogênico veiculado em alimentos

A ocorrencia de doencas transmitidas por alimentos (DTAs) tem sido foco de discussoes nos ultimos anos, devido a preocupacao mundial com estrategias que permitam seu controle e, consequentemente, garantam a colocacao de produtos seguros no mercado consumidor. A Salmonella spp. e um dos microrganismos mais amplamente distribuidos na natureza, sendo o homem e os animais seus principais reservatorios naturais, com ocorrencia de sorotipos regionais, reconhecidos como salmoneloses, e considerado como um dos principais agentes envolvidos em surtos de origem alimentar em paises desenvolvidos. O aumento da incidencia da salmonelose provocada por alimentos contaminados demonstra que, na atualidade, apesar dos avancos tecnologicos alcancados, este problema ainda ocorre mundialmente. As aves e bovinos sao responsaveis pela maior disseminacao desse agente patogenico. A ampla distribuicao da Salmonella spp. entre os animais, a existencia de portadores assintomaticos e sua permanencia no ambiente e nos alimentos contribuem para que este microrganismo assuma um papel de grande relevância na saude publica mundial e, portanto, programas permanentes de controle e erradicacao devem ser adotados.The occurrence of foodborne diseases has been a matter of discussion over the last years due to the worldwide concern with strategies for their control and for ensuring that safe food products reach the consumer. Salmonella spp. is among the most widespread microorganisms in nature, having man and animals as main natural reservoirs. With occurrence of regional serotypes causing salmonellosis, this pathogen is considered one of the main agents responsible for outbreaks of foodborne disease in the developing countries. The increasing incidence of salmonellosis caused by contaminated food has shown that, despite the recent technological improvements, this problem still occurs in all countries. Cattle and poultry are the main responsible for the transmission of this pathogenic agent. Due to its wide distribution in animals, the existence of asymptomatic carriers and its presence in foodstuff and in the environment Salmonella spp. represents a significant public health problem worldwide calling for permanent control programs and eradication strategies.

A carbon nanotube screen-printed electrode for label-free detection of the human cardiac troponin T.

Label-free immunosensor based on amine-functionalized carbon nanotubes screen-printed electrode is described for detection of the cardiac troponin T, an important marker of acute myocardial infarction. The disposable sensor was fabricated by tightly squeezing an adhesive carbon ink containing carbon nanotubes onto a polyethylene terephthalate substrate forming a thin film. The use of carbon nanotubes increased the reproducibility and stability of the sensor, and the amine groups permitted nonrandom immobilization of antibodies against cardiac troponin T. Amperometric responses were obtained by differential pulse voltammetry in presence of a ferrocyanide/ferricyanide redox probe after troponin T incubation. The calibration curve indicated a linear response of troponin T between 0.0025 ng mL(-1) and 0.5 ng mL(-1), with a good correlation coefficient (r=0.995; p<0.0001, n=7). The limit of detection (0.0035 ng mL(-1) cardiac troponin T) was lower than any previously described by immunosensors and was comparable with conventional analytical methods. The high reproducibility and clinical range obtained using this immunosensor support its utility as a potential tool for point-of-care acute myocardial infarction diagnostic testing.

A thiophene-modified screen printed electrode for detection of dengue virus NS1 protein.

A thiophene-modified screen printed electrode (SPE) for detection of the Dengue virus non-structural protein 1 (NS1), an important marker for acute phase diagnosis, is described. A sulfur-containing heterocyclic compound, the thiophene was incorporated to a carbon ink to prepare reproducible screen printed electrodes. After cured, the thiophene SPE was coated by gold nanoparticles conjugated to Protein A to form a nanostrutured surface. The Anti-NS1 antibodies immobilized via their Fc portions via Protein A, leaving their antigen specific sites free circumventing the problem of a random antibodies immobilization. Amperometric responses to the NS1 protein of dengue virus were obtained by cyclic voltammetries performed in presence of ferrocyanide/ferricyanide as redox probe. The calibration curve of immunosensor showed a linear response from 0.04 µg mL(-1) to 0.6 µg mL(-1) of NS1 with a good linear correlation (r=0.991, p<0.05). The detection limit (0.015 µg mL(-1) NS1) was lower than conventional analytical methods. In this work, thiophene monomers incorporated in the carbon ink enhanced the electroanalytical properties of the SPEs, increasing their reproducibility and sensitivity. This point-of-care testing represents a great potential for use in epidemic situations, facilitating the early diagnosis in acute phase of dengue virus.