Search

trans-Sialidase and cruzipain are important virulence factors from Trypanosoma cruzi, the etiological agent of Chagas disease, that have highly antigenic domains in their structure and were reported as potential tools for diagnosis of the illness. The aim of the present study is to assess the possibility of using cruzipain and the catalytic domain of trans-sialidase in a Surface Plasmon Resonance-based immunosensor for the diagnosis of chronic Chagas disease. Immunoassays carried out with canine sera verified that cruzipain allows the detection of anti-Trypanosoma cruzi antibodies whereas recombinant trans-sialidase did not yield specific detections, due to the high dilutions of serum used in the immunoassays that hinder the possibility to sense the specific low titer antibodies. The developed cruzipain-based biosensor, whose price per assay is comparable to a commercial enzyme-linked immunosorbent assay (ELISA), was successfully applied for the rapid quantification of specific antibodies against Trypanosoma cruzi in fresh human sera showing an excellent agreement with ELISA Fil: Chain, Cecilia Yamil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Pires Souto, Dênio Emanuel. Universidade Federal do Paraná; Brasil Fil: Sbaraglini, Maria Laura. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Labriola, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Daza Millone, Maria Antonieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Ramirez, Eduardo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Cisneros, José Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Lopez Albizu, Maria Constanza. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; Argentina Fil: Scollo, Karenina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; Argentina Fil: Kubota, Lauro T.. Universidade Estadual de Campinas; Brasil Fil: Ruiz, Andrés Mariano. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; Argentina Fil: Vela, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina

SGTs (small glutamine-rich TPR-containing proteins) are dimeric proteins that belong to the class of co-chaperones characterized by the presence of TPR domains (containing tetratricopeptide repeats). Human (SGTA) and yeast (Sgt2) SGTs are characterized by three distinct domains: an N-terminal dimerization domain, a central TPR-domain important for binding to other proteins (chaperones included) and a C-terminal domain involved in hydrophobic interactions. Both these SGTs are involved in the cellular PQC (protein quality control) system, as they interact with chaperones and have functions that aid stress recovery. However, there are differences between them, such as structural features and binding specificities, that could be better understood if other orthologous proteins were studied. Therefore, we produced and characterized a putative SGT protein, designated AaSGT, from the mosquito Aedes aegypti, which is a vector of several diseases, such as dengue and Zika. The protein was produced as a folded dimer which was stable up to 40 °C and was capable of binding to AaHsp90 and fully protecting a model protein, α-synuclein, from aggregation. The conformation of AaSGT was investigated by biophysical tools and small angle X-ray scattering, which showed that the protein had an elongated conformation and that its C-terminal domain was mainly disordered. The results with a C-terminal deletion mutant supported these observations. Altogether, these results are consistent with those from other functional SGT proteins and add to the understanding of the PQC system in Aedes aegypti, an important aim that may help to develop inhibitory strategies against this vector of neglected diseases.

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

An immunosensor was developed using a SAM of an alkanethiol associated with PAMAM(G4) dendrimers based on surface plasmon resonance (SPR) to enhance the sensitivity for troponin T detection in blood samples. The feasibility of using three-dimensional platforms based on dendrimers for the development of immunosensors was demonstrated by evaluating three different generations of these dendrimers (G3, G4, and G5) to detect troponin T. The results showed the efficiency of these 3D platforms in anchoring biomolecules, amplifying the detection of troponin T. The sandwich assay showed good performance for troponin T detection, using secondary monoclonal antibodies, in the concentration range of 5 - 300 ng mL-1 (0.14 - 8.67 nmol L-1), R2 = 0.991, with the LOD of 3.6 ng mL-1. The sandwich assay's applicability was demonstrated by evaluating a secondary polyclonal antibody's performance in the concentration range of 3 - 30 ng mL-1, R2 = 0.998, with the LOD of 0.98 ng mL-1. The immunosensor was applied to determine troponin T in blood plasma samples from healthy patients, with an average recovery of 88 to 104%. The performance of the SPR-based immunosensor indicates reliable results and is expected to contribute to the rapid diagnosis of heart attack, with reduced costs.

The immune system can recognize microbes and sterile tissue damage. Among the damage-associated molecular patterns (DAMPs), uric acid is considered a major component which can trigger inflammation. It represents a breakpoint in the evolutionary history of humans as our ancestors lost the uricase gene, the enzyme responsible for its cleavage. High soluble uric acid (sUA) concentration is able to increase IL-1β in murine, but not human macrophages. We observed that sUA increased the mRNA expression of Naip1 in murine macrophages, and, therefore, we hypothesized that the recognition of sUA can be made by a Naip1-Nlrp3 inflammasome platform. Additionally, we used genome-wide transcriptome analysis, functional analyses and structural modeling predictions and observed that virus-transduction of murine Naip1 into human macrophages induced IL-1β after sUA stimulus, besides leading to fatty acid production and an inflammation-related response. Moreover, pharmacologic inhibition and genetic loss of Nlrp3 led to decreased IL-1β production upon sUA stimulus. Surface plasmon resonance and quartz crystal microbalance showed that sUA is able to interact with Naip1. Naip could be a lost receptor for sUA in the evolutionary process and a better understanding of the immune modulatory function of sUA could lead to design rational novel anti-hyperuricemic therapies.

This work describes the development of a high sensitive photoelectrochemical immunosensor for the detection of anti-Leishmania infantum antibodies. The proposed sensor is based on cadmium sulfide films and gold nanoparticles deposited on indium tin oxide coated glass slide (AuNP/CdS/ITO). The band gap and flat band characteristics of AuNP/CdS platform were evaluated by UV–vis spectrophotometry and electrochemical impedance spectroscopy. The effects of the nature, concentration, and pH of the buffer solution as well as the effects of the applied potential on the response of the photoelectrochemical platform to a donor molecule were investigated. Under optimized conditions, the AuNP/CdS/ITO platform modified with recombinant L. infantum antigens (C8/MPA-AuNP/CdS/ITO) shows a linear response range from 1 up to 300 nmol L −1 to anti-C8 antibodies. Finally, the capability of the proposed photoelectrochemical immunosensor to discriminate positive and negative canine serum samples was evaluated.

A photoelectrochemical immunosensor for the detection of anti-Leishmania infantum antibodies based on zinc oxide and cadmium sulfide films electrodeposited on an ITO-coated glass slide (CdS/ZnO/ITO) has been proposed. The effects of light/dark conditions and the kinetics of CdS sensitizer regeneration were evaluated by scanning electrochemical microscopy in feedback mode. The platform was modified using two different peptides (PEP 13 and PEP 16) from two different proteins of high specificity and selectivity toward recognition of L. infantum antibodies, producing Peps/CdS/ZnO/ITO. This photoelectrochemical immunosensor provides a cheap and promising method of discriminating between positive and negative canine serum samples. Keywords: Photoelectrochemical immunosensor, Visceral leishmaniasis, Visible LED light, Cadmium sulfide, Zinc oxide

The use of biosensors in point-of-care (POC) testing devices has attracted considerable attention in the past few years, mainly because of their high specificity, portability, and relatively low cost. Coupling these devices with miniaturized electrochemical transducers has shown great potential toward simple, rapid, and cost-effective analysis that can be performed in the field, especially for healthcare, environmental monitoring, and food quality control. For this reason, the number of publications in this field has grown exponentially over the past decade, making it a trending topic in current research. Although great improvement has been achieved in the field of electrochemical biosensing, there are still some challenges to overcome, especially concerning the improvement of sensing materials and miniaturization. In this Review, we summarize some of the most recent advances achieved in POC electrochemical biosensor applications, focusing on new materials and modifiers for biorecognition developed to improve sensitivity, specificity, stability, and response time.

DnaJ/Hsp40 chaperones deliver unfolded proteins and stimulate the ATPase activity of DnaK/Hsp70 via their J-domain. However, the interaction is transient, creating a challenge for detailed analysis. We investigated whether it would be possible to gain further understanding of this interaction by engineering a chimeric polypeptide where the J-domain of Hsp40 was covalently attached to the substrate binding domain (SBD) of Hsp70 by a flexible linker. The rationale is to increase the proximity between the interacting partners to promote their natural interaction and facilitate the characterization of the interaction. The resulting chimera, termed J-SBD, was properly folded and had properties not present in the full-length Hsp70 or in the SBD alone, for instance a higher protective effect against aggregation and being a monomer. Substrate binding also appear to exceed that of SBD alone as revealed by a decreased binding to bis-ANS, a probe for hydrophobic patches. This hypothesis is supported by the structural model created by small angle X-ray scattering, suggesting that the lid subdomain (SBDα) is partially opened in the J-SBD. Collectively, our results suggest a model in which J-domain binding may shift the Hsp70 equilibrium towards the monomer state, exposing hydrophobic sites prone to substrate accommodation.

The co-chaperone CHIP (carboxy terminus of Hsc70 interacting protein) is very important for many cell activities since it regulates the ubiquitination of substrates targeted for proteasomal degradation. However, information on the structure-function relationship of CHIP from plants and how it interacts and ubiquitinates other plant chaperones is still needed. For that, the CHIP ortholog from Sorghum bicolor (SbCHIP) was identified and studied in detail. SbCHIP was purified and produced folded and pure, being capable of keeping its structural conformation up to 42 °C, indicating that cellular function is maintained even in a hot environment. Also, SbCHIP was able to bind plant Hsp70 and Hsp90 with high affinity and interact with E2 enzymes, performing E3 ligase activity. The data allowed to reveal the pattern of plant Hsp70 and Hsp90 ubiquitination and described which plant E2 enzymes are likely involved in SbCHIP-mediated ubiquitination. Aditionally, we obtained information on the SbCHIP conformation, showing that it is a non-globular symmetric dimer and allowing to put forward a model for the interaction of SbCHIP with chaperones and E2 enzymes that suggests a mechanism of ubiquitination. Altogether, the results presented here are useful additions to the study of protein folding and degradation in plants.

In this work, an SPR immunosensor was developed to elucidate the reaction kinetics between a protein of unknown function in Leishmania infantum (hypothetical C1 protein) and specific antibodies of the visceral leishmaniasis (VL). A platform, which is based on layer-by-layer assembly was formed by cysteamine in combination with a fourth-generation poly(amidoamine) dendrimer (PAMAM(G4)) on gold surface for the immobilisation of the protein. This film resulted in amplification of the signal of SPR. Then, a kinetic model based on a bivalent ligation suggested that the reaction between the C1 protein and the anti-C1 antibody occurs in two steps. The value of the equilibrium dissociation constant (KD1×KD2=1.64×10−7 mol L−1) demonstrated high binding affinity between the biomolecules. Furthermore, low limits of detection (LOD=7.37 nmol L−1) and quantification (LOQ=7.83 nmol L−1) were presented with the proposed SPR immunosensor. Afterwards, the addition of real samples consisting of positive and negative canine sera for VL was accompanied by high sensitivity and selectivity by SPR immunosensor. Therefore, this study quantitatively demonstrated the strong antigenic character of a hypothetical protein and consequently its potential use in the immunodiagnosis of the VL.

This work describes the synthesis of the 1,2,3-triazole amino acid-derived-3-O-galactosides 1-6 and the 1,2,3-triazole di-lactose-derived glycoconjugate 7 as potential galectin-3 inhibitors. The target compounds were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-derived amino acids N3-ThrOBn, N3-PheOBn, N3-N-Boc-TrpOBn, N3-N-Boc-LysOBn, N3-O-tBu-AspOBn and N3-l-TyrOH, and the corresponding alkyne-based sugar 3-O-propynyl-GalOMe, as well as by click chemistry reaction between the azido-lactose and 2-propynyl lactose. Surface plasmon resonance (SPR) assays showed that all synthetic glycoconjugates 1-7 bound to galectin-3 with high affinity, but the highest binders were the amino acids-derived glycoconjugates 2 (KD 7.96μM) and 4 (KD 4.56μM), and the divalent lactoside 7 (KD1 0.15μM/KD2 19μM). Molecular modeling results were in agreement with SPR assays, since more stable interactions with galectin-3 were identified for glycoconjugates 2, 4 and 7. Regarding compounds 2 and 4, they established specific cation-π (Arg144) and ionic (Asp148) interactions, whereas glycoconjugate 7 was capable to bridge two independent galectin-3 CRDs, creating a non-covalent cross-link between two monomers and, thus, reaching a submicromolar affinity towards galectin-3.

Protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, which needs urgent progress in its immunological diagnosis. Surface plasmon resonance (SPR) is a promising technique for the development of immunosensors with biomedical applications. In this work, a SPR-based immunosensor has been developed by the first time for real time and label free immunoassay for detection of anti -T. cruzi antibodies in serum samples. T. cruzi antigen was successfully immobilized on a SPR sensor chip via activated mixed self-assembled monolayer (SAM) of 3-mercaptopropionic acid (3-MPA) and 11-mercaptoundecanoic acid (11-MUA), by amide coupling. After the sensor construction, a pool of human sera infected with T. cruzi was added to its surface and the antibodies were detected in sera diluted up to 1280 times, indicating excellent sensitivity of the technique for detection of antigen–antibody interaction. The addition of a pool of negative human serum at dilutions lower than 1:160 to the sensor surface was accompanied by a null or very low response. Then, the following operational parameters of the immunoassay were optimized and defined: time of immobilization and antigen concentration at 20 min and 30 mg mL −1 , serum dilution at 1:320, preventing of nonspecific bindings with solution of BSA 1.0% and surface regeneration by injection of SDS 1.0%. The immunoassay, here termed SPRCruzi, showed high capability in the discrimination of positive and negative sera, including those infected with other pathogens usually sources of false positives results in conventional serodiagnosis. Therefore, the proposed immunosensor was successfully developed and the immunoassay allowed a simple, effective, faster and specific detection of anti- T. cruzi antibodies, which represents an encouraging field for the progress of the diagnosis of Chagas disease.

The Triangulum--Andromeda (TriAnd) overdensity is a distant structure of the Milky Way located in the second Galactic quadrant well below the Galactic plane. Since its discovery, its nature has been under discussion, whether it could be old perturbations of the Galactic disk or the remains of a disrupted former dwarf galaxy. In this study, we investigate the kinematics and chemical composition in 13 stars selected as TriAnd candidates from 2MASS photometry. The sample was observed using the GRACES high-resolution spectrograph attached to the Gemini North telescope. Based on radial velocities obtained from the spectra and the astrometric data from GAIA, three different kinematic criteria were used to classify our sample stars as belonging to the TriAnd overdensity. The TriAnd confirmed members in our sample span a range in metallicities, including two metal-poor stars ([Fe/H] $\sim$ -1.3 dex). We show that the adopted kinematical classification also chemically segregates TriAnd and non-TriAnd members of our sample, indicating a unique chemical pattern of the TriAnd stars. Our results indicate different chemical patterns for the [Na/Fe], [Al/Fe], [Ba/Fe], and [Eu/Fe] ratios in the TriAnd stars when compared to the chemical pattern of the local disk; the paucity of studies chemically characterizing the outer disk population of the Milky Way is the main obstacle in establishing that the TriAnd population is chemically similar to field stars in the outer disk. But TriAnd chemical pattern is reminiscent of that found in outer disk open clusters, although the latter are significantly more metal-rich than TriAnd., Comment: 15 pages, 12 figures, Accepted to ApJ

In the present paper, the use of a gold electrode modified by 4-nitrothiophenol/4-mercaptobenzoic acid binary self-assembled monolayer (4NTP/4MBA SAM) for the simultaneous determination of epinephrine (EP) and uric acid (UA) is described. Electron transfer (ET) kinetics through 4NTP/4MBA SAM was studied using scanning electrochemical microscopy (SECM) and electrochemical impedance spectroscopy (EIS). The SECM strategy was used to measure the heterogeneous rate constant for activated 4NTP/4MBA SAM and non-activated 4NTP/4MBA SAM using [ Fe ( CN ) 6 ] 4 - / 3 - as probe. The apparent surface pKap value of the 4NTP/4MBA SAM modified electrode was estimated by EIS (pKap = 5.0). The electrochemical response of the modified electrode toward epinephrine (EP) and uric acid (UA) were investigated by differential pulse voltammetry (DPV). The results showed an efficient electrochemical response and surface activity of the electrode for the electro-oxidation of EP and UA, which leads to improvement of selectivity of the electrode response. Two well-defined voltammetric peaks, in a buffered solution at pH 5.5, were obtained by differential pulse voltammetry (DPV) at 0.120 and 0.475 V vs Ag/AgCl for EP and UA, respectively. Linear calibration plots were obtained in the range of 0.1–2.0 μmol L−1 for EP and 1–175 μmol L−1 for UA, with sensitivity of 1.2 μA L μmol−1 and 0.0117 μA L μmol−1, respectively.

Polyaniline/O,O′-bis (2-aminoethyl) polyethylene glycol-functionalized multiwalled carbon nanotube (PANI/PEG–MWCNT) composite-modified electrode was successfully prepared by electropolymerization. The ionic transport in PANI/PEG–MWCNT film and its effects on the composite performance are presented. Both protonic and anionic participation in the charge compensation processes were calculated and they indicated that the presence of the PEG–MWCNT in the PANI film suppress the anionic transportation and improve the composite ability in fixing horseradish peroxidase enzyme. Finally, the adsorption between the negatively charged PANI/PEG–MWCNT nanocomposite and the positively charged Horseradish peroxidase resulted in a high sensitivity (1.01 μA L cm−2 μmol−1) to hydrogen peroxide. This sensor exhibited a good reproducibility and stability at an applied potential of −100 mV vs Ag/AgCl.

Orientador: Lauro Tatsuo Kubota Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química Resumo: A leishmaniose é referida pela organização mundial da saúde como uma das principais doenças parasitárias, acometendo cerca de 2 milhões de pessoas anualmente em 98 países diferentes. A leishmaniose visceral (LV) é a forma mais grave e devido às limitações dos ensaios convencionais (ELISA, por exemplo) utilizados para seu diagnóstico, grandes motivações estão voltadas ao desenvolvimento de novas metodologias para detecção dessa parasitose. No presente trabalho, as técnicas de ressonância de plásmons de superfície (SPR) e microbalança de cristal de quartzo (QCM) foram utilizadas no desenvolvimento de imunossensores para detecção de anticorpos específicos da LV. Em relação à etapa de funcionalização de superfícies metálicas durante a construção dos biossensores, maior eficiência foi observada através de plataformas multivalentes formadas por dendrímeros ou dendrons combinados ou não a monocamadas auto-organizadas (SAMs) de alcanotióis. Nesta etapa, destaca-se a síntese orgânica de um dendron inédito do tipo HS-dendron-(PEG-COOH)9. Em relação à caracterização dos biossensores, enfatiza-se uma abordagem particularmente adotada pelo nosso grupo, através da qual foi possível mediante curvas de refletância de SPR obtidas em diferentes comprimentos de onda (670 e 785 nm) determinar simultaneamente a espessura (4,64 nm) e o índice de refração (1,475) de uma proteína recombinante quimérica imobilizada sobre o substrato sensor. Através de estudos cinéticos, foi possível mensurar o potencial antigênico de novas proteínas de L. infantum, verificando elevada afinidade de ligação de duas proteínas (KD = 8,27 x 10-10 mol L-1 para a proteína quimérica recombinante (CP10); e KD1 x KD2 = 1,64x10-7 mol L-1 para a proteína recombinante hipotética C1) frente a anticorpos específicos da LV. Tais resultados, somados à capacidade de detecção de casos assintomáticos (> 80%) observados nos ensaios de ELISA, evidenciaram a viabilidade de aplicação dessas proteínas no imunodiagnóstico da LV. Análises com amostras reais (soros caninos) apontaram vantagens dos biossensores de SPR e QCM em relação aos testes convencionais, tais como, maior sensibilidade, especificidade, rapidez e a não necessidade de marcação. Portanto, os resultados obtidos neste trabalho de tese são substanciais para contribuição significativa no diagnóstico e programas de controle da LV Abstract: Leishmaniasis is referred to by the world health organization as one of the main parasitical diseases, affecting approximately 2 million people annually in 98 different countries. The visceral leishmaniasis (VL) is the most serious form of the disease and due to the limitations of the conventional assays (ELISA, for example) used for its diagnosis, high motivations are turned to the development of new methodologies for the detection of this parasitosis. In this current work, the techniques of surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) were used in the development of immunosensors for the detection of specific antibodies of VL. Regarding the step of functionalization of metallic surfaces during the construction of the biosensors, a higher efficiency was observed through multivalent platforms formed by dendrimers or dendrons combined or not to self-assembled monolayers (SAMs) of alkanethiols. In this step, emphasis is given to the organic synthesis of an unprecedented dendron of the type HS-dendron-(PEG-COOH)9. With respect to the characterization of the biosensors, an approach adopted particularly by our group is highlighted, by which it was possible, using reflectance curves of SPR obtained at different wavelengths (670 and 785 nm), to determine simultaneously the thickness (4,64 nm) and the refractive index (1,475) of a recombinant chimeric protein immobilized on the sensor substrate. Through kinetic studies, it was possible to measure the antigenic potential of new proteins of L. infantum, verifying high bonding affinity of two proteins (KD = 8,27 x 10-10 mol L-1 for the recombinant chimeric protein (CP10); and KD1 x KD2 = 1,64x10-7 mol L-1 for the recombinant chimeric protein C1) towards specific antibodies of the VL. Such results, added to the capacity of detection of asymptomatic cases (> 80%) observed in the ELISA assays, gave evidence to the viability of the application of these proteins in the immunodiagnostics of the VL. Analyses with real samples (canine serums) pointed out advantages of the SPR and QCM biosensors when compared to conventional tests, such as higher sensitivity, specificity, speed and the fact that marking is not necessary. Therefore, the results obtained in this thesis work are substantial for significant contribution in the diagnosis and control programs of VL Doutorado Química Analítica Doutor em Ciências FAPESP 2012/24493-3

Instituto de Qumica, Universidade Federal de Uberlndia, 38700-128 Patos de Minas – MG, Brasil*e-mail: wallanst@yahoo.com.brReceived: January 10, 2012;&Accepted: February 17, 2012AbstractThis work presents a simple, fast and low-cost method for simultaneous determination of acetylsalicylic acid (ASA),without alkaline hydrolysis and caffeine (CF) in pharmaceutical formulations using a boron-doped diamond as theworking electrode through differential pulse voltammetry. A good repeatability was reached for 20 measurements,with a low relative standard deviation of less than 1.0%. The calibration curves presented a great linear correlationcoefficient for both drugs (R=0.999) with a limit of detection of 1.610

In this work we demonstrate the use of a dielectric barrier discharge plasma for the treatment of SU-8. The resulting hydrophilic surface displays a 5° contact angle and (0.40 ± 0.012) nm roughness. Using this technique we also present a proof of concept of IgG and prostate specific antigen biodetection on a thin layer of SU-8 over gold via surface plasmon resonance detection.

BACKGROUND: We defined the methodological criteria for the interpretation of the results provided by a novel immunoassay based on surface plasmon resonance (SPR) to detect antibodies anti-Trypanosoma cruzi in human sera (SPRCruzi). Then, we evaluated its applicability as a diagnostic tool for Chagas disease. METHODS: To define the cut-off point and serum dilution factor, 57 samples were analyzed at SPRCruzi and the obtained values of SPR angle displacement (ΔθSPR) were submitted to statistical analysis. Adopting the indicated criteria, its performance was evaluated into a wide panel of samples, being 99 Chagas disease patients, 30 non-infected subjects and 42 with other parasitic/infectious diseases. In parallel, these samples were also analyzed by ELISA. RESULTS: Our data demonstrated that 1:320 dilution and cut-off point at ∆θSPR=17.2 m° provided the best results. Global performance analysis demonstrated satisfactory sensitivity (100%), specificity (97.2%), positive predictive value (98%), negative predictive value (100%) and global accuracy (99.6%). ELISA and SPRCruzi showed almost perfect agreement, mainly between chagasic and non-infected individuals. However, the new immunoassay was better in discriminate Chagas disease from other diseases. CONCLUSION: This work demonstrated the applicability of SPRCruzi as a feasible, real time, label free, sensible and specific methodology for the diagnosis of Chagas disease.

This work describes the highly sensitive detection of organophosphorus pesticides employing the cobalt(II) 4,4,4,4-tetrasulfo-phthalocyanine (CoTSPc) macrocycle complex, carbon nanotubes (CNT), and 1-methyl-3-octylimidazolium tetrafluoroborate (OMIM[BF4]). The technique is based on enzyme acetylcholinesterase (AChE) inhibition. The composite was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and amperometry. The AChE was immobilized on the composite electrode surface by cross-linking with glutaraldehyde and chitosan. The synergistic action of the CoTSPc/CNT/OMIM[BF4] composite showed excellent electrocatalytic activity, with a low applied potential for the amperometric detection of thiocholine (TCh) at 0.0 V vs. Ag/AgCl. The calculated catalytic rate constant, k(cat), was 3.67 × 10(3) mol(-1) L s(-1). Under the optimum conditions, the inhibition rates of these pesticides were proportional to their concentrations in the ranges of 1.0 pmol L(-1) to 1.0 nmol L(-1) (fenitrothion), 2.0 pmol L(-1) to 8.0 nmol L(-1) (dichlorvos), and 16 pmol L(-1) to 5.0 nmol L(-1) (malathion).

In the present study, the surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) techniques were employed to kinetically evaluate the binding affinity of a new recombinant chimeric protein (CP10) toward anti-Leishmania infantum antibodies for the immunodiagnostics of the visceral leishmaniasis (VL). This chimeric protein was formed by the union in a same artificial coding DNA of ten different peptides, which showed themselves reactive toward positive canine serum for VL. Using the CP10 in enzyme-linked immunosorbent assays (ELISA), it was possible to detect 80% of the asymptomatic infected dogs. After this, SPR and QCM immunosensors were constructed by the covalent immobilization of the CP10 on a self-assembled monolayer (SAM) formed by adsorption of alkanethiol on gold substrates. The thickness (6.80 nm) and the refractive index (1.475) of the protein on the SAM were simultaneously determined through SPR curves measured in different wavelengths (670 and 785 nm). Interactions between the CP10 and its specific IgGs (anti-CP10 antibodies) were characterized by the electrochemical impedance spectroscopy, SPR and QCM techniques. The equilibrium dissociation constant obtained by SPR (K(D) = 8.27 x 10(-10) mol.L(-1)) and QCM (K(D) = 2.42 x 10(- 10) mol.L(-1)) demonstrated high binding affinity of the CP10 toward anti-CP10 antibodies. In this sense, this work quantitatively proves the strong antigenic character of a new recombinant chimeric protein, giving evidence to potential contribution for the use of this protein in programs of control of the VL.

In this work ,a surface Plasmon resonance (SPR) immune sensor was developed using an 11-mercaptoundecanoic acid (11-MUA) modified gold SPR sensor chip for the detection of anti- Leishmania infantum antibodies. The soluble antigens of L. infantum were securely immobilized on an SPR gold disk byan 11-MUA self-assembled monolayer. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) techniques were employed in the characterization of the antigenim mobilization. After the immune sensor construction, canine serum positive for visceral leishmaniasis was added to its surface and showed significant variation in the SPR angle, indicating excellent sensitivity of the technique for antigen–antibody interaction detection. Moreover, the addition of negative serum was accompanied by as maller response, demonstrating that the immunosensor shows good specificity against anti-L. infantum antibodies. Therefore, this work demonstrates the success ful development of an SPR sensor for anti- L. infantum antibodies detection in short time, showing a great perspective as asensing system of visceral leishmania sisinende micregions.