A Redox-Probe-Free Immunosensor Based on Electrocatalytic Prussian Blue Nanostructured Film One-Step-Prepared for Zika Virus Diagnosis.

The Zika virus (ZIKV) is a great concern for global health due to its high transmission, including disseminating through blood, saliva, urine, semen and vertical transmission. In some cases, ZIKV has been associated with microcephaly, neurological disorders, and Guillain−Barré syndrome. There is no vaccine, and controlling the disease is a challenge, especially with the co-circulation of the Dengue virus, which causes a severe cross-reaction due to the similarity between the two arboviruses. Considering that electrochemical immunosensors are well-established, sensitive, and practical tools for diagnosis, in this study we developed a sensor platform with intrinsic redox activity that facilitates measurement readouts. Prussian blue (PB) has a great ability to form electrocatalytic surfaces, dispensing redox probe solutions in voltammetric measurements. Herein, PB was incorporated into a chitosan−carbon nanotube hybrid, forming a nanocomposite that was drop-casted on a screen-printed electrode (SPE). The immunosensor detected the envelope protein of ZIKV in a linear range of 0.25 to 1.75 µg/mL (n = 8, p < 0.01), with a 0.20 µg/mL limit of detection. The developed immunosensor represents a new method for electrochemical measurements without additional redox probe solutions, and it is feasible for application in point-of-care diagnosis.

Transient Expression of Dengue Virus NS1 Antigen in Nicotiana benthamiana for Use as a Diagnostic Antigen.

Dengue is a viral disease that represents a significant threat to global public health since billions of people are now at risk of infection by this mosquito-borne virus. The implementation of extensive screening tests is indispensable to control this disease, and the Dengue virus non-structural protein 1 (NS1) is a promising antigen for the serological diagnosis of dengue fever. Plant-based systems can be a safe and cost-effective alternative for the production of dengue virus antigens. In this work, two strategies to produce the dengue NS1 protein in Nicotiana benthamiana leaves were evaluated: Targeting NS1 to five different subcellular compartments to assess the best subcellular organelle for the expression and accumulation of NS1, and the addition of elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags to NS1. The transiently expressed proteins in N. benthamiana were quantified by Western blot analysis. The NS1 fused to ELP and targeted to the ER (NS1 ELP-ER) showed the highest yield (445 mg/kg), approximately a forty-fold increase in accumulation levels compared to the non-fused protein (NS1-ER), representing the first example of transient expression of DENV NS1 in plant. We also demonstrated that NS1 ELP-ER was successfully recognized by a monoclonal anti-dengue virus NS1 glycoprotein antibody, and by sera from dengue virus-infected patients. Interestingly, it was found that transient production of NS1-ER and NS1 ELP-ER using vacuum infiltration of whole plants, which is easier to scale up, rather than syringe infiltration of leaves, greatly improved the accumulation of NS1 proteins. The generated plant made NS1, even without extensive purification, showed potential to be used for the development of the NS1 diagnostic tests in resource-limited areas where dengue is endemic.

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.

[Spatial distribution and breeding site characteristics of Aedes albopictus and Aedes aegypti in Fortaleza, State of Ceará]. / Distribuição espacial e características dos criadouros de Aedes albopictus e Aedes aegypti em Fortaleza, Estado do Ceará

INTRODUCTION: Since the registration of Aedes albopictus in the municipality of Fortaleza in 2005, it has shown rapid dispersion. A study aiming to identify its occurrence areas, breeding sites and associations with Aedes aegypti and other culicids was carried out during 2008. METHODS: Between January and July 2008, samples of immature forms of culicids were gathered from properties located in districts of Fortaleza. Certain features of the breeding sites were highlighted, such as location (indoors or outdoors), presence of covering (protection against impact of sunlight and rain), water turbidity (water clear or not clear), constituent material, volume, height above ground level and simultaneous presence of different species of culicids in the same breeding site. RESULTS: Indoor location was an important factor for the breeding sites for Aedes albopictus [PR = 0.52; 95% CI: 0.33-0.81]. On the other hand, water turbidity and reservoir covering were not shown to be differentiators regarding infestation [p > 0.05]. Absence of water turbidity was significant for Aedes aegypti infestation in breeding sites [PR = 1.14; 95% CI: 1.06-1.22]. CONCLUSIONS: Absence of one of the species from the breeding sites enhanced the infestation by another species. Breeding sites that had not been infested by Aedes albopictus had a prevalence of infestation by Aedes aegypti that was 2.05 times greater [95% CI: 1.72-2.44]. There was no significant association between volume and height of the breeding sites and infestation by both species. Both species were found to be dispersed throughout the municipality, occupying a wide diversity of breeding sites. However, a slight physical separation could be identified, with higher infestation with Aedes albopictus outdoors.

Distribuição espacial e características dos criadouros de Aedes albopictus e Aedes aegypti em Fortaleza, Estado do Ceará / Spatial distribution and breeding site characteristics of Aedes albopictus and Aedes aegypti in Fortaleza, State of Ceará

INTRODUÇÃO: desde seu registro, em 2005, no município de Fortaleza, o Aedes albopictus tem exibido uma rápida dispersão. Um estudo visando à identificação das áreas de sua ocorrência, os seus criadouros e a associação com Aedes aegypti e outros culicídeos foi realizado durante o ano de 2008. MÉTODOS: foram coletadas, de janeiro a julho de 2008, amostras de formas imaturas de culicídeos provenientes de imóveis situados nos bairros de Fortaleza, ressaltando-se algumas características dos criadouros, tais como localização (intradomicílio ou peridomicílio), presença de cobertura (proteção contra a incidência de raios solares e chuva), turbidez da água (água límpida e não límpida), material constituinte, volume, altura em relação ao nível do solo e presença simultânea de diferentes espécies de culicídeos no mesmo criadouro. RESULTADOS: a localização no intradomicílio foi um fator importante para os criadouros do Aedes albopictus [RP=0,52 IC95 por cento (0,33-0,81)], por outro lado, a turbidez da água e a cobertura do depósito não se mostraram como diferenciadores para a infestação [p>0,05]. Para o Aedes aegypti a ausência de turbidez da água foi significativa para a infestação nos criadouros [RP=1,14 IC95 por cento (1,06-1,22)]. CONCLUSÕES: a ausência de uma das espécies nos criadouros favorecia sua infestação por outra; criadouros não infestados por Aedes albopictus tinham uma prevalência de infestação de 2,05 [IC95 por cento1,72-2,44] vezes maior pelo Aedes aegypti. Não houve associação significativa entre volume e altura do criadouro e infestação por ambas as espécies. As duas espécies encontram-se dispersas por todo o município, ocupando os mais diversos tipos de criadouros. No entanto, pode-se identificar uma ligeira separação física, com uma infestação maior do Aedes albopictus no peridomicílio.

INTRODUCTION: Since the registration of Aedes albopictus in the municipality of Fortaleza in 2005, it has shown rapid dispersion. A study aiming to identify its occurrence areas, breeding sites and associations with Aedes aegypti and other culicids was carried out during 2008. METHODS: Between January and July 2008, samples of immature forms of culicids were gathered from properties located in districts of Fortaleza. Certain features of the breeding sites were highlighted, such as location (indoors or outdoors), presence of covering (protection against impact of sunlight and rain), water turbidity (water clear or not clear), constituent material, volume, height above ground level and simultaneous presence of different species of culicids in the same breeding site. RESULTS: Indoor location was an important factor for the breeding sites for Aedes albopictus [PR = 0.52; 95 percent CI: 0.33-0.81]. On the other hand, water turbidity and reservoir covering were not shown to be differentiators regarding infestation [p > 0.05]. Absence of water turbidity was significant for Aedes aegypti infestation in breeding sites [PR = 1.14; 95 percent CI: 1.06-1.22]. CONCLUSIONS: Absence of one of the species from the breeding sites enhanced the infestation by another species. Breeding sites that had not been infested by Aedes albopictus had a prevalence of infestation by Aedes aegypti that was 2.05 times greater [95 percent CI: 1.72-2.44]. There was no significant association between volume and height of the breeding sites and infestation by both species. Both species were found to be dispersed throughout the municipality, occupying a wide diversity of breeding sites. However, a slight physical separation could be identified, with higher infestation with Aedes albopictus outdoors.

Surface plasmon resonance immunosensor for human cardiac troponin T based on self-assembled monolayer.

The cardiac troponin T (cTnT) is specific biomarker important for trials of acute myocardial infarctions (AMI). In this paper, a SPR sensor in real time to detect the biomarker was developed on a commercially available surface plasmon resonance AUTOLAB SPIRIT. The cTnT receptor molecule was covalently immobilized on a gold substrate via a self-assembled monolayer (SAM) of thiols by using cysteamine-coupling chemistry. This biosensor presented a linear response range for cTnT between 0.05 and 4.5 ng/mL (r=0.997, p<<0.01) with a good reproducibility (CV=4.4%). The effect of the cysteamine (CYS) concentrations on the SAM coated gold sensor was studied as a function of the amount of the immobilized cTnT monoclonal antibodies. Analysis using serum samples undiluted was carried out at room temperature showing a well agreement with the ECLIA methods and the sensor surface could be regenerated by using a solution of 1% (w/v) sodium dodecyl sulphate (SDS) without losing the sensor immunoreactivity. These studies open new perspectives of using SAM to develop regenerable immunosensor with a good reproducibility allowing its use in the clinical applications.

An SPR immunosensor for human cardiac troponin T using specific binding avidin to biotin at carboxymethyldextran-modified gold chip.

BACKGROUND: The cardiac troponin T (TnT) is a cardiospecific, highly sensitive marker for myocardial damage and is immediately released to bloodstream during the acute myocardial infarction (AMI). Surface plasmon resonance (SPR) immunosensor was developed for the quick detection of human cTnT in real time. METHODS: An SPR sensor streptavidin terminated self-assembled monolayer, which was used to binding biotinylated anti troponin T monoclonal antibodies, was developed. RESULTS: The cTnT was determined from successive injections with a linear range from 0.03 up to 6.5 ng/ml. The detection limit was 0.01 ng/ml corresponding to a resonant angle change of 1.28 millidegrees. The system presented a good repeatability with 3.4% of variation between run after regeneration of the coated surface with a solution of 1% SDS. The effects of blocking of non-specific adsorption using different solutions were studied. It was possible to measure the cTnT without dilution of the human serum with good specificity and reproducibility. CONCLUSION: This sensor is practical and offer quick response in interval of 800 s.