Development of a disposable and highly sensitive paper-based immunosensor for early diagnosis of Asian soybean rust.

Soybean is one of the most important crops and plays a key role in the whole food chain production. Soybean crops are very susceptible to the fungus Phakopsora Pachyrhizi, the agent responsible by the Asian soybean rust. The spore of the fungus is easily disseminated by wind with adequate environment, leaf wetness, high humidity and temperatures, the crop can be totally lost within few days. A high sensitive, specific and easy test is the key for early diagnosing the soybean rust and therefore save the crop. Here we present a paper-based immunosensor for early stage diagnosis of soybean rust that can be performed by unskilled operators on-site. Nitrocellulose membrane was chosen as the substrate to stick the antigen due to its high binding properties. Polyclonal antibodies labeled with fluorescent nanoparticles were employed as the recognizers. An analytical curve with spiked samples shows a linear response range from 0.0032 to 3.2 µg/mL. This immunosensor presents a very low detection limit of 2.2 ng/mL, which corresponds approximately to 8-12 spores/mL. The paper-based sensor reachs the detection range of ELISA and PCR based test systems, and outranges the available commercial test kits by two order of magnitude. We believe this immunosensor has a great potential as a point-of-care device for the early diagnosis of Asian soybean rust.

One-step synthesis of polymer core-shell particles with a carboxylated ruthenium complex: a potential tool for biomedical applications.

Luminescent core-shell particles are structures widely applied to biomedical purposes with the potential of combining multiple features within one single particle. The development of particles that are easily synthesised and tunable for each application, combining biocompatibility, easy bioconjugation and a high detection signal as a label, is highly desired. In this work, we describe a one-step synthesis of poly[styrene-co-(2-hydroxyethyl methacrylate)], PSHEMA, core-shell particles containing [Ru(4,4'-dicarboxilate-2,2'-bpy)3] luminescent complexes. These particles show monodispersity, biocompatibility, easy functionalization and dye incorporation to focus on bioapplications, such as cell-tracking and diagnostics. The monomers assemble during the polymerization and produce core-shell structures with hydrophilic-hydrophobic character. This allows the concentration of hydrophilic ruthenium complexes onto the shell and incorporation of hydrophobic molecules (e.g. diphenylanthracene) due to the hydrophobic character of styrene. The incorporation of the Ru complex resulted in higher photostability compared to the free dye. Furthermore, carboxylic groups on the particle surface originated from carboxylated ligands of Ru complexes were used to immobilize biomolecules. The particles were successfully used as a diagnostic label for dengue fever (DF) infection. Using the complexes in the immunospot assay the test provided a detection limit (DL) of 187 ng mL-1 for the viral non-structural glycoprotein NS1. The particles showed a considerable decrease in the DL and allowed the diagnosis of the infection 24 hours earlier compared to common available assays based on gold nanoparticles. In addition, the particles were tested with an adherent grown fibroblast cell line and showed potential biocompatibility.

Immunospot assay based on fluorescent nanoparticles for Dengue fever detection.

Dengue fever is one of the most neglected tropical diseases and of highest international public health importance, with 50 million cases worldwide every year. Early detection can decrease mortality rates from more than 20% to less than 1% and the relevant early diagnosis analyte is the viral non-structural glycoprotein, NS1. Currently, enzyme linked immunosorbent assay (ELISA) is the method of choice to detect NS1. However, this is a time consuming method, requiring 3-5h, and it is the bottleneck for routine of clinical analysis laboratory in epidemic periods, when hundreds of samples should be tested. Here we describe an easy method combining principles of fluorophore linked immunosorbent assay (FLISA) and enzyme linked immunospotting (ELISPOT). For detection, we used mouse anti-NS1 IgG labeled with fluorescent nanoparticles. The presented procedure needs only 4 µL of serum samples and requires 45-60 min. The detection limit, 5.2 ng/mL, is comparable to ELISA tests. The comparison of 83 samples with a commercial ELISA revealed a sensitivity of 81% and specificity of 88%. The use of fluorescent nanoparticles provides a higher sensitivity than an assay using usual fluorescent dye molecules, besides avoiding bleaching effects. Based on the results, the proposed method provides fast, specific and sensitive results, and proves to be a suitable method for Dengue NS1 detection in impoverished regions or epidemic areas.

Enhancement of the detection limit for lateral flow immunoassays: evaluation and comparison of bioconjugates.

There is an increasing demand for convenient and accurate point-of-care tools that can detect and diagnose different stages of a disease in remote or impoverished settings. In recent years, lateral flow immunoassays (LFIA) have been indicated as a suitable medical diagnostic tool for these environments because they require little or no sample preparation, provide rapid and reliable results with no electronic components and thus can be manufactured at low costs and operated by unskilled personnel. However, even though they have been successfully applied to acute and chronic disease detection, LFIA based on gold nanoparticles, the standard marker, show serious limitations when high sensitivity is needed, such as early stage disease detection. Moreover, based on the lack of comparative information for label performance, significant optimization of the systems that are currently in use might be possible. To this end, in the presented work, we compare the detection limit between the four most used labels: colloidal-gold, silver enhanced gold, blue latex bead and carbon black nanoparticles. Preliminary results were obtained by using the biotin-streptavidin coupling as a model system and showed that carbon black had a remarkably low detection limit of 0.01 µg/mL in comparison to 0.1 µg/mL, 1 µg/mL and 1mg/mL for silver-coated gold nanoparticles, gold nanoparticles and polystyrene beads, respectively. Therefore, as a proof of concept, carbon black was used in a detection system for Dengue fever. This was achieved by immobilizing monoclonal antibodies for the nonstructural glycoprotein (NS1) of the Dengue virus to carbon black. We found that the colorimetric detection limit of 57 ng/mL for carbon black was ten times lower than the 575 ng/mL observed for standard gold nanoparticles; which makes it sensitive enough to diagnose a patient on the first days of infection. We therefore conclude that, careful screening of detection labels should be performed as a necessary step during LFIA development in order to enhance the detection limit in a final test system.

Electrostatic contributions in the increased compatibility of polymer blends.

Successful blending of different polymers to make a structural or functional material requires overcoming limitations due to immiscibility and/or incompatibility that arise from large polymer-polymer interfacial tensions. In the case of latex blends, the combination of capillary adhesion during the blended dispersion drying stage with electrostatic adhesion in the final product is an effective strategy to avoid these limitations, which has been extended to a number of polymer blends and composites. This work shows that adhesion of polymer domains in blends made with natural rubber and synthetic latexes is enhanced by electrostatic adhesion that is in turn enhanced by ion migration, according to the results from scanning electric potential microscopy. The additional attractive force between domains improves blend stability and mechanical properties, broadening the possibilities and scope of latex blends, in consonance with the "green chemistry" paradigm. This novel approach based on electrostatic adhesion can be easily extended to multicomponent systems, including nonpolymers.

Clay platelet partition within polymer blend nanocomposite films by EFTEM.

Transmission electron microscopy (TEM) is the main technique used to investigate the spatial distribution of clay platelets in polymer nanocomposites, but it has not often been successfully used in polymer blend nanocomposites because the high contrast between polymer phases impairs the observation of clay platelets. This work shows that electron spectral imaging in energy-filtered TEM (EFTEM) in the low-energy-loss spectral crossover region allows the observation of platelets on a clear background. Separate polymer domains are discerned by imaging at different energy losses, above and below the crossover energy, revealing the material morphology. Three blends (natural rubber [NR]/poly(styrene-butyl acrylate) [P(S-BA)], P(S-BA)/poly(vinyl chloride) [PVC], and NR/starch) were studied in this work, showing low contrast between the polymer phases in the 40-60 eV range. In the NR/P(S-BA) and P(S-BA)/PVC blend nanocomposites, the clay platelets accumulate in the P(S-BA) phase, while in the P(S-BA)/PVC nanocomposites, clay is also found at the interfaces. In the NR/starch blend, clay concentrates at the interface, but it also penetrates the two polymer phases. These observations reveal that nanostructured soft materials can display complex morphochemical patterns that are discerned thanks to the ability of EFTEM to produce many contrast patterns for the same sample.

Molecular mapping by low-energy-loss energy-filtered transmission electron microscopy imaging.

Structure-function relationships in supramolecular systems depend on the spatial distribution of molecules, ions, and particles within complex arrays. Imaging the spatial distribution of molecular components within nanostructured solids is the objective of many recent techniques, and a powerful tool is electron spectroscopy imaging in the transmission electron microscope (ESI-TEM) in the low-energy-loss range, 0-80 eV. This technique was applied to particulate and thin film samples of dielectric polymers and inorganic compounds, providing excellent distinction between areas occupied by various macromolecules and particles. Domains differentiated by small changes in molecular composition and minor differences in elemental contents are clearly shown. Slight changes in the molecules produce intensity variations in molecular spectra that are in turn expressed in sets of low-energy-loss images, using the standard energy-filtered transmission electron microscopy (EFTEM) procedures. The molecular map resolution is in the nanometer range and very close to the bright-field resolution achieved for the same sample, in the same instrument. Moreover, contrast is excellent, even though sample exposure to the electron beam is minimal.