The application of microbeads to microfluidic systems for enhanced detection and purification of biomolecules.

This paper describes microbead-based microfluidic systems. Several aspects of bead assays in microfluidics make them advantageous for bioassays in simple microchannels, including enhanced surface-to-volume ratio, improved molecular recognition reaction efficiency, and the wide range of surface functionalization available with commercial microbeads. Two-level SU-8 molds are used to fabricate PDMS microchannels that can hydrodynamically trap different types of microbeads, with characteristic dimensions of tens of microns. The use of these microbead-based microfluidic systems in the biosensing of antibodies, toxins and nucleic acids, as well as in antibody purification will be presented and discussed in this paper.

A point-of-use microfluidic device with integrated photodetector array for immunoassay multiplexing: Detection of a panel of mycotoxins in multiple samples.

For a point-of-use analytical device to be successful in real-world applications, it needs to be rapid, simple to operate and, ideally, able to multiplex the detection of several analytes and samples. Mycotoxin detection in food and feedstock in particular has become increasingly relevant as these toxins, such as ochratoxin A (OTA), aflatoxin B1 (AFB1) and deoxynivalenol (DON), are subject to strict regulations and recommendations in the European Union. A novel, simple, negative pressure-driven device with manually operated magnetic valves was developed and the simultaneous immunodetection of these three mycotoxins was demonstrated via the laminar flow patterning of probes in an area of ≈0.12mm2 and subsequent chemiluminescence generation via HRP-labeled antibodies. The three mycotoxins were detected in less than 20min at concentrations of 100ng/mL for OTA and DON and 3ng/mL for AFB1, spiked in a sample under analysis and simultaneously compared to a toxin-free reference and a standard contaminated with critical target concentrations. The on-chip optical detection was performed in a single acquisition step by integrating a microfabricated array of 25×25µm2 hydrogenated amorphous silicon (a-Si:H) photosensors below the microfluidic chip. The device presented in this work is simple and effective for point-of-use multiplexing of immunoassays and was applied in this work to the screening of mycotoxins.

On-chip sample preparation and analyte quantification using a microfluidic aqueous two-phase extraction coupled with an immunoassay.

Immunoassays are fast and sensitive techniques for analyte quantification, and their use in point-of-care devices for medical, environmental, and food safety applications has potential benefits of cost, portability, and multiplexing. However, immunoassays are often affected by matrix interference effects, requiring the use of complex laboratory extraction and concentration procedures in order to achieve the required sensitivity. In this paper we propose an integrated microfluidic device for the simultaneous matrix clean-up, concentration and detection. This device consists of two modules in series, the first performing an aqueous two-phase extraction (ATPE) for matrix extraction and analyte pre-concentration, and the second an immunoassay for quantification. The model analyte was the mycotoxin ochratoxin A (OTA) in a wine matrix. Using this strategy, a limit of detection (LoD) of 0.26 ng mL(-1) was obtained for red wine spiked with OTA, well below the regulatory limit for OTA in wines of 2 ng mL(-1) set by the European Union. Furthermore, the linear response on the logarithmic concentration scale was observed to span 3 orders of magnitude (0.1-100 ng mL(-1)). These results are comparable to those obtained for the quantification of OTA in plain buffer without an integrated ATPE (LoD = 0.15 ng mL(-1)). The proposed method was also found to provide similar results for markedly different matrices, such as red and white wines. This novel approach based on aqueous two-phase systems can help the development of point-of-care devices that can directly deal with real samples in complex matrices without the need for extra extraction processes and equipment.

Aqueous two-phase systems for enhancing immunoassay sensitivity: simultaneous concentration of mycotoxins and neutralization of matrix interference.

Immunoassays have a broad application range, from environmental and food toxicology to biomedical analysis, providing rapid and simple methods for analyte quantification. Immunoassays, however, are often challenging at nM and sub nM concentrations and are affected by detrimental matrix interference effects, as is the case of the detection of ochratoxin A (OTA) and Aflatoxin B1 (AFB1). These are widespread mycotoxins found in food and feed, with serious potential implications for human health. This work demonstrates the use of polymer-salt aqueous two phase systems (ATPSs) for the simultaneous concentration of mycotoxins and neutralization of matrix interference. In particular, polyethylene glycol (PEG)-phosphate salt ATPSs were used to enhance the detection sensitivity of OTA and AFB1 in wines and beer by an indirect competitive ELISA. Using this methodology it was possible to quantify both analytes spiked in red wine with limits-of-detection (LoD) down to 0.19 ng/mL and 0.035 ng/mL, respectively, with results comparable to those obtained using solutions of toxins in phosphate buffered saline (PBS) buffer (0.7 ng/mL and 0.009 ng/mL, respectively). Furthermore, a very low matrix-to matrix variability was observed, with LoD and half inhibitory concentration (IC50) values of 5.17 ± 1.08 and 33.2 ± 3.5 ng/mL (±SD) obtained in the detection of OTA spiked in red and white wines, beer or PBS buffer. These results indicate the potential of ATPS as a fast and simple concentration step and in providing matrix-independent analyte quantification for enhanced immunoassay sensitivity below regulatory levels.

Atividade antimicrobiana e sinérgica das frações das folhas de Schinopsis brasiliensis Engl. frente a clones multirresistentes de Staphylococcus aureus / Antimicrobial and synergic activity of fractions from the leaves of Schinopsis brasiliensis Engl. against Staphylococcus aureus multiresistant clones

No presente estudo objetivou-se avaliar a atividade antimicrobiana e sinérgica de 4 frações das folhas de Schinopsis brasiliensis Engl (F1', F2', F1" e F2") frente às cepas Staphylococcus aureus MRSA multirresistentes. Os métodos utilizados foram poços de difusão em ágar, concentração mínima inibitória (CMI) - diluição em ágar, e bioautografia. Nos resultados bioautográficos observou-se três halos de inibição relacionados, no mínimo, à quatro constituintes ativos; sendo dois deles isolados das folhas (galato de metila e ácido gálico). A F2" (200∝g/mL) apresentou halos de inibição de 16 e 19mm frente as cepas de S. aureus multirresistente e Klebsiella pneumoniae, e CMI 100∝g/mL, respectivamente. Quanto as análises das associações das frações F1" ou F2" (25 e 50∝g/mL) com a tetraciclina e oxacilina, mostraram ações aditiva e sinérgica para a F2" (50∝g/mL), embora não suficiente para que a CMI atingisse valores inferiores a 2 e 4∝g/mL, necessário para serem classificadas como cepas sensíveis a oxacilina e tetraciclina, respectivamente. "Assim, conclui-se que a F2" das folhas de S. brasiliensis apresentou potencial antimicrobiano frente às cepas de S. aureus MRSA multirresistentes e que as associações das frações com os antibióticos testados não apresentaram benefícios não justificando o uso concomitante.

The aim of this study was to evaluate the antimicrobial and synergic activity of 4 leaf fractions of Schinopsis brasiliensis Engl (F1', F2', F1" and F2") against multidrug-resistant Staphylococcus aureus strains. The used methods were agar well diffusion, minimum inhibitory concentration (MIC) - agar dilution, and bioautography. The bioautographic results showed three inhibition zones that corresponded to at least four active compounds, two of which (methyl gallate and gallic acid) have already been isolated from leaves. The F2" (200∝g/mL) fraction showed inhibition zones of 16 mm and 19 mm against S. aureus multidrug-resistant and Klebsiella pneumoniae strains and a MIC value of 100∝g/mL, respectively. The analyses of associations of fraction F1" or F2" (25 and 50∝g/mL) with tetracycline and oxacillin showed additive and synergistic action for F2" (50∝g/mL), although it was not enough to decrease the MIC values to less than 2 and 4∝g/mL, necessary to classify the strains as susceptible to oxacillin and tetracycline, respectively. Thus, it was concluded that F2" from the leaves of S. brasiliensis showed antimicrobial potential against multidrug-resistant MRSA strains, and the associations of the fractions with the tested antibiotics showed no benefits, not justifying their concomitant use.