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Novo P.,INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology | Moulas G.,INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology | Prazeres D.M.F.,University of Lisbon | Prazeres D.M.F.,IBB Institute for Biotechnology And Bioengineering | And 3 more authors.
Sensors and Actuators, B: Chemical | Year: 2013

Ochratoxin A (OTA) is a mycotoxin produced by naturally occurring fungi in crops such as grapes and cereals. OTA has demonstrated toxicity and carcinogenicity in animals and therefore biosensing systems are required for its detection and quantification in commercialized goods. This work demonstrates an integrated analytical system that conjugates an indirect competitive enzyme-linked immunosorbent assay strategy developed in PDMS microfluidics with integrated microfabricated hydrogenated amorphous silicon photodiodes for chemiluminescence detection. A limit of detection of 0.85 ng/mL was obtained for OTA detection in a PBS solution using a straight-channel configuration. Comparable limits of detection were obtained for beer extracts but for red wine extracts a higher limit of detection of OTA of 28 ng/mL was obtained. A two-channel U-shaped microfluidic device was developed to perform the simultaneous analysis of a reference solution and of an ochratoxin A contaminated solution. This configuration efficiently reduced measurement errors resulting in an improvement of one order of magnitude in the limits of detection of OTA in beer and red wine extracts. The results show that the miniaturized system can be developed into one stage of a future highly sensitive, portable, and fully integrated "toxin-chip" for monitoring food safety applications. © 2012 Elsevier B.V. All rights reserved.


Gualdino A.,INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology | Chu V.,INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology | Conde J.P.,INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology | Conde J.P.,University of Lisbon
Journal of Applied Physics | Year: 2013

Thin-film silicon micro resonators are fabricated by surface micromachining at temperatures that are CMOS and large area substrate-compatible. Disk resonators offer large working surfaces and a large number of vibrational modes. The vibrational modes of micromechanical disk resonators made from hydrogenated amorphous silicon thin films were studied in this work. The dynamic behavior of these structures is shown to be mechanically described to be in the transition between a membrane and a plate due to the influence of residual stresses generated during the film growth and to thermal mismatch with underlying layers. Non-degenerate modes are observed as a consequence of the radial symmetry and their effective stiffness is related to the anchor geometry and the parity of the number of diametric nodal lines. The experimentally measured frequencies were compared with the simulated values from finite element modeling with good agreement. Investigation of the intrinsic quality factors shows that there is a dependence of the energy dissipation per cycle with the mode order that is related to the clamping anchors. Thermal annealing experiments show that enhanced quality factors can be obtained using low temperature annealing for a limited period of time. © 2013 AIP Publishing LLC.


Silva D.F.C.,INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology | Silva D.F.C.,IBB Institute for Biotechnology And Bioengineering | Azevedo A.M.,University of Lisbon | Azevedo A.M.,IBB Institute for Biotechnology And Bioengineering | And 7 more authors.
Journal of Chromatography A | Year: 2012

The use of monoclonal antibodies (mAbs) in medical treatments and in laboratory techniques has a very important impact in the battle against many diseases, namely in the treatment of cancer, autoimmune diseases and neural disorders. Thus these biopharmaceuticals have become increasingly important, reinforcing the demand for efficient, scalable and cost-effective techniques for providing pure antibodies. Aqueous two-phase systems (ATPS) have shown potential for downstream processing of mAbs. In this work, an ATPS in a microfluidic platform was designed and tested for mAbs extraction. The system demonstrated the potential to be an effective tool to accelerate bioprocess design and optimization. The partition of immunoglobulin G (IgG) tagged with fluorescein isothiocyanate (FITC) in an ATPS of polyethylene-glycol (PEG)/phosphate buffer with NaCl was investigated using a PDMS microfluidic device fabricated using soft lithography techniques. Different structures were tested with different values of microchannel length (3.14-16.8. cm) and flow rates of the salt (1-2 μL/min) and PEG-rich phases (0.2-0.5 μL/min). A stable interphase between the phases was obtained and the phenomena of diffusion and of partition of the IgG from the salt-rich phase to the PEG-rich phase were measured by fluorescence microscopy. Process simulation allowed the modeling of the IgG diffusion and partitioning behavior observed in the microstructure. The reduction to the microscale does not greatly affect the antibody extraction yield when compared with macroscale results, but it does reduce the operation time, demonstrating the potentiality of this approach to process optimization. © 2012 Elsevier B.V.


Pereira A.T.,INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology | Pereira A.T.,IBB Institute for Biotechnology And Bioengineering | Novo P.,INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology | Prazeres D.M.F.,IBB Institute for Biotechnology And Bioengineering | And 4 more authors.
Biomicrofluidics | Year: 2011

Miniaturization of immunoassays through microfluidic technology has the potential to decrease the time and the quantity of reactants required for analysis, together with the potential of achieving multiplexing and portability. A lab-on-chip system incorporating a thin-film amorphous silicon (a-Si:H) photodiode microfabricated on a glass substrate with a thin-film amorphous silicon-carbon alloy directly deposited above the photodiode and acting as a fluorescence filter is integrated with a polydimethylsiloxane-based microfluidic network for the direct detection of antibody-antigen molecular recognition reactions using fluorescence. The model immunoassay used consists of primary antibody adsorption to the microchannel walls followed by its recognition by a secondary antibody labeled with a fluorescent quantum-dot tag. The conditions for the flow-through analysis in the microfluidic format were defined and the total assay time was 30 min. Specific molecular recognition was quantitatively detected. The measurements made with the a-Si:H photodiode are consistent with that obtained with a fluorescence microscope and both show a linear dependence on the antibody concentration in the nanomolar-micromolar range. © 2011 American Institute of Physics.


PubMed | Leibniz Institute for Analytical Sciences, INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology, University of Lisbon and IBB Institute for Biotechnology And Bioengineering
Type: | Journal: Journal of chromatography. A | Year: 2014

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.


PubMed | University of Lisbon and INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology
Type: Journal Article | Journal: Journal of biomedical materials research. Part B, Applied biomaterials | Year: 2015

Although the plasma technology has long been applied to treat contact lenses, the effect of this treatment on the performance of drug-loaded contact lenses is still unclear. The objective of this work is to study the effect of nitrogen plasma treatment on two drug-loaded polymeric formulations which previously demonstrated to be suitable for therapeutic contact lenses: a poly-hydroxyethylmethacrylate (pHEMA) based hydrogel loaded with levofloxacin and a silicone-based hydrogel loaded with chlorhexidine. Modifications of the surface and the optical properties, and alterations in the drug release profiles and possible losses of the antimicrobial activities of the drugs induced by the plasma treatment were assessed. The results showed that, depending on the system and on the processing conditions, the plasma treatment may be beneficial for increasing wettability and refractive index, without degrading the lens surface. From the point of view of drug delivery, plasma irradiation at moderate power (200 W) decreased the initial release rate and the amount of released drug, maintaining the drug activity. For lower (100 W) and higher powers (300 W), almost no effect was detected because the treatment was, respectively, too soft and too aggressive for the lens materials.


PubMed | INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology, University of Lisbon and IBB Institute for Biotechnology And Bioengineering
Type: | Journal: Journal of chromatography. A | Year: 2014

Aqueous two phase systems (ATPS) offer great potential for selective separation of a wide range of biomolecules by exploring differences in solubility in each of the two phases. However, their use has been greatly hindered due to poor theoretical understanding of the principles behind ATPS formation and the empirical and time-consuming techniques used for the determination of optimal extraction parameters including the binodal curves. In this work, characteristic ATPS binodal curves were determined by a novel technique in which the formation of an ATPS system is measured in a microfluidic device. Two solutions containing separate ATPS solution precursors were loaded into the side inlets of a three inlet microfluidic channel while milli-Q water was loaded into the middle inlet. By varying the flow rates of the three solutions, a wide range of concentrations inside the microchannel could be rapidly tested using limited volumes. Using optical microscopy, depending on the concentrations inside the microchannel, three different states could be observed at the end of the microchannel (i) the presence of an interface; (ii) no presence of an interface; or (iii) the presence of an unstable interface. The binodal curve was calculated using the points corresponding to unstable interfaces and compared to binodal curves obtained through the standard turbidometric titration method for both PEG/salt and PEG/dextran systems.


PubMed | INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology
Type: Journal Article | Journal: Lab on a chip | Year: 2014

Parkinsons disease (PD) is a common age-associated neurodegenerative disorder. The protein -synuclein (aSyn) is a key factor in PD both due to its association with familial and sporadic cases and because it is the main component of the pathological protein aggregates known as Lewy bodies. However, the precise cellular effects of aSyn aggregation are still elusive. Here, we developed an elastomeric microfluidic device equipped with a chemical gradient generator and 9 chambers containing cell traps to study aSyn production and aggregation in Saccharomyces cerevisiae. This study involved capturing single cells, exposing them to specific chemical environments and imaging the expression of aSyn by means of a GFP fusion (aSyn-GFP). Using a galactose (GAL) gradient we modulated aSyn expression and, surprisingly, by tracking the behavior of single cells, we found that the response of individual cells in a population to a given stimulus can differ widely. To study the combined effect of environmental factors and aSyn expression levels, we exposed cells to a gradient of FeCl3. We found a dramatic increase in the percentage of cells displaying aSyn inclusions from 27% to 96%. Finally, we studied the effects of ascorbic acid, an antioxidant, on aSyn aggregation and found a significant reduction in the percentage of cells bearing aSyn inclusions from 87% to 37%. In summary, the device developed here offers a powerful way of studying aSyn biology with single-cell resolution and high throughput using genetically modified yeast cells.


PubMed | INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology
Type: Journal Article | Journal: Biosensors & bioelectronics | Year: 2012

Two-dimensional fluorescence spectroscopy (2D FS) provides a non-invasive means to assess cell condition without the introduction of changes to the cell environment. The method relies on the measurement of the excitation-emission fluorescence intensity matrix of key intrinsic fluorophores, like aromatic amino acids, enzyme cofactors, and vitamins. Commonly used detection systems are complex, with multiple bandpass filters, and are hard to miniaturize. Here, an amorphous silicon photodetector array system integrated with amorphous silicon-carbon alloy filters designed to detect three key fluorophores - tryptophan (Trp), reduced nicotine adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) - is demonstrated. These intrinsic fluorophores were detected in pure solutions and also in suspended yeast cells. The array system was used to monitor changes in intrinsic fluorophore concentration when a yeast cell solution was subject to a thermal shock stress.


PubMed | INESC Microsistemas e Nanotecnologias and IN Institute of Nanoscience and Nanotechnology
Type: Journal Article | Journal: Lab on a chip | Year: 2013

Microfluidics and miniaturization of biosensors are fundamental for the development of point-of-care (PoC) diagnostic and analytical tools with the potential of decreasing reagent consumption and time of analysis while increasing portability. However, interfacing microfluidics with fluid control systems is still a limiting factor in practical implementation. We demonstrate an innovative capillary microfluidic design that allows sequential insertion of controlled volumes of liquids into a microfluidic channel with general applicability. The system requires only the placing of liquids at the corresponding inlets. Subsequently, the different solutions flow inside the microfluidic device sequentially and autonomously without the use of valves using integrated capillary pumps. The capillary microfluidic system is demonstrated with a model immunoassay.

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