Browsing by Author "Loureiro, Joana A."
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- Biosensor for direct bioelectrocatalysis detection of nitric oxide using nitric oxide reductase incorporated in carboxylated single-walled carbon nanotubes/lipidic 3 bilayer nanocompositePublication . Gomes, Filipa O.; Maia, Luísa B.; Loureiro, Joana A.; Pereira, Maria Carmo; Delerue-Matos, Cristina; Moura, Isabel; Moura, José J.G.; Morais, SimoneAn enzymatic biosensor based on nitric oxide reductase (NOR; purified from Marinobacter hydrocarbonoclasticus) was developed for nitric oxide (NO) detection. The biosensor was prepared by deposition onto a pyrolytic graphite electrode (PGE) of a nanocomposite constituted by carboxylated single-walled carbon nanotubes (SWCNTs), a lipidic bilayer [1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG)] and NOR. NOR direct electron transfer and NO bioelectrocatalysis were characterized by several electrochemical techniques. The biosensor development was also followed by scanning electron microscopy and Fourier transform infrared spectroscopy. Improved enzyme stability and electron transfer (1.96 × 10-4 cm.s-1 apparent rate constant) was obtained with the optimum SWCNTs/(DOPE:DOTAP:DSPE-PEG)/NOR) ratio of 4/2.5/4 (v/v/v), which biomimicked the NOR environment. The PGE/[SWCNTs/(DOPE:DOTAP:DSPE-PEG)/NOR] biosensor exhibited a low Michaelis-Menten constant (4.3 μM), wide linear range (0.44-9.09 μM), low detection limit (0.13 μM), high repeatability (4.1% RSD), reproducibility (7.0% RSD), and stability (ca. 5 weeks). Selectivity tests towards L-arginine, ascorbic acid, sodium nitrate, sodium nitrite and glucose showed that these compounds did not significantly interfere in NO biosensing (91.0 ± 9.3%-98.4 ± 5.3% recoveries). The proposed biosensor, by incorporating the benefits of biomimetic features of the phospholipid bilayer with SWCNT's inherent properties and NOR bioelectrocatalytic activity and selectivity, is a promising tool for NO.
- Nanostructured label–free electrochemical immunosensor for detection of a Parkinson's disease biomarkerPublication . Carneiro, Pedro; Loureiro, Joana A.; Delerue-Matos, Cristina; Morais, Simone; Pereira, Maria do CarmoAggregation of α-synuclein has been recognized as a critical event in the pathogenesis of Parkinson's disease whose prevalence is increasing with great socio-economic challenges for future generations. Here, we developed a sensitive and specific electrochemical immunosensor for the detection and quantification of this biomarker, based on the voltammetric study of a redox indicator signal, which decreases upon the analyte recognition by the antibody due to the electronic resistance increase. The proposed immunosensor is based on a screen-printed carbon electrode modified in a layer-by-layer approach, which through extensive characterization led to the successful nanostructuration of the transducer, through the drop-cast of 3.0 μL of a 0.1 mg mL−1 single-walled carbon nanotubes suspension followed by electrodeposition of gold nanoparticles in a 3 mM HAuCl4 solution under a −0.2 V potential for 150 s. Monoclonal antibodies were immobilized on the gold nanoparticles surface through chemical modification at an optimal concentration of 200 μg mL−1. Using the proposed immunosensor, α-synuclein was detected in the range of 0.01–10 ng mL−1 with a 4.1 and 12.6 pg mL−1 limits of detection and quantification, respectively. Recovery values of 96.7, 106.2 and 102.9% were attained for the tested concentrations spiked in fetal bovine serum while also presenting excellent specificity and stability throughout one month. The nanostructured immunosensor provided a great interface for electronic transduction and biological recognition events, which enabled fast, sensitive and specific detection of α-synuclein while being based on a simple and inexpensive technology requiring small sample volumes, crucial characteristics for application in point-of-care testing.