RESUMO
The outbreak of COVID-19, a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is regarded as the most severe of the documented coronavirus pandemics. The measurement and monitoring of SARS-CoV-2 antibody levels by serological tests are relevant for a better epidemiological and clinical understanding of COVID-19. The aim of this work was to design a method called the SARS-CoV-2 antibody detection method (SARS-CoV-2 AbDM) for fluorescence immunodetection of anti-SARS-CoV-2 IgG and IgM on both plate and microfluidic chip. For this purpose, a system with magnetic beads that immobilize the antigen (S protein and RBD) on its surface was used to determine the presence and quantity of antibodies in a sample in a single reaction. The SARS-CoV-2 AbDM led to several advantages in the performance of the tests, such as reduced cost, possibility of performing isolated or multiple samples, potential of multiplex detection, and capacity to detect whole blood samples without losing resolution. In addition, due to the microfluidic chip in conjunction with the motorized actuated platform, the time, sample quantity, and operator intervention during the process were reduced. All these advantages suggest that the SARS-CoV-2 AbDM has the potential to be developed as a PoC that can be used as a tool for seroprevalence monitoring, allowing a better understanding of the epidemiological and clinical characteristics of COVID-19 and contributing to more effective and ethical decision-making in strategies to fight against the COVID-19 pandemic.
RESUMO
Lipase cartridges are currently the mainstay of treatment to improve fat absorption related to pancreatic insufficiency (PI) in patients receiving enteral nutrition feedings. Enzyme immobilization is an essential prerequisite for designing lipase cartridges systems for efficient enzymatic fat hydrolysis. A microfluidic approach has been adopted to produce lipase (LIP) caged in hyperbranched polyglycerol microcapsules (HPGly). The resulting HPGly-LIP microcapsules are spherical and had an average diameter of 29 µm with monomodal size distribution. The optimum conditions determined by artificial neural networks were HPGly concentration of 10 wt.%, LIP loading of 20% (wt) and total flow rate in microfluidic cell of 1.0 mL/h. Under these conditions, the maximum capacity of the LIP that can be microencapsulated is around 85% with respect to the HPGly concentration of 10 wt.% and total flow rate in microfluidic cell of 1.0 mL/h. This resultant HPGly-LIP exhibited Michaelis-Menten coefficients of 1.138,14 mM (Km) and 0.49 U/mg (Vmax) showing higher activity compared to free LIP. Finally, the robust HPGly-LIP microcapsules showed excellent recyclability. The in vitro Analysis of the HPGly-LIP cytotoxicity showed that microcapsules had no cytotoxic effect to L929 fibroblasts cells and behaved very similar to the negative control. These features will be useful for the facile construction of biocatalytic systems with high efficiency, excellent recyclability and adequate biocompatibility for treatment of patients with PI receiving enteral nutrition feedings.
Assuntos
Insuficiência Pancreática Exócrina , Lipase , Cápsulas , Glicerol , Humanos , Microfluídica , PolímerosRESUMO
The purpose of this study was to develop a silica nanoparticle-based immunosensor with laser-induced fluorescence (LIF) as a detection system. The proposed device was applied to quantify the immunoreactive trypsin (IRT) in cystic fibrosis (CF) newborn screening. A new ultrasonic procedure was used to extract the IRT from blood spot samples collected on filter papers. After extraction, the IRT reacted immunologically with anti-IRT monoclonal antibodies immobilized on a microfluidic glass chip modified with 3-aminopropyl functionalized silica nanoparticles (APSN-APTES-modified glass chips). The bounded IRT was quantified by horseradish peroxidase (HRP)-conjugated anti-IRT antibody (anti-IRT-Ab) using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as enzymatic mediator. The HRP catalyzed the oxidation of nonfluorescent ADHP to highly fluorescent resorufin, which was measured by LIF detector, using excitation lambda at 561nm and emission at 585nm. The detection limits (LODs) calculated for LIF detection and for a commercial enzyme-linked immunosorbent assay (ELISA) test kit were 0.87 and 4.2ngml(-1), respectively. The within- and between-assay variation coefficients for the LIF detection procedure were below 6.5%. The blood spot samples collected on filter papers were analyzed with the proposed method, and the results were compared with those of the reference ELISA method, demonstrating a potential usefulness for the clinical assessment of IRT during the early neonatal period.