RESUMO
The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has accelerated the development of biosensors based on new materials and techniques. Here, we present our effort to develop a fast and affordable optical biosensor using photoluminescence spectroscopy for anti-SARS-CoV-2 antibody detection. The biosensor was fabricated with a thin layer of the semiconductor polymer Poly[(9,9-di-n-octylfluorenyl-2,7-diyl)-alt-2,2'-bithiophene-5,5'-diyl)] (F8T2) as a signal transducer material. We mounted the biosensors by depositing a layer of F8T2 and an engineered version of RBD from the SARS-CoV-2 spike protein with a tag to promote hydrophobic interaction between the protein and the polymeric surface. We validated the biosensor sensitivity with decreasing anti-RBD polyclonal IgG concentrations and challenged the biosensor specificity with human serum samples from both COVID-19 negative and positive individuals. The antibody binding to the immobilized antigen shifted the F8T2 photoluminescence spectrum even at the low concentration of 0.0125 µg/mL. A volume as small as one drop of serum (100 µL) was sufficient to distinguish a positive from a negative sample without requiring multiple washing steps and secondary antibody reactions.
Assuntos
Técnicas Biossensoriais , COVID-19 , Doenças Transmissíveis , Anticorpos Antivirais , Técnicas Biossensoriais/métodos , COVID-19/diagnóstico , Humanos , Polímeros , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
The potential for thermal denaturation to cause enzyme losses during solid-state fermentation processes for the production of enzymes was examined, using the protease of Penicillium fellutanum as a model system. The frequency factor and activation energies for the first-order denaturation of this enzyme were determined as 3.447 x 10(59) h(-1) and 364,070 Jmol(-1), respectively. These values were incorporated into a mathematical model of enzyme deactivation, which was used to investigate the consequences of subjecting this protease to temporal temperature profiles reported in the literature for mid-height in a 34 cm high packed-bed bioreactor of 150 mm diameter. In this literature source, temperature profiles were measured for 5, 15 and 25 liters per minute of air and enzyme activities were measured as a function of time. The enzyme activity profiles predicted by the model were distributed similarly, one relative to the other, as had been found in the experimental study, with substantial amounts of denaturation being predicted when the substrate temperature exceeded 40 degrees C, which occurred at the lower two airflow rates. A mathematical model of a well-mixed bioreactor was used to explore the difficulties that would be faced at large scale. It suggests that even with airflows as high as one volume per volume per minute, up to 85% of the enzyme produced by the microorganism can be denatured by the end of the fermentation. This work highlights the extra care that must be taken in scaling up solid-state fermentation processes for the production of thermolabile products.