Banca de QUALIFICAÇÃO: ANDRE OLIVEIRA SANTOS
Uma banca de QUALIFICAÇÃO de DOUTORADO foi cadastrada pelo programa.STUDENT : ANDRE OLIVEIRA SANTOS
DATE: 15/03/2024
TIME: 09:00
LOCAL: Será realizada de forma remota. Link: http://meet.google.com/bcx-tikf-ymw
TITLE:
KEY WORDS:
Fungal lectins; Agaricus bisporus; Lactose; Photoelectrochemical biosensors.
PAGES: 107
BIG AREA: Ciências Biológicas
AREA: Bioquímica
SUMMARY:
Diets rich in lactose-based products provide vitamins and minerals important for our health, but people with lactose intolerance face serious adverse effects. Strict quality control of these products and their commercial formulations is important for adherence to lactose-free diets, as well as for the development of more appropriate nutritional strategies. The development of lectin-based biosensors for carbohydrate analysis has been gaining increasing attention among the biotechnological applications of these macromolecules. Basidiomycota macrofungi, especially wild mushrooms, are promising sources of lectins. Among them, the mushroom Agaricus bisporus has a lectin (ABL) with high biotechnological potential, containing specific interaction sites for simple and complex sugars, including lactose, enabling the development of highly sensitive photoelectrochemical biosensors for this carbohydrate. In this work, two devices containing ABL as a biorecognition element for lactose were developed. The high lectin activity found in the extracts of this macrofungus (640 UH mL−1), even at critical pH (4–10) and temperatures (20–100 °C), allowed its direct use as a source of ABL. Theoretical and experimental evidence revealed favorable and biocompatible electrostatic conditions for immobilizing ABL. In the first biosensor, a glass platform coated with fluorine-doped tin oxide, modified with poly(methylene blue) and ABL, was used, resulting in the ABL/PMB/FTO biosensor. PMB increased the photoactivity of the device, allowing the identification of lectin-carbohydrate interactions with even greater sensitivity. The dose-response curves studied by electrochemical impedance spectroscopy (EIS) showed a well-fitted sigmoidal profile by the Hill equation, expanding the working dynamic range (lactose from 15–540 nM; detection limit of 20.2 pM) and avoiding undesirable sample dilution or pre-concentration procedures. Under optimized photoelectrochemical conditions, the ABL/PMB/FTO biosensor showed remarkable signal stability, precision, specificity, and selectivity for lactose analysis in food products. In the second biosensor, a vitreous carbon support modified with poly(betacyclodextrin), nanocellulose, cerium molybdate nanoparticles, and ABL was used, resulting in the GCE/β-CD/CeMo/ABL biosensor. Additionally, principal component analysis (PCA) was adopted for the selection of data from multiple frequencies in the construction of association curves. Computational simulations for the immobilization of ABL from its extract showed that this procedure also contributed to reducing the flat band potential to a magnitude equivalent to that obtained with GCE/β-CD/CeMo irradiation. For PCA analyses, normalized impedance module data enabled better separation between lactose levels and less dispersion between measurements, compared to phase angle data. Association curves for frequencies between 3.1 and 31.6 Hz, fitted by the biphasic association model, showed two ABL-lactose association events. However, with data obtained from the ABL/PMB/FTO biosensor combined with PCA analysis and physicochemical calculations, it was possible to determine with a good level of certainty three association events, with dissociation constants K_(D,1)=0.46 nM, K_(D,2)=5.45 nM, K_(D,3)=127.55 nM. The GCE/β-CD/CeMo biosensor exhibited high sensitivity (detection limit of 0.8 pM) and selectivity for lactose, (no interference from glucose, fructose, inositol, sucrose, and maltose), constituting an important analytical advantage of this device. This research fosters interest in ABL-based biosensors and the added value of Agaricus bisporus crude extract for the development of greener and more sustainable biotechnological approaches.
COMMITTEE MEMBERS:
Externo à Instituição - SUELY SOUZA LEAL DE CASTRO
Externo à Instituição - FRANCISCO WIRLEY PAULINO RIBEIRO
Interno - HEBERTY DI TARSO FERNANDES FACUNDO