Banca de DEFESA: CARLOS HENRIQUE DA SILVA MENDES

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : CARLOS HENRIQUE DA SILVA MENDES
DATE: 22/02/2022
TIME: 09:00
LOCAL: Videoconferência
TITLE:

DEVELOPMENT OF ELECTROANALYTICAL METHODS FOR DETECTION AND QUANTIFICATION OF DNA METHYLATION USING CARBON ELECTRODES

KEY WORDS:

5-methyl-cytosine, 7-methyl-guanine, DNA methylation, biomarkers of human diseases, Glassy carbon electrode, Voltammetry, Impedance. 

PAGES: 100
BIG AREA: Ciências Exatas e da Terra
AREA: Química
SUBÁREA: Química Analítica
SPECIALTY: Eletroanalítica
SUMMARY:

The electro-oxidation of 5-methyl-cytosine (5-mCyt) and 7-methyl-guanine (7-mGua) in aqueous support electrolytes were investigated at carbon electrodes, glassy carbon electrode (GCE) and boron-doped diamond electrode (BDDE), using electrochemical techniques, cyclic voltammetry (CV), square wave voltammetry (SWV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS), as well as compared with the anodic behaviour of other similar bases. The redox properties of methylated bases (5-mCyt and 7-mGua) were also explored to develop electroanalytical methods for detection and quantification of DNA methylation. The electrochemical results indicated that 5-mCyt is more reactive towards Cyt, since its anodic process occurs in the methyl group, at more negative potential values (~ 150 mV), with the removal of one electron and one proton generating intermediate radicals that react with water to form hydroxylated products, such as 5-hydroxy-mCyt, and/or dimerize. Regarding 7-mGua, the results showed that its electro-oxidation mechanism occurs, similarly to Gua, on carbon at the C8 position with the removal of one electron and one proton, forming a highly reactive intermediate radical that can dimerize and/or react with water, producing 8-hydroxy-7-mGua, which can then be electro-oxidized to form an electroactive product in an acidic medium, 8-oxo-7-mGua. Furthermore, the results indicated that, unlike Cyt, the methyl group attached to N7 is not electroactive, but hinders the electro-oxidation of 7-mGua since it occurs at more positive potential values (~ 200 mV) in relation to Gua. The electro-oxidation mechanisms of 5-mCyt and 7-mGua have been proposed. Using DPV, experimental conditions, such as working electrode, electrode size, composition of the supporting electrolyte, pH and influence of possible interferents (Gua, adenine, thymine and cytosine), were investigated for detection and quantification of 5-mCyt and 7-mGua, with low detection limits. Under the best conditions, the proposed DPV method, with a 3.0 mm diameter GCE anodically pretreated, provided a linear analytical curve for 5-mCyt in phosphate buffer solution (pH = 7.0) in the concentration range of 3 at 15 µmol L−1, with a limit of detection (LOD) of 0.11 µmol L−1 and high correlation coefficient (r = 0.997). A DPV method with GCE (1.6 mm) was proposed for the determination of 7-mGua with LOD of 4.2 µmol L-1 and r = 0.989 at pH = 4.5 and with LOD of 3.8 µmol L- 1 and r = 0.995 at pH = 7.0. The detection and quantification of 7-mGua in the presence of interferents, such as Gua, Ade and Thy, was also performed satisfactorily in acetate (pH = 4.5) and phosphate (pH = 7.0) buffer. New simple and sensitive electroanalytical methods, using anodically pretreated GCEs, for the quantification of DNA methylation are then presented and can be applied to real samples of hydrolyzed DNA.

BANKING MEMBERS:
Interno - 2140782 - ALEX SOUZA MORAES
Externo à Instituição - ERIC DE SOUZA GIL - UFG
Interno - 3133784 - MARCILIO MARTINS DE MORAES
Presidente - 2452141 - SEVERINO CARLOS BEZERRA DE OLIVEIRA
Externo à Instituição - VAGNER BEZERRA DOS SANTOS - UFPE