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Construção de sensores de DNA para a identificação de polimorfismos associados à metabolização de fármacos antiepiléticos
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Authors
Abstract(s)
A epilepsia é uma doença neurológica crónica, caracterizada por descargas neuronais anómalas
que provocam crises recorrentes, afetando cerca de 50 milhões de pessoas em todo o mundo,
frequentemente associadas a comorbilidades psiquiátricas. A eficácia da terapêutica com
fármacos antiepiléticos (FAE) apresenta grande variabilidade entre indivíduos, influenciada por
fatores genéticos, farmacocinéticos e interações medicamentosas.
A farmacogenética possibilita a individualização da terapêutica com base em variantes genéticas
que modulam a resposta aos fármacos. O gene CYP2D6, responsável pela metabolização de
diversos psicofármacos, apresenta o polimorfismo rs1065852, que caracteriza o alelo
CYP2D6*10, associado ao fenótipo de metabolizador intermédio, traduzindo-se numa atividade
enzimática reduzida e num risco aumentado de resposta clínica subótima.
Neste contexto, foi desenvolvido um genossensor eletroquímico descartável para a deteção
rápida e específica do polimorfismo rs1065852. A abordagem consistiu na imobilização de uma
sequência de oligonucleotidos tiolada a, composta por 25 pares de bases (pb), em elétrodos
serigrafados de ouro, através da criação de uma bicamada automontada constituída por DNA
tiolado (grupo -SH) e mercaptohexanol (MCH), seguida de reação de hibridação em formato
sandwich com uma sequência complementar de fita simples de DNA marcada com fluoresceína
e monitorizada por técnicas voltamétricas, nomeadamente a cronoamperometria.
O genossensor desenvolvido demonstrou ter elevada sensibilidade, seletividade e
reprodutibilidade (RSD de 17,65% e 23,79%). A sequência de DNA alvo totalmente
complementar (Target T) gerou um sinal eletroquímico 56,81% superior ao da sequência de DNA
alvo com um polimorfismo (Target C). Estes resultados evidenciam que o genossensor constitui
uma ferramenta analítica rápida, precisa e robusta, com potencial para aplicação na
farmacogenética clínica, permitindo a otimização individualizada da terapêutica da epilepsia.
Epilepsy is a chronic neurological disorder characterized by abnormal neuronal discharges that cause recurrent seizures, affecting approximately 50 million people worldwide and often associated with psychiatric comorbidities. The efficacy of antiepileptic drug (AED) therapy shows considerable interindividual variability, influenced by genetic factors, pharmacokinetics, and drug interactions. Pharmacogenetics enables the personalization of therapy based on genetic variants that modulate drug response. The CYP2D6 gene, responsible for the metabolism of various psychotropic drugs, presents the rs1065852 polymorphism, which defines the CYP2D6 10 allele. This allele is associated with an intermediate metabolizer phenotype, resulting in reduced enzymatic activity and an increased risk of suboptimal clinical response. In this context, a disposable electrochemical genosensor was developed for the rapid and specific detection of the rs1065852 polymorphism. The approach involved immobilizing a 25- base thiolated oligonucleotide sequence on gold screen-printed electrodes via a self-assembled monolayer composed of thiolated DNA (-SH group) and mercaptohexanol (MCH), followed by a sandwich hybridization reaction with a complementary single-stranded DNA sequence labeled with fluorescein and monitored using voltammetric techniques, specifically chronoamperometry. The developed genosensor demonstrated high sensitivity, selectivity, and reproducibility (RSD of 17.65% and 23.79%). The fully complementary DNA target sequence (Target T) produced an electrochemical signal 56.81% higher than that of the DNA target sequence containing the polymorphism (Target C). These results indicate that the genosensor represents a rapid, precise, and robust analytical tool with potential for clinical pharmacogenetics, enabling individualized optimization of epilepsy therapy.
Epilepsy is a chronic neurological disorder characterized by abnormal neuronal discharges that cause recurrent seizures, affecting approximately 50 million people worldwide and often associated with psychiatric comorbidities. The efficacy of antiepileptic drug (AED) therapy shows considerable interindividual variability, influenced by genetic factors, pharmacokinetics, and drug interactions. Pharmacogenetics enables the personalization of therapy based on genetic variants that modulate drug response. The CYP2D6 gene, responsible for the metabolism of various psychotropic drugs, presents the rs1065852 polymorphism, which defines the CYP2D6 10 allele. This allele is associated with an intermediate metabolizer phenotype, resulting in reduced enzymatic activity and an increased risk of suboptimal clinical response. In this context, a disposable electrochemical genosensor was developed for the rapid and specific detection of the rs1065852 polymorphism. The approach involved immobilizing a 25- base thiolated oligonucleotide sequence on gold screen-printed electrodes via a self-assembled monolayer composed of thiolated DNA (-SH group) and mercaptohexanol (MCH), followed by a sandwich hybridization reaction with a complementary single-stranded DNA sequence labeled with fluorescein and monitored using voltammetric techniques, specifically chronoamperometry. The developed genosensor demonstrated high sensitivity, selectivity, and reproducibility (RSD of 17.65% and 23.79%). The fully complementary DNA target sequence (Target T) produced an electrochemical signal 56.81% higher than that of the DNA target sequence containing the polymorphism (Target C). These results indicate that the genosensor represents a rapid, precise, and robust analytical tool with potential for clinical pharmacogenetics, enabling individualized optimization of epilepsy therapy.
Description
Keywords
Epilepsy Pharmacogenetics CYP2D6 rs1065852 Electrochemical Sensor Epilepsia Farmacogenética Sensor eletroquímico