Development of an electrochemical genosensor for the detection of BDNF Val66Met polymorphism

• Autor(a) principal: Carvalho, Renato Manuel de Caldevilla
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.22/24806
• topic: Depression; Chronoamperometry; Electrochemical genosensor; Val66Met polymorphism

Major depressive disorder (MDD) is one of the most prevalente psychiatric conditions, with a problematic etiopethogenesis. Antidepressant drugs remain the gold standard for the treatment of depressed patients. Yet, their efficacy in clinical treatments remain a cause of concern for the medical community, as 30 to 40% of patients do not respond sufficiently to these drugs. Gentic variety is a crucial factor involved in MDD, influencing the remission rates of patients undergoing antidepressant treatment, and literature has found the BDNF gene to be na essential player on this situation. This gene codes for a neurotrophin of the same name, wich has been shown to be essential in neuroplasticity and neurogenesis. Moreover, the rs6265 (Val66Met) genetic polymorphism has been widely studied as a cause of variation in patient’s antodepressant response rates. Personalizing antidepressant tratments is becoming increasingly more required. However, conventional genotyping methodologies have some intrinsic drawbacks, such as high equipment costs and lengthy experimente periods. Consequently, there is na increasing demand for quicker, less expensive approaches to genotype these individuals. Genosensors meet these requirements and can be advantageous in the detection of polymorphisms. In this thesis, a novel low-cost electrochemical genosensensing platform, capable of detecting the Val66Met polymorphism, was developed and optimized. The working principle of the genosensor is its capability to recognize and detect the DNA hybridization reactions between two complementary DNA sequences. Two specific target DNA sequences of interest from the Val66Met polymorphism (one for the Val variant and another for the Met variant) were designed and selected in-silico. The construction of the genosensor involved 4 main steps: pre-treatment; sensing phase; sandwich DNA hybridization assay and electrochemical detection. After the optimization of a variety of experimental conditions, namely DNA capture probe concentration, MCH concentration and incubation time, homogeneous and heterogeneous hybridization steps and anti-FITC-POD antibody concentration and incubation time, calibration curves were plotted, revealing a linear correlation between the electrochemical current and DNA target concentration from 0.1 to 2.0 nM. The genosensor also showed good ability to discriminate between both target sequences, with a limit of quantifiation of 0.057 nM. The developed genosensor demonstrated to be a promising and effective tool for detection od Val66Met genetic polymorphism.