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Authors
Abstract(s)
O cancro da próstata (PCa) define-se por ser o tipo de cancro mais
frequentemente diagnosticado em todo o mundo, entre pessoas do sexo masculino. De
acordo com a International Agency for Research on Cancer (IARC), em Portugal a taxa
de incidência desta doença oncológica ronda em torno dos 20%, podendo aumentar esta
percentagem para 22.4% em homens de idade mais avançada. A Six-Transmembrane
Epithelial Antigen of the Prostate 1 (STEAP1) é uma proteína integral com seis domínios
transmembranares, que desempenha funções cruciais na comunicação celular, em
processos de adesão celular, atuando como proteína transportadora e canal iónico, e na
estimulação do crescimento das células, o que poderá contribuir para a progressão e
agressividade de um tumor. Inúmeros estudos demonstraram que a expressão da
STEAP1 é maioritariamente restringida à próstata, encontrando-se em níveis de
expressão mais elevados em tecidos neoplásicos da próstata, comparativamente a tecidos
saudáveis. Assim, a STEAP1 pode ser considerada um biomarcador altamente específico
para o PCa.
Posto isto, o principal objetivo deste trabalho foca-se no desenvolvimento de um
biossensor eletroquímico com base em polímeros de impressão molecular (do inglês,
molecularly-imprinted polymer, MIP) para a deteção e monitorização do PCa, através
da deteção da STEAP1. Para a deteção específica e seletiva da proteína foi utilizado um
material MIP obtido através da eletropolimerização da mistura da STEAP1 com um
polímero (ácido pirrol-2-carboxílico). Após a eletropolimerização da mistura na
superfície do elétrodo de trabalho, a proteína foi removida da matriz polimérica através
de um tratamento enzimático por meio da tripsina, dando origem às cavidades molde
específicas para a deteção posterior da STEAP1.
De forma a controlar as modificações realizadas à superfície do biossensor, foi
utilizada a análise por espectroscopia de Raman e técnicas analíticas, nomeadamente
impedância eletroquímica (do inglês, electrochemical impedance spectroscopy EIS) e
voltametria cíclica (do inglês, cyclic voltammetry CV). Adicionalmente, recorreu-se à
microscopia eletrónica de superfície (do inglês, surface electron microscopy SEM) para
a análise morfológica da superfície do biossensor ao longo das diferentes modificações.
A análise do desempenho analítico do biossensor foi realizada recorrendo às
técnicas de EIS e de voltametria de onda quadrada (do inglês, square wave voltammetry
SWV) através da avaliação da resposta obtida com soluções padrão entre 0.00013 µg/mL e 13 µg/mL de STEAP1 preparadas em PBS e num meio mais complexo, o pellet de
extratos celulares LNCaP.
De forma geral, o biossensor apresentou um bom desempenho analítico,
demonstrando boa sensibilidade e linearidade de resposta, em ambos os meios. A
avaliação da seletividade do biossensor foi realizada recorrendo a três possíveis espécies
interferentes, através da incubação destes em mistura com uma dada concentração de
STEAP1, sendo posteriormente avaliada a variação da resposta.
Face ao referido, o biossensor desenvolvido neste trabalho apresentou
características essenciais como seletividade, sensibilidade e reprodutibilidade para a sua
aplicação em pesquisa médica e clínica na deteção da STEAP1 para um diagnóstico e
prognóstico não evasivo do cancro da próstata.
Prostate cancer (PCa) is defined as the most frequently diagnosed cancer worldwide among males. In Portugal, the incidence rate of this disease is around 20%, and this percentage may increase to 22.4% in older men. Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral protein with six transmembrane domains, which plays crucial roles in cell communication, cell adhesion processes, acting as a carrier protein and ion channel, and in stimulating cell growth, which may contribute to tumor progression and aggressiveness. Numerous studies have shown that STEAP1 expression is mostly restricted to the prostate, being found at higher expression levels in prostate neoplastic tissues compared to healthy tissues. Thus, STEAP1 can be considered a highly specific biomarker for PCa. Therefore, the main objective of this work is focused on the development of an electrochemical biosensor based on molecularly imprinted polymer (MIP) for the detection and monitoring of PCa, through the detection of STEAP1. For the specific and selective detection of the protein was used a MIP material obtained by electropolymerization of the mixture of STEAP1 with a polymer (pyrrol-2-carboxylic acid). After electropolymerization the mixture on the working electrode surface, the protein was removed from the polymeric network by enzymatic treatment with trypsin, giving rise to the specific template cavities for further STEAP1 detection. In order to control the modifications made to the biosensor surface, Raman spectroscopy and analytical techniques, namely electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used. In addition, surface electron microscopy (SEM) was used for the morphological analysis of the biosensor surface throughout the different modifications. The analytical performance of the biosensor was analyzed using EIS and square wave voltammetry (SWV) techniques by evaluating the response obtained with standard solutions between 0.00013 µg/mL and 13 µg/mL of STEAP1 prepared in PBS and in a more complex medium, the pellet of LNCaP cellular extracts. Overall, the biosensor showed good analytical performance, demonstrating good sensitivity and linearity of response, in both media. The evaluation of the biosensor selectivity was performed using three possible interfering species, by incubating them in mixture with a given concentration of STEAP1, and then evaluating the response variation In view of the above, the biosensor developed in this work presented essential features such as selectivity, sensitivity, and reproducibility for its application in medical and clinical research in STEAP1 detection for non-evasive PCa diagnosis and prognosis.
Prostate cancer (PCa) is defined as the most frequently diagnosed cancer worldwide among males. In Portugal, the incidence rate of this disease is around 20%, and this percentage may increase to 22.4% in older men. Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral protein with six transmembrane domains, which plays crucial roles in cell communication, cell adhesion processes, acting as a carrier protein and ion channel, and in stimulating cell growth, which may contribute to tumor progression and aggressiveness. Numerous studies have shown that STEAP1 expression is mostly restricted to the prostate, being found at higher expression levels in prostate neoplastic tissues compared to healthy tissues. Thus, STEAP1 can be considered a highly specific biomarker for PCa. Therefore, the main objective of this work is focused on the development of an electrochemical biosensor based on molecularly imprinted polymer (MIP) for the detection and monitoring of PCa, through the detection of STEAP1. For the specific and selective detection of the protein was used a MIP material obtained by electropolymerization of the mixture of STEAP1 with a polymer (pyrrol-2-carboxylic acid). After electropolymerization the mixture on the working electrode surface, the protein was removed from the polymeric network by enzymatic treatment with trypsin, giving rise to the specific template cavities for further STEAP1 detection. In order to control the modifications made to the biosensor surface, Raman spectroscopy and analytical techniques, namely electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used. In addition, surface electron microscopy (SEM) was used for the morphological analysis of the biosensor surface throughout the different modifications. The analytical performance of the biosensor was analyzed using EIS and square wave voltammetry (SWV) techniques by evaluating the response obtained with standard solutions between 0.00013 µg/mL and 13 µg/mL of STEAP1 prepared in PBS and in a more complex medium, the pellet of LNCaP cellular extracts. Overall, the biosensor showed good analytical performance, demonstrating good sensitivity and linearity of response, in both media. The evaluation of the biosensor selectivity was performed using three possible interfering species, by incubating them in mixture with a given concentration of STEAP1, and then evaluating the response variation In view of the above, the biosensor developed in this work presented essential features such as selectivity, sensitivity, and reproducibility for its application in medical and clinical research in STEAP1 detection for non-evasive PCa diagnosis and prognosis.
Description
Keywords
Biossensor Cancro da Próstata Polímeros de Impressão Molecular Steap1