Publicação
Development of a novel aptamer-based multi-sensor device for the detection of osteopontin
| Resumo: | Breast cancer is the most prevalent cancer in women worldwide and its mortality is closely associated with the development of metastasis of the primary tumor. It is currently the fifth cause of death from cancer (522,000 deaths), with 1.67 million new breast cancer cases estimated in 2012. An early diagnosis is crucial to improve patients survival and disease prognostic. Hence, sensitive and specific methods ought to be developed and improved towards this purpose. The use of aptamers as bioreceptors in the design of electrochemical aptasensors has offered new prospects in diagnostic assays in the areas of protein disease biomarkers detection. In this thesis, the possibility of using an electrochemical aptamer-based biosensor for the detection of osteopontin (OPN) has been studied. Aptamers are artificial oligonucleotides (DNA or RNA) that bind their targets with high affinity and specificity. OPN is a glycoprotein present in many tissues and body fluids that is considered a potential biomarker for breast cancer. Initially, an RNA aptamer against OPN, previously reported, was used as the bioreceptor element in the design of an aptasensor. The OPN-binding RNA aptamer was immobilized onto a screen printed gold electrode through a biotin-streptavidin interaction. The electrochemical RNA aptasensor showed a good sensitivity to human OPN and for thrombin (a protein commonly found in human serum). Thus, other specific bioreceptors that could enable the selective detection of human OPN were selected. A DNA aptamer was isolated through the SELEX methodology. This aptamer was characterized by fluorescence assays and presented a good affinity to OPN, thus it was used as the bioreceptor in the design of a new electrochemical DNA aptasensor. This aptasensor exhibited a better performance regarding the sensitivity and selectivity for the detection of human OPN as compared to the above mentioned RNA aptasensor that used the same electrode surface, immobilization method and voltammetry techniques. Finally, these two aptamers (DNA and RNA) were used to develop a labelfree aptasensor array that could simultaneously detect OPN using a dual-screen printed gold electrode. In conclusion, the electrochemical aptasensors herein developed can be an alternative to the standard methods currently used to detect/quantify protein disease biomarkers and could be a useful tool in the diagnosis of breast cancer disease. |
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| Autores principais: | Meirinho, Sofia G. |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2016 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | português |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Breast cancer is the most prevalent cancer in women worldwide and its mortality is closely associated with the development of metastasis of the primary tumor. It is currently the fifth cause of death from cancer (522,000 deaths), with 1.67 million new breast cancer cases estimated in 2012. An early diagnosis is crucial to improve patients survival and disease prognostic. Hence, sensitive and specific methods ought to be developed and improved towards this purpose. The use of aptamers as bioreceptors in the design of electrochemical aptasensors has offered new prospects in diagnostic assays in the areas of protein disease biomarkers detection. In this thesis, the possibility of using an electrochemical aptamer-based biosensor for the detection of osteopontin (OPN) has been studied. Aptamers are artificial oligonucleotides (DNA or RNA) that bind their targets with high affinity and specificity. OPN is a glycoprotein present in many tissues and body fluids that is considered a potential biomarker for breast cancer. Initially, an RNA aptamer against OPN, previously reported, was used as the bioreceptor element in the design of an aptasensor. The OPN-binding RNA aptamer was immobilized onto a screen printed gold electrode through a biotin-streptavidin interaction. The electrochemical RNA aptasensor showed a good sensitivity to human OPN and for thrombin (a protein commonly found in human serum). Thus, other specific bioreceptors that could enable the selective detection of human OPN were selected. A DNA aptamer was isolated through the SELEX methodology. This aptamer was characterized by fluorescence assays and presented a good affinity to OPN, thus it was used as the bioreceptor in the design of a new electrochemical DNA aptasensor. This aptasensor exhibited a better performance regarding the sensitivity and selectivity for the detection of human OPN as compared to the above mentioned RNA aptasensor that used the same electrode surface, immobilization method and voltammetry techniques. Finally, these two aptamers (DNA and RNA) were used to develop a labelfree aptasensor array that could simultaneously detect OPN using a dual-screen printed gold electrode. In conclusion, the electrochemical aptasensors herein developed can be an alternative to the standard methods currently used to detect/quantify protein disease biomarkers and could be a useful tool in the diagnosis of breast cancer disease. |
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