Desenvolvimento e aperfeiçoamento de protocolos de conjugação de nanomarcadores luminescentes com sistemas biológicos para aplicação em imunoensaios
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Data
2011-11-25
Autores
Gelamos, João Paulo [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Neste trabalho foram desenvolvidos e aprimorados protocolos de conjugação entre nanopartículas do luminóforo Y2O3:Er,Yb, aminofuncionalizadas e a proteína Streptavidina, para atuarem como marcador em imunoensaios. A streptavidina faz parte do sistema de auto-reconhecimento biotina-avidina mais aplicado em ensaios biológicos, sendo que, devido ao seu alto custo, a Albumina Sérica Bovina foi escolhida como proteína de trabalho em substituição à Avidina. Na etapa final, no entanto, utilizou-se a Streptavidina para comparativamente finalizar as discussões. Desta forma, o desenvolvimento do protocolo considerado padrão foi feito aplicando-se o crosslinker homobifuncional glutaraldeído, e a partir deste, estudou-se o comportamento da ligação luminóforo-proteína utilizando-se outros crosslinkers heterobifuncionais, no caso sulfo-N-succinimidil 4-maleimido- butirato sal de sódio (Sulfo-GMBS) e cloridrato de N-(3-dimetilaminopropil)-N'-etilcarbodiimida (EDC). Tal alteração teve como princípio o aperfeiçoamento do protocolo de conjugação, estabelecendo comparativos entres os dados experimentais já obtidos e as possíveis vantagens resultantes dos crosslinkers heterobifuncionais, já que neste caso a possibilidade de ocorrer autoconjugação, ligação cruzada intramolecular e/ou polimerização entre as nanopartículas aminofuncionalizadas pode ser nula. Com relação às nanopartículas, estas, antes da conjugação, foram caracterizadas por espectroscopia de luminescência, microscopia eletrônica de varredura e/ou transmissão, assim como titulação potenciométrica para quantificação dos grupos NH2 após funcionalização. No desenvolvimento e aperfeiçoamento dos protocolos de conjugação todas as etapas foram monitoradas por Espectroscopia de Absorção na Região...
In this work conjugation protocols between the aminofunctionalized nanophosphor Y2O3: Er, Yb, and the protein Streptavidin was developed and improved to be possible applied as markers in immunoassays. The Streptavidin is part of the biotin-avidin self-recognition system applied to most biological assays, and, due to its high cost, Bovine Serum Albumin protein was chosen to be used in most part of the developing protocol as a substitute for avidin. In the final step, however, the Streptavidin was used in comparison to the other results in order to finalize the study. Thus, the development of the standard protocol was done by applying the homobifunctional crosslinker glutaraldehyde, and from this, it was studied the behavior of protein-nanophosphors binding using the heterobifunctional crosslinkers N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and Sulfo-N-succinimidyl 4-maleimidobutyrate sodium salt (Sulfo-GMBS). Such a change had the challenge to improve conjugation protocol, establishing the comparison between experimental data already obtained and the possible advantages due to the use of heterobifunctional crosslinkers, considering that in this case the possibility of occurring self-conjugation, intermolecular crosslinking and/or polymerization between the aminofuctionalized nanoparticles can be null. With respect to the nanoparticles before the conjugation step, they were characterized by luminescence spectroscopy, scanning and/or transmission electron microscopy as well as potentiometric titration for NH2 groups quantification after functionalization. During the development and the improvement of the conjugation protocols all steps were monitored by UV-VIS absorption spectroscopy to verify the behavior of molecular species present before and after the reactions... (Complete abstract click electronic access below)
In this work conjugation protocols between the aminofunctionalized nanophosphor Y2O3: Er, Yb, and the protein Streptavidin was developed and improved to be possible applied as markers in immunoassays. The Streptavidin is part of the biotin-avidin self-recognition system applied to most biological assays, and, due to its high cost, Bovine Serum Albumin protein was chosen to be used in most part of the developing protocol as a substitute for avidin. In the final step, however, the Streptavidin was used in comparison to the other results in order to finalize the study. Thus, the development of the standard protocol was done by applying the homobifunctional crosslinker glutaraldehyde, and from this, it was studied the behavior of protein-nanophosphors binding using the heterobifunctional crosslinkers N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and Sulfo-N-succinimidyl 4-maleimidobutyrate sodium salt (Sulfo-GMBS). Such a change had the challenge to improve conjugation protocol, establishing the comparison between experimental data already obtained and the possible advantages due to the use of heterobifunctional crosslinkers, considering that in this case the possibility of occurring self-conjugation, intermolecular crosslinking and/or polymerization between the aminofuctionalized nanoparticles can be null. With respect to the nanoparticles before the conjugation step, they were characterized by luminescence spectroscopy, scanning and/or transmission electron microscopy as well as potentiometric titration for NH2 groups quantification after functionalization. During the development and the improvement of the conjugation protocols all steps were monitored by UV-VIS absorption spectroscopy to verify the behavior of molecular species present before and after the reactions... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Análise espectral, Espectroscopia de luminescencia, Nanotecnologia, Immunoassays, Luminescence spectroscopy
Como citar
GELAMOS, João Paulo. Desenvolvimento e aperfeiçoamento de protocolos de conjugação de nanomarcadores luminescentes com sistemas biológicos para aplicação em imunoensaios. 2011. 91 f. Dissertação (mestrado) - Universidade Estadual Paulista, Instituto de Biociências, Letras e Ciências Exatas, 2011.