Biossensor para identificação da doença de alzheimer
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Data
2021-10-07
Autores
Bovolato, Ana Livia de Carvalho
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A doença de Alzheimer (DA) é a uma doença neurodegenerativa complexa, responsável pela maioria dos casos de comprometimento cognitivo progressivo em pacientes idosos. O processo degenerativo pode iniciar 20 anos antes da manifestação clínica da doença. O diagnóstico precoce e preciso da DA continua laborioso e quase sempre interfaceado com o diagnóstico de exclusão. Neste projeto é proposto o desenvolvimento de um biossensor para a detecção da doença de Alzheimer baseado na detecção de autoanticorpos anti-ATP-sintase subunidade Beta e anti-FRMD8 presentes em amostra periféricas, soro, e em LCR (líquido cefalorraquidiano) de pacientes com a doença de Alzheimer. Para tal foi construído dois sistemas baseados em imunoensaios: 1) imobilizou-se ATP-Sintase subunidade Beta em lâmina de ouro modificadas com 11-MUA e Quitosana e 2) imobilizou-se o peptídeo FRMD8 associado ao lipossomo de DPPG em superfície de ouro modificada com 11-MUA e PEI. Em ambos os casos se quantificou os respectivos anticorpos comerciais diluídos nas proporções não diluído, 0,01mg/mL (puro), 1:10 (0,001mg/mL), 1:100 (0,0001mg/mL), 1:1000 (10ng/mL) e 1:10000 (1ng/mL), com o intuito de gerar uma curva de calibração. Para realizar as medidas foi feita a técnica de SERS (Espectroscopia Raman Aprimorada por Superfície) usando sondas SERS de nanopartículas de ouro modificadas com Anti-IgG porção Fc. Como análise comparativa, ambos os resultados foram comparados com amostras de indivíduos não portadores de DA (como controle Alzheimer, CA) e com anti-HIV p17 como controle experimental (CE). Os sensores foram hábeis de gerar curvas de calibração na qual o biossensor baseado em FRMD8 foi capaz de detectar até 1ng/mL e o biossensor baseado em ATP-Sintase foi capaz de detectar até 10ng/mL. Na validação, apenas o biossensor baseado em ATP Sintase se demostrou eficaz na semi-quatificação e diferenciação entre controles e amostras e através de análise de PCA foi estabelecer e criar um Score SERS e correlacionar com o Score MEEM. Um ponto extremamente importante a se destacar nesse trabalho é que não há biomarcador “perfeito” para a DA devido ao seu múltiplo interfaceamento, sendo, a melhor alternativa, uma análise de múltiplos marcadores.
Alzheimer's disease (AD) is a complex neurodegenerative disease, responsible for most cases of progressive cognitive impairment in elderly patients. The degenerative process can start 20 years before the clinical manifestation of the disease. The early and accurate diagnosis of AD remains laborious and is almost always intertwined with diagnoses of exclusion. This project proposes the development of a biosensor for the detection of Alzheimer's disease based on the detection of antiATP-synthase Beta and anti-FRMD8 autoantibodies present in peripheral samples, serum, and in CSF (cerebrospinal fluid) from patients with Alzheimer's disease. For this purpose, two systems based on immunoassays were constructed: 1) ATP-Synthase Beta subunit was immobilized on gold plate modified with 11-MUA and Chitosan and 2) FRMD8 peptide associated with DPPG liposome was immobilized on a modified gold surface with 11-MUA and PEI. In both cases, the respective commercial antibodies were quantified in the proportions undiluted, 0.01mg/mL (pure), 1:10 (0.001mg/mL), 1:100 (0.0001mg/mL), 1:1000 (1ng/mL) and 1:10000 (1ng/mL), to generate a calibration curve. The SERS (Surface Enhanced Raman Spectroscopy) technique was performed using SERS probes of gold nanoparticles modified with Anti-IgG Fc portion. As a comparative analysis, both results were compared with samples from individuals without AD (as control Alzheimer, CA) and with anti-HIV p17 as experimental control (EC). The sensors were able to generate calibration curves in which the FRMD8-based biosensor was able to detect up to 1ng/mL and the ATP-Synthase-based biosensor was able to detect up to 10ng/mL. In the validation test, only the ATP Synthase-based biosensor proved to be effective in semi-qualification and differentiation between controls and samples, and, through PCA analysis, it was established and created SERS Score and correlated with the MEEM Score. An extremely important point to be highlighted in this work is that there is no “perfect” biomarker for AD due to its multiple interfacing, the best alternative being an analysis of multiple markers.
Alzheimer's disease (AD) is a complex neurodegenerative disease, responsible for most cases of progressive cognitive impairment in elderly patients. The degenerative process can start 20 years before the clinical manifestation of the disease. The early and accurate diagnosis of AD remains laborious and is almost always intertwined with diagnoses of exclusion. This project proposes the development of a biosensor for the detection of Alzheimer's disease based on the detection of antiATP-synthase Beta and anti-FRMD8 autoantibodies present in peripheral samples, serum, and in CSF (cerebrospinal fluid) from patients with Alzheimer's disease. For this purpose, two systems based on immunoassays were constructed: 1) ATP-Synthase Beta subunit was immobilized on gold plate modified with 11-MUA and Chitosan and 2) FRMD8 peptide associated with DPPG liposome was immobilized on a modified gold surface with 11-MUA and PEI. In both cases, the respective commercial antibodies were quantified in the proportions undiluted, 0.01mg/mL (pure), 1:10 (0.001mg/mL), 1:100 (0.0001mg/mL), 1:1000 (1ng/mL) and 1:10000 (1ng/mL), to generate a calibration curve. The SERS (Surface Enhanced Raman Spectroscopy) technique was performed using SERS probes of gold nanoparticles modified with Anti-IgG Fc portion. As a comparative analysis, both results were compared with samples from individuals without AD (as control Alzheimer, CA) and with anti-HIV p17 as experimental control (EC). The sensors were able to generate calibration curves in which the FRMD8-based biosensor was able to detect up to 1ng/mL and the ATP-Synthase-based biosensor was able to detect up to 10ng/mL. In the validation test, only the ATP Synthase-based biosensor proved to be effective in semi-qualification and differentiation between controls and samples, and, through PCA analysis, it was established and created SERS Score and correlated with the MEEM Score. An extremely important point to be highlighted in this work is that there is no “perfect” biomarker for AD due to its multiple interfacing, the best alternative being an analysis of multiple markers.
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Palavras-chave
Biomarcadores, Biossensores, Doença de Alzheimer, Analise espectral, SERS, Alzheimer's disease, Biosensors, Biomarkers