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Desenvolvimento de um biossensor piezoelétrico para determinação de ácido úrico em amostras biológicas: prevenção e controlo da gota úrica
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O presente trabalho versa o desenvolvimento de um biossensor piezoelétrico para a determinação do ácido úrico em amostras de urina na perspetiva do controlo e prevenção de gota úrica. A motivação principal deste trabalho prende-se com o desenvolvimento de uma metodologia alternativa aos procedimentos de diagnósticos estabelecidos, em particular um imunossensor piezoelétrico, para determinação precoce de ácido úrico na urina, caracterizada, essencialmente, por um procedimento simplificado, uma resposta rápida, pela possível reutilização e por um baixo custo associado. O elemento de reconhecimento biológico ou biorecetor do biossensor desenvolvido, consistiu em anticorpos policlonais contra o ácido úrico. Este elemento de reconhecimento foi imobilizado na superfície do transdutor selecionado para o biossensor desenvolvido que consistiu num cristal de quartzo, piezoelétrico, com 14 mm de diâmetro, recoberto com um elétrodo de ouro em ambas as superfícies e exibindo uma frequência de ressonância nativa de 10 MHz. Como complemento do sistema de transdução, foi utilizada uma microbalança de cristal de quartzo, a qual permitiu a medição da variação da frequência de vibração do cristal piezoelétrico decorrente da ligação da espécie de interesse ao biorecetor. O procedimento de imobilização do biorecetor incluiu a preparação inicial de uma monocamada automontada de ácido 11-mercaptoundecanóico na superfície do sensor, ativada com uma mistura de N-Hidroxissuccinimida e 1-etil-3-(3-dimetilaminopropil) carbodiimida. As condições experimentais inerentes à formação da biocamada foram otimizadas com base numa análise de variância (ANOVA) de acordo com um planeamento fatorial do tipo 23. Neste sentido, foram estudados 3 fatores, designadamente a concentração de ácido 11-mercaptoundecanóico, a relação entre o 1-etil-3-(3-dimetilaminopropil) carbodiimida e o N-Hidroxissuccinimida e a diluição da solução de anticorpo comercial e 2 níveis para cada fator. Devido a limitações do planeamento, foram realizados alguns ensaios adicionais visando a otimização da resposta analítica biossensor e que versaram, nomeadamente, o efeito do tempo de ligação dos anticorpos policlonais à monocamada automontada e do tempo de incubação do antigénio ao biorecetor. Em termos da sua performance analítica, o biossensor desenvolvido exibiu um intervalo de resposta linear entre 10 mM e 500 mM de ácido úrico, uma sensibilidade de 0,578 Hz g-1 mL, um limite de deteção de 5,0 g/mL, um limite de quantificação de 18,0 g/mL e um tempo de resposta de ~20min. O biossensor desenvolvido foi utilizado na determinação da concentração de ácido úrico em amostras de urina fortificadas com uma solução de antigénio comercial. Obteve-se um erro relativo inferior a 10% para todas as concentrações de controlo, nomeadamente 20 g/mL, 75 g/mL e 150 g/mL de ácido úrico, o que traduz uma exatidão bastante significativa das medidas efetuadas.
Abstract The present work deals with the development of a piezoelectric biosensor for the determination of uric acid in urine samples in the perspective of control and prevention of gout. The main motivation of this work was the development of an alternative methodology to the established diagnostic procedures, in particular a piezoelectric immunosensor, for the early determination of uric acid in urine, characterized essentially by a simplified procedure, a fast response, the possible reuse and a low associated cost. The biological recognition element or bioreceptor of the developed biosensor, consisted of polyclonal antibodies against uric acid. This recognition element was immobilized on the surface of the transducer selected for the developed biosensor which consisted of a quartz crystal, piezoelectric, 14 mm in diameter, covered with a gold electrode on both surfaces and exhibiting a native resonance frequency of 10 MHz. As a complement of the transduction system, a quartz crystal microbalance was used, which allowed the measurement of the variation of the vibration frequency of the piezoelectric crystal resulting from the binding of the species of interest to the bioreceptor. The immobilization procedure of the biorecetor included the initial preparation of a self-assembled monolayer of acid 11-mercaptoundecanoic acid on the sensor surface, activated with a mixture of N-Hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The experimental conditions inherent to the formation of the biolayer were optimized based on an analysis of variance (ANOVA) according to a 23 factorial planning. In this sense, 3 factors were studied, namely the concentration of 11-mercaptoundecanoic acid, the ratio between 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide and the dilution of the commercial antibody solution and 2 levels for each factor. Due to planning limitations, some additional assays were performed aiming to optimise the biosensor analytical response, namely the effect of the binding time of the polyclonal antibodies to the self-assembled monolayer and the incubation time of the antigen to the biosensor. In terms of its analytical performance, the developed biosensor exhibited a linear response range between 10 mM and 500 mM uric acid, a sensitivity of 0,578 Hz g-1 mL, a limit of detection of 5,0 g/mL, a limit of quantification of 18,0 g/mL and a response time of ~20min. The developed biosensor was used in the determination of uric acid concentration in urine samples fortified with a commercial antigen solution. A relative error lower than 10% was obtained for all control concentrations, namely 20 g/mL, 75 g/mL and 150 g/mL of uric acid, which translates into a very significant accuracy of the measurements performed.
Abstract The present work deals with the development of a piezoelectric biosensor for the determination of uric acid in urine samples in the perspective of control and prevention of gout. The main motivation of this work was the development of an alternative methodology to the established diagnostic procedures, in particular a piezoelectric immunosensor, for the early determination of uric acid in urine, characterized essentially by a simplified procedure, a fast response, the possible reuse and a low associated cost. The biological recognition element or bioreceptor of the developed biosensor, consisted of polyclonal antibodies against uric acid. This recognition element was immobilized on the surface of the transducer selected for the developed biosensor which consisted of a quartz crystal, piezoelectric, 14 mm in diameter, covered with a gold electrode on both surfaces and exhibiting a native resonance frequency of 10 MHz. As a complement of the transduction system, a quartz crystal microbalance was used, which allowed the measurement of the variation of the vibration frequency of the piezoelectric crystal resulting from the binding of the species of interest to the bioreceptor. The immobilization procedure of the biorecetor included the initial preparation of a self-assembled monolayer of acid 11-mercaptoundecanoic acid on the sensor surface, activated with a mixture of N-Hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The experimental conditions inherent to the formation of the biolayer were optimized based on an analysis of variance (ANOVA) according to a 23 factorial planning. In this sense, 3 factors were studied, namely the concentration of 11-mercaptoundecanoic acid, the ratio between 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide and the dilution of the commercial antibody solution and 2 levels for each factor. Due to planning limitations, some additional assays were performed aiming to optimise the biosensor analytical response, namely the effect of the binding time of the polyclonal antibodies to the self-assembled monolayer and the incubation time of the antigen to the biosensor. In terms of its analytical performance, the developed biosensor exhibited a linear response range between 10 mM and 500 mM uric acid, a sensitivity of 0,578 Hz g-1 mL, a limit of detection of 5,0 g/mL, a limit of quantification of 18,0 g/mL and a response time of ~20min. The developed biosensor was used in the determination of uric acid concentration in urine samples fortified with a commercial antigen solution. A relative error lower than 10% was obtained for all control concentrations, namely 20 g/mL, 75 g/mL and 150 g/mL of uric acid, which translates into a very significant accuracy of the measurements performed.
Descrição
Palavras-chave
Imunossensor Transdução piezoelétrica Ácido úrico Urina Immunosensor Piezoelectric transduction Uric acid Urine