The potentiality and limitations of electrochemical impedance spectroscopic methods for molecular film applications
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Data
2018-03-27
Autores
Lucas Garrote, Beatriz
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Universidade Estadual Paulista (Unesp)
Resumo
A pesquisa em biomedicina está direcionando seus esforços para conseguir desenvolver dispositivos analíticos rápidos, simples e point-of-care para o diagnóstico de doenças. Os biossensores eletroquímicos label-free baseados na espectroscopia de impedância eletroquímica (EIE) são uma ferramenta interessante para este fim. EIE é uma técnica mais sensível do que outras técnicas eletroquímicas, como as amperométricas, potenciometricas ou voltamétricas. Em um sistema EIE com configuração faradaica, as variações na superfície do eletrodo são monitoradas, geralmente, pela resistência de transferência de carga (Rct) e pode-se correlacionar as variações no valor do Rct com a concentração do analito específico. Assim, EIE é uma técnica muito usada para biossensoriamento. Neste trabalho foi proposto um sistema baseado em EIE para a detecção da proteína interleukina-6 (IL-6). O eletrodo de trabalho foi modificado com uma monocamada auto-organizada (SAM) de alcanotióis. Nela é imobilizado covalentemente o receptor biológico (Ab IL-6). Durante o desenvolvimento do trabalho, foram enfrentados alguns problemas experimentais que comprometeram a resposta analítica do sistema. Para encontrar a causa da falta de resposta, foi analisado cada etapa de construção do biossensor. Após esses estudos foi concluído que as razões da ausência de resposta eram os defeitos ou pinholes presentes na SAM, devido ao fato de que EIE é uma técnica baseada na transferência de carga e a presença de defeitos na monocamada pode facilitar a migração iônica e/ou eletrônica. Para resolver isso, foi proposto um segundo sistema para a detecção da proteína IL-6 baseado na ECE (espectroscopia de capacitância eletroquímica), usando uma monocamada auto-organizada formada por um peptídeo redox. De novo, a sensibilidade do sistema não foi suficiente para a detecção da proteína. De acordo com trabalhos publicados previamente pelo nosso grupo de pesquisa, foi possível estabelecer uma correlação entre a sensibilidade do sistema e o peso molecular do antígeno e do receptor biológico. Assim, foi observado que sistemas similares mostram maior sensibilidade quanto maior é o analito. Da mesma forma, foi observado que sistemas com receptores biológicos pequenos e analitos maiores mostram maior sensibilidade. Essas duas afirmações podem ter afetado a sensibilidade do sistema proposto, pois a proteína IL-6 é uma proteína de 26 kDa e é a menor proteína já testada pelo nosso grupo em um sistema ECE e o receptor biológico foi um anticorpo de 150 kDa, 5,8 vezes maior do que o analito. Paralelamente, foi realizado um estudo eletroquímico preliminar de um sistema com duas sondas redox, em solução e confinada na superfície. O objetivo foi estudar o fenômeno de amplificação de sinal observado quando ambas as sondas redox formam parte do sistema e entender o processo eletroquímico. Foi observada uma transformação de um sistema capacitivo, quando só a sonda redox confinada na superfície está presente para um sistema impedimétrico, quando a sonda redox em solução participa no processo. A presença das duas sondas redox diminuiu a capacitância redox da monocamada eletroativa e a resistência de ressonância e aumentou a energia do sistema e a frequência de relaxação, devido a que foi favorecido a difusão dos elétrons com a solução.
Biomedicine research is directing its effort to achieve fast, simple, point–of–care analytical devices for disease diagnostic. Label-free electrochemical biosensors based on electrochemical impedance spectroscopy (EIS) are an interesting tool for this purpose. For analytical objective, EIS is more sensitive than other electrochemical techniques, such as amperometric, potentiometric or voltammetry. EIS measures the complex resistance of the system. In faradaic EIS system, variations on the electrode surface are monitored by the charge transfer resistance (Rct), so correlations between the variation in Rct value and the concentration of a specific target could be made. Thus, EIS is a widely used technique for biosensing. Here, an EIS based system for the detection of the protein interleukin-6 (IL-6) was proposed. The working electrode was modified with a self-assembled monolayer (SAM) of alkanethiols, in which the biological receptor (Ab IL-6) was covalently immobilized. During the development of the system experimental problems compromised the analytical response. It was analysed every step of the biosensor construction. It was concluded that defects or pinholes on the SAM were the responsible for the absence of response, since EIS is a technique based on charge transfer and the presence of pinholes on the self-assembled monolayer could allow to free ionic and/or electronic migration. Because of this, it was proposed a second system for IL-6 detection based on electrochemical capacitance spectroscopy (ECS) and using a redox peptide SAM. Again, the sensitive of the system was not high enough to the protein detection. According to previous work published by our group, it was possible to correlate the sensitivity of the system with the molecular weight of the biological receptor and the specific target. It was shown that systems with bigger specific target and similar receptors resulted in more sensitive devices. Moreover, it was observed that system with small receptors and bigger targets showed higher sensitivity. IL-6 is a 26 kDa protein and is the smaller protein tested in an ECS based system by our group, and the biological receptor of the system was an antibody (150 kDa), 5.8 times higher than the biological receptor. Thus, the absence of response of the ECS system was attributed to the small molecular weight of the IL-6 protein and the high difference in weight between the biological receptor and the target. At the same time, it was performed a preliminary electrochemical study of a system with a redox probe confined on the electrode surface and a second one in solution. The objective was study the signal amplifier behaviour observed when two redox probes are involved on the system and understand the electrochemical process. It was observed the variation from a capacitive-based system, with the confined redox probe, to an impedance one, when the redox probe in solution was present. The presence of two redox probes decreased the redox capacitance of the electroactive film and the resonance resistance of the electrons between the electrode surface and the film; moreover, increased the energy of the system and the frequency of relaxation, due to the increase of the electron diffusion with the solution.
Biomedicine research is directing its effort to achieve fast, simple, point–of–care analytical devices for disease diagnostic. Label-free electrochemical biosensors based on electrochemical impedance spectroscopy (EIS) are an interesting tool for this purpose. For analytical objective, EIS is more sensitive than other electrochemical techniques, such as amperometric, potentiometric or voltammetry. EIS measures the complex resistance of the system. In faradaic EIS system, variations on the electrode surface are monitored by the charge transfer resistance (Rct), so correlations between the variation in Rct value and the concentration of a specific target could be made. Thus, EIS is a widely used technique for biosensing. Here, an EIS based system for the detection of the protein interleukin-6 (IL-6) was proposed. The working electrode was modified with a self-assembled monolayer (SAM) of alkanethiols, in which the biological receptor (Ab IL-6) was covalently immobilized. During the development of the system experimental problems compromised the analytical response. It was analysed every step of the biosensor construction. It was concluded that defects or pinholes on the SAM were the responsible for the absence of response, since EIS is a technique based on charge transfer and the presence of pinholes on the self-assembled monolayer could allow to free ionic and/or electronic migration. Because of this, it was proposed a second system for IL-6 detection based on electrochemical capacitance spectroscopy (ECS) and using a redox peptide SAM. Again, the sensitive of the system was not high enough to the protein detection. According to previous work published by our group, it was possible to correlate the sensitivity of the system with the molecular weight of the biological receptor and the specific target. It was shown that systems with bigger specific target and similar receptors resulted in more sensitive devices. Moreover, it was observed that system with small receptors and bigger targets showed higher sensitivity. IL-6 is a 26 kDa protein and is the smaller protein tested in an ECS based system by our group, and the biological receptor of the system was an antibody (150 kDa), 5.8 times higher than the biological receptor. Thus, the absence of response of the ECS system was attributed to the small molecular weight of the IL-6 protein and the high difference in weight between the biological receptor and the target. At the same time, it was performed a preliminary electrochemical study of a system with a redox probe confined on the electrode surface and a second one in solution. The objective was study the signal amplifier behaviour observed when two redox probes are involved on the system and understand the electrochemical process. It was observed the variation from a capacitive-based system, with the confined redox probe, to an impedance one, when the redox probe in solution was present. The presence of two redox probes decreased the redox capacitance of the electroactive film and the resonance resistance of the electrons between the electrode surface and the film; moreover, increased the energy of the system and the frequency of relaxation, due to the increase of the electron diffusion with the solution.
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Palavras-chave
Electrochemical impedance spectroscopy, Electrochemical capacitance spectroscopy, Biosensor, Molecular film, Biosensoring interface, Espectroscopia de impedância eletroquímica, Espectroscopia de capacitância eletroquímica, Biossensor, Filmes moleculares, Interface biossensora