Superfícies capacitivas e impedimétricas como plataforma eletroquímica para determinação de biomarcadores clinicamente relevantes
Carregando...
Data
2015-07-31
Autores
Fernandes, Flávio Cesar Bedatty
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Neste trabalho está descrito a aplicação de superfícies moleculares nanoestruturadas com grupos redox confinados e elementos de reconhecimento específico (anticorpos) para detecção de biomarcadores clinicamente importantes como: proteína C-reativa (CRP), fosfatase prostática ácida humana (hPAP) e alfasinucleína. Em princípio, os grupos redox confinados contribuem para um carregamento interfacial substancial dependente do potencial que pode ser monitorado sensivelmente e resolvido na frequência por espectroscopia de impedância derivada em capacitância (ECE). O sinal de carregamento interfacial, aqui nomeado como capacitância redox (𝐶�������𝑟�������), o qual tem a magnitude do sinal relacionado com a densidade de centros redox ocupados e a sua natureza quântica é sensível a modificações na superfície. Assim, o sinal 𝐶�������𝑟������� é capaz de traduzir sensívelmente eventos de ligações e reconhecimentos de alvos específicos sobre a superfície receptiva. Este novo método de transdução foi aplicado aqui usando superfícies de diferentes composições químicas construídos a partir de diferentes tipos de tióis como pentadecanotiol, 16-mercaptohexadecanóico ácido ou PEGlate conjudagados com 11-ferrocenilundecanotiol em diferentes proporções. No primeiro estudo a CRP e a hPAP foram detectadas de uma forma específica, com uma faixa linear clinicamente útil e limites de detecção (L.D.) significantemente melhores do que outros trabalhos eletroquímicos previamente reportados na literatura (200 pmol L-1 e 11 ρmol L-1 para CRP e hPAP, respectivamente). Adicionalmente, a ECE e a espectroscopia de impedância eletroquímica (EIE) foram comparadas analiticamente mostrando resultados equivalentes para detecção de CRP por ambas as técnicas com a vantagem que a ECE não requer a adição de uma probe redox em solução. Também, as formas de imobilização hidrofóbica e covalente da espécie receptiva foram comparadas e a imobilização covalente mostrou resultados mais robustos do que a imobilização hidrofóbica do anticorpo. A última vantagem proposta neste trabalho foi a aplicação das análises de função de imitância (𝐼�������𝑚�������𝐹�������𝑠�������) que é baseada no uso de um portfólio de funções de imitância matematicamente derivadas e componentes relacionados, capazes, a partir do mesmo conjunto de dados, de permitir o aumento da sensibilidade das análises de detecção de biomarcadores e primordialmente encurtar o tempo de ensaios em comparação a analises impedimétricas realizadas usando abordagens faradaicas, não-faradaicas ou capacitiva redox tradicionais. O conceito de 𝐼�������𝑚�������𝐹������� foi primeiramente validado para metodos EIE para detecção de CRP e o desempenho analítico das 𝐼�������𝑚�������𝐹�������𝑠������� foi superior ao parâmetro tradicional 𝑅�������𝑐�������𝑡������� resultando em ensaios aproximadamente 12 vezes mais sensíveis e 10 vezes mais rápidos. O conceito de 𝐼�������𝑚�������𝐹������� foi também combinado com ECE para detecção de alfa-sinucleína e CRP alcançando valores de L.D. de 121,1 ± 2,7 pmol L-1 e 7,4 ± 3,1 pmol L-1, respectivamente. O ponto mais promissor nos resultados foi o curto tempo requerido para acquisição de dados, em que menos de 3 minutos por medidas em triplicatas foram necessários. Estes ensaios são label free, ultrassensíveis, específicos e acompanhados por uma boa faixa linear.
In this work we described the applications of nanostructured molecular surfaces containing redox tagged groups and specific recognition elements (here antibodies) for detection of clinically important biomarkers such as: C-reactive protein (CRP); human prostatic acid phosphatase (hPAP) and alpha-synuclein. In principle, the redox confined groups contributes to a substantial potential-dependent interfacial charging that can be sensitively monitored and frequency-resolved by impedancederived capacitance spectroscopy (ECS). The interfacial charging signal, named herein as redox capacitance (𝐶������𝑟������), which has the magnitude of signal related with the density of the redox centers occupied and its quantum nature is sensitive to surfaces modifications. Thus, the 𝐶������𝑟������ signal is able to sensitively transduce the recognition and binding events of specific targets on the receptive surface. This novel transduction method was exemplified here using surfaces of different chemistries constructed from different kinds of thiols as pentadecanethiol, 16-mercaptohexadecanoic acid or PEGlate conjugated to 11-ferrocenyl–undecanethiol in different proportions. In the first study CRP and hPAP were detected in a specific way, with a clinically useful linear range and detection limits (L.D.) significantly better than others electrochemical works previously reported in the literature (200 pmol L-1 and 11 ρmol L-1 for CRP and hPAP, respectively). Additionally, the ECS and electrochemical spectroscopic impedance (EIS) were analytically compared showing equivalent results for CRP detection by both techniques with advantage that ECS does not requires addition of a redox probe in solution. Also, hydrophobic and covalent immobilizations of the receptive specie were compared herein and the covalent immobilization showed more robust results than hydrophobic antibody immobilization. The last advantage proposed in this work was the application of the immittance function analysis (𝐼������𝑚������𝐹������𝑠������) which is based on the use of a portfolio of mathematically derived immittance functions and related components, capable, from the same raw data sets, of enabling increased assay sensitivity of biomarkers and markedly shorter assay times in comparison to impedance analyses performed using faradaic, non-faradaic or redox impedimetric/capacitive traditional approaches. The concept of 𝐼������𝑚������𝐹������ was firstly validated regarding EIS methods for CRP detection and the analytical performance of 𝐼������𝑚������𝐹������𝑠������ was superior to the traditional parameter 𝑅������𝑐������𝑡������, resulting in assays 12 fold more sensitive and approximately 10 fold faster. The 𝐼������𝑚������𝐹������ concept was also combined to ECS for alpha-synuclein and CRP detection archiving values of L.D. of 121.1 ± 2.7 pmol L-1 and 7.4 ± 3.1 pmol L-1, respectively. The most promising point in this result was the short time required to data acquisition, wherein less than 3 minutes per measurements in triplicate were required. These assays are label free, ultrasensitive, specific and accompanied by a good linear range.
In this work we described the applications of nanostructured molecular surfaces containing redox tagged groups and specific recognition elements (here antibodies) for detection of clinically important biomarkers such as: C-reactive protein (CRP); human prostatic acid phosphatase (hPAP) and alpha-synuclein. In principle, the redox confined groups contributes to a substantial potential-dependent interfacial charging that can be sensitively monitored and frequency-resolved by impedancederived capacitance spectroscopy (ECS). The interfacial charging signal, named herein as redox capacitance (𝐶������𝑟������), which has the magnitude of signal related with the density of the redox centers occupied and its quantum nature is sensitive to surfaces modifications. Thus, the 𝐶������𝑟������ signal is able to sensitively transduce the recognition and binding events of specific targets on the receptive surface. This novel transduction method was exemplified here using surfaces of different chemistries constructed from different kinds of thiols as pentadecanethiol, 16-mercaptohexadecanoic acid or PEGlate conjugated to 11-ferrocenyl–undecanethiol in different proportions. In the first study CRP and hPAP were detected in a specific way, with a clinically useful linear range and detection limits (L.D.) significantly better than others electrochemical works previously reported in the literature (200 pmol L-1 and 11 ρmol L-1 for CRP and hPAP, respectively). Additionally, the ECS and electrochemical spectroscopic impedance (EIS) were analytically compared showing equivalent results for CRP detection by both techniques with advantage that ECS does not requires addition of a redox probe in solution. Also, hydrophobic and covalent immobilizations of the receptive specie were compared herein and the covalent immobilization showed more robust results than hydrophobic antibody immobilization. The last advantage proposed in this work was the application of the immittance function analysis (𝐼������𝑚������𝐹������𝑠������) which is based on the use of a portfolio of mathematically derived immittance functions and related components, capable, from the same raw data sets, of enabling increased assay sensitivity of biomarkers and markedly shorter assay times in comparison to impedance analyses performed using faradaic, non-faradaic or redox impedimetric/capacitive traditional approaches. The concept of 𝐼������𝑚������𝐹������ was firstly validated regarding EIS methods for CRP detection and the analytical performance of 𝐼������𝑚������𝐹������𝑠������ was superior to the traditional parameter 𝑅������𝑐������𝑡������, resulting in assays 12 fold more sensitive and approximately 10 fold faster. The 𝐼������𝑚������𝐹������ concept was also combined to ECS for alpha-synuclein and CRP detection archiving values of L.D. of 121.1 ± 2.7 pmol L-1 and 7.4 ± 3.1 pmol L-1, respectively. The most promising point in this result was the short time required to data acquisition, wherein less than 3 minutes per measurements in triplicate were required. These assays are label free, ultrasensitive, specific and accompanied by a good linear range.
Descrição
Palavras-chave
Biossensores, Análise eletroquímica, Capacitância redox, Funções de imitância, Química de superfície