Desenvolvimento de biossensor impedimétrico/capacitivo para detecção de biomarcadores de importância clínica
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Data
2017-03-24
Autores
Santos, Adriano dos [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A técnica de espectroscopia de capacitância eletroquímica foi recentemente utilizada com sucesso para detecção de biomarcadores de interesse clínico, como o caso da proteína C-reativa (CRP), que está relacionada com doenças cardíacas e processos inflamatórios. Nesta tese, esta técnica foi utilizada para desenvolver dispositivos eletroanalíticos com possíveis aplicações para a detecção de trombose, utilizando a proteína biomarcadora D-dímero; câncer de próstata, por meio da detecção da enzima fosfatase ácida prostática (PAP), e glicoproteína HRP, para possível detecção de células tumorais e como modelo para o estudo da interação lectina-glicoproteína. Além, no último caso, utilizaram-se também as funções de imitância como sinal transdutor como possível aplicação em glycoarray. Para a construção da superfície receptora sobre eletrodo de ouro, foram utilizadas duas moléculas diferentes de tiol (R-SH, sendo R uma cadeia carbônica genérica). A primeira, que contém em sua estrutura o grupo terminal carboxílico, foi utilizada com o objetivo de imobilizar o material biológico para reconhecimento do analito (i.e., anticorpos ou lectina) via protocolo EDC/NHS. A segunda, com o grupo redox terminal (11-ferrocenil-undecanotiol, 11Fc), foi utilizada com o intuito de fornecer o sinal transdutor. As monocamadas bifuncionais obtidas pela adsorção conjunta de ambas as moléculas apresentam densidade molecular superficial na ordem de 5 x 10-10 mol/cm2. A imobilização do anticorpo para detecção da PAP (anti-PAP) foi investigada por QCM, sendo possível verificar que a saturação ocorre em aproximadamente 1 h, atingindo densidade molecular superficial de 3,7 mg/m2, sugerindo orientação “end-on”. Utilizando o sinal de capacitância eletroquímica, foi possível desenvolver um biossensor para detecção de PAP com limite de detecção (LD) e quantificação (LQ) de 9 pmol/L e 28 pmol/L, respectivamente, aplicado a uma faixa de concentração clinicamente útil de PAP entre 50-1000 pmol/L em tampão fosfato salino (PBS, pH 7,4). O desvio-padrão relativo obtido foi de 9,5%, e apresentou especificidade quando avaliado frente à fetuína como controle negativo. O mesmo ensaio foi realizado em soro comercial não diluído, apresentando faixa linear de 50-5000 pmol/L, com limites de detecção e quantificação maiores quando comparados com resultados em PBS, de 52 pmol/L e 132 pmol/L, respectivamente. A etapa de imobilização do anticorpo para detecção de D-dímero (anti-DD) também foi avaliada por QCM, obtendo-se densidade molecular superficial de 3,0 mg/m2, sugerindo que o filme pode apresentar orientação dos anticorpos numa composição intermediária entre “end-on” e “side on”. D-dímero foi detectado tanto em solução PBS (pH 7,4) como em soro não diluído, numa faixa de concentração de 5 pmol/L - 5 nmol/L, com LD e LQ de 50 fmol/L e 165 fmol/L (em PBS) e 7 pmol/L e 21 pmol/L (em soro), respectivamente. A técnica de espectroscopia de capacitância eletroquímica foi capaz de avaliar a interação entre a lectina ArtinM e glicoproteína HRP em concentrações 1500 vezes menores do que a piezelétrica (QCM). A abordagem possibilitou detectar HRP na faixa de concentração de 0,5-100 nmol/L com LD de 200 pmol/L e com desvio-padrão relativo de 7%. As funções de imitância também foram utilizadas como sinal transdutor para o estudo da interação ArtinM-HRP, apresentando a mesma sensibilidade que a espectroscopia de capacitância eletroquímica, com a vantagem de oferecer ensaios com maior rapidez. Os resultados obtidos nesta tese evidenciam o potencial uso da capacitância eletroquímica e das funções de imitância para o desenvolvimento de dispositivos biossensores aplicados ao diagnóstico clínico (trombose e câncer de próstata) e em glycoarray.
The electrochemical capacitance spectroscopy technique has been recently applied to detect biomarkers of clinical interest, such as C-reactive protein (CRP) which is related to heart disease and inflammatory processes. Herein, this technique was used to develop electroanalytical approaches with potential applications for thrombosis diagnosis by detecting D-dimer biomarker protein; prostatic cancer, using prostatic acid phosphatase (PAP) enzyme as target; and glycoprotein assay, for possible detection of tumor cells and as a model for studying lectin-glycoprotein interaction (using ArtinM lectin and HRP glycoprotein). In addition, in the latter case, immittance functions were used as transducer signals as possible application for glycoarray. For the construction of the receptor surface on gold electrodes, two different thiol molecules (R-SH, being R a generic carbonic chain) were used. The first, which contains the carboxylic terminal group, was applied to immobilize the biological material for target recognition (i.e., either antibodies or lectin) via EDC/NHS protocol. The second molecule contains a terminal redox group (11-ferrocenyl-undecanethiol, 11Fc) in order to provide the transducer signal. The bifunctional monolayers obtained herein present high surface coverage, ≈ 5 x 10-10 mol/cm2. The immobilization of the antibody to PAP detection (anti-PAP) was investigated by QCM, and it was verified that saturation occurs in approximately 1 h, yielding a surface coverage of ≈ 3.7 mg/m2, suggesting “end on” orientation. Using the electrochemical capacitance signal, it was possible to develop an approach for PAP detection with limit of detection (LD) and quantification (LQ) of 9 pmol/L and 28 pmol/L, respectively, applied to a PAP clinically useful concentration range of 50-1000 pmol/L in phosphate buffered saline (PBS, pH 7.4). The relative standard deviation obtained was 9.5%, and showed specificity when evaluated against fetuin as a negative control. The same assay was performed in undiluted commercial serum in a linear range of 50-5000 pmol/L, with higher LD and LQ when compared to PBS, with results of 52 and 132 pmol/L, respectively. Anti-DD immobilization was also evaluated by QCM, presenting similar saturation time, 1 h, when compared with anti-PAP coupling. The surface coverage, ≈ 3.0 mg/m2, suggests an intermediate antibody SAM orientation between “end on” and “side on”. D-dimer was detected in both PBS (pH 7.4) and undiluted serum at a concentration range of 5 pmol/L - 5 nmol/L, with LD and LQ of 50 fmol/L and 165 fmol/L (in PBS) and 7 pmol/L and 21 pmol/L (in serum), respectively. Higher values of LD and LQ were obtained in serum when compared to those in PBS in combination with increase in relative standard deviation, from 11% (in PBS) to 14% (in serum) due to the matrix effect. The capacitance spectroscopy technique was also able to evaluate the interaction between ArtinM lectin and HRP glycoprotein at concentrations 1,500 times lower than those used in classical techniques, such as piezoelectric (QCM). The approach allowed detecting HRP in the concentration range of 0.5-100 nmol/L with LD of 200 pmol/L and with a relative standard deviation of 7%. Immittance functions were also used as a transducer signal for the study of the ArtinM-HRP interaction, presenting the same sensitivity as electrochemical capacitance, with the advantage of offering quicker assays. The results obtained in this thesis show the potential use of electrochemical capacitance and immittance functions for the development of biosensor devices applied for clinical diagnosis (prostatic cancer and thromboses) and glycoarray.
The electrochemical capacitance spectroscopy technique has been recently applied to detect biomarkers of clinical interest, such as C-reactive protein (CRP) which is related to heart disease and inflammatory processes. Herein, this technique was used to develop electroanalytical approaches with potential applications for thrombosis diagnosis by detecting D-dimer biomarker protein; prostatic cancer, using prostatic acid phosphatase (PAP) enzyme as target; and glycoprotein assay, for possible detection of tumor cells and as a model for studying lectin-glycoprotein interaction (using ArtinM lectin and HRP glycoprotein). In addition, in the latter case, immittance functions were used as transducer signals as possible application for glycoarray. For the construction of the receptor surface on gold electrodes, two different thiol molecules (R-SH, being R a generic carbonic chain) were used. The first, which contains the carboxylic terminal group, was applied to immobilize the biological material for target recognition (i.e., either antibodies or lectin) via EDC/NHS protocol. The second molecule contains a terminal redox group (11-ferrocenyl-undecanethiol, 11Fc) in order to provide the transducer signal. The bifunctional monolayers obtained herein present high surface coverage, ≈ 5 x 10-10 mol/cm2. The immobilization of the antibody to PAP detection (anti-PAP) was investigated by QCM, and it was verified that saturation occurs in approximately 1 h, yielding a surface coverage of ≈ 3.7 mg/m2, suggesting “end on” orientation. Using the electrochemical capacitance signal, it was possible to develop an approach for PAP detection with limit of detection (LD) and quantification (LQ) of 9 pmol/L and 28 pmol/L, respectively, applied to a PAP clinically useful concentration range of 50-1000 pmol/L in phosphate buffered saline (PBS, pH 7.4). The relative standard deviation obtained was 9.5%, and showed specificity when evaluated against fetuin as a negative control. The same assay was performed in undiluted commercial serum in a linear range of 50-5000 pmol/L, with higher LD and LQ when compared to PBS, with results of 52 and 132 pmol/L, respectively. Anti-DD immobilization was also evaluated by QCM, presenting similar saturation time, 1 h, when compared with anti-PAP coupling. The surface coverage, ≈ 3.0 mg/m2, suggests an intermediate antibody SAM orientation between “end on” and “side on”. D-dimer was detected in both PBS (pH 7.4) and undiluted serum at a concentration range of 5 pmol/L - 5 nmol/L, with LD and LQ of 50 fmol/L and 165 fmol/L (in PBS) and 7 pmol/L and 21 pmol/L (in serum), respectively. Higher values of LD and LQ were obtained in serum when compared to those in PBS in combination with increase in relative standard deviation, from 11% (in PBS) to 14% (in serum) due to the matrix effect. The capacitance spectroscopy technique was also able to evaluate the interaction between ArtinM lectin and HRP glycoprotein at concentrations 1,500 times lower than those used in classical techniques, such as piezoelectric (QCM). The approach allowed detecting HRP in the concentration range of 0.5-100 nmol/L with LD of 200 pmol/L and with a relative standard deviation of 7%. Immittance functions were also used as a transducer signal for the study of the ArtinM-HRP interaction, presenting the same sensitivity as electrochemical capacitance, with the advantage of offering quicker assays. The results obtained in this thesis show the potential use of electrochemical capacitance and immittance functions for the development of biosensor devices applied for clinical diagnosis (prostatic cancer and thromboses) and glycoarray.
Descrição
Palavras-chave
Espectroscopia de capacitância eletroquímica, Biossensores, Funções de imitância, Biomarcadores, ArtinM, Electrochemical capacitance spectroscopy, Biosensors, Immittance functions, Biomarkers