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Publication
Optimisation of an aptamer-biosensor to detect C-reactive protein
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Advisor(s)
Abstract(s)
A proteína C reactiva (PCR) é um biomarcador de doenças caracterizadas por uma
resposta inflamatória aguda; tais como: doenças coronárias, diabetes mellitus ou
carcinomas. A detecção desta proteína em fluidos corporais, em especial em soro
humano pode ainda determinar risco de desenvolvimento futuro de doenças coronárias
em indivíduos aparentemente saudáveis.
Foi realizado a optimização de um novo biosensor aplicando a técnica de Voltametria
de Pulso Diferencial (DPV). A detecção foi realizada em solução tampão aquosa
utilizando eléctrodos de carbono e o produto enzimático electroactivo da reacção de 1-
naftil fosfato com alcalina fosfatase. Neste trabalho foi optimizado, em solução tampão,
o tempo de incubação e o valor de concentração da alcalina fosfatase conjugada com
streptavidina. As condições finais foram testadas em solução de soro humano livre de
PCR diluído 1:10.
A proteína C-reactiva foi retirada do meio líquido aplicando a técnica de ensaio
imunológica “sandwich”. O ensaio requereu como suporte sólido esferas magnéticas
cobertas por streptavidina e como captores biológicos um aptamero de RNA e um
anticorpo monoclonal, ambos biotinilados.
O tempo optimizado de incubação foi de 10 min e a concentração óptima de fosfatase
alcalina acoplada a streptavidina foi de 0.22U/ml. Estas condições em meio tampão
demonstraram um limite de detecção de pelo menos 0.1mg/l de PCR com um
coeficiente de variação igual a ±14.45%. Para as mesmas condições, mas em soro
humano livre de PCR, o limite de detecção foi de pelo menos 0.5mg/l de concentração
proteica e o coeficiente de variância de ±7.32%.
O biosensor desenvolvido demonstrou elevada exactidão, reprodutibilidade, relativa
sensibilidade e rápida concretização do ensaio quando comparado com outros métodos
de detecção tradicionais. Deste modo o novo biosensor desenvolvido apresenta-se como
um excelente candidato à detecção da PCR no campo do diagnóstico médico.
C-reactive protein is a biomarker for diseases where an acute inflammatory response is present. Examples are coronary disease, diabetes mellitus and cancer. The presence of the CRP protein in body fluids, especially in serum may also predict risk of future coronary complications in apparently healthy people. A new aptamer biosensor based on the electrochemical differential pulse voltammetry detection method was optimised. The detection was done in solution using screenprinted carbon electrodes and the properties of the electroactive enzymatic product of 1- naphytl phosphate with alkaline phosphatase. In this work the optimisation of the time of incubation and concentration of the alkaline phosphatase enzyme conjugated with streptavidin was performed in buffer medium and the optimised conditions tested in CRP-free human serum with a total dilution of 1:10. The C-reactive protein was captured from the media, using a sandwich immunoassay technique. The solid phase used for the realisation of the sandwich technique in solution was magnetic beads covered with streptavidin. The sandwich assay was performed with an RNA aptamer and a monoclonal antibody, both biotinylated. The optimised time of incubation was 10 minutes and the optimised concentration of the streptavidin-alkaline phosphatase conjugate 0.22U/ml. These conditions in buffer medium showed a detection limit of at least 0.1mg/l of CRP concentration with a mean of coefficient of variance equal to ±14.45%. For the same conditions in CRP-free human serum, the detection limit was at least 0.5mg/l of protein concentration and the coefficient of variance ±7.32%. The developed aptamer biosensor presented elevated accuracy, reproducibility, relative sensitivity due to some matrix effects and less consumed laboratory time when compared with traditional tests. For these reasons the new aptamer biosensor developed appears to be an excellent candidate to detect CRP in the diagnostic field.
C-reactive protein is a biomarker for diseases where an acute inflammatory response is present. Examples are coronary disease, diabetes mellitus and cancer. The presence of the CRP protein in body fluids, especially in serum may also predict risk of future coronary complications in apparently healthy people. A new aptamer biosensor based on the electrochemical differential pulse voltammetry detection method was optimised. The detection was done in solution using screenprinted carbon electrodes and the properties of the electroactive enzymatic product of 1- naphytl phosphate with alkaline phosphatase. In this work the optimisation of the time of incubation and concentration of the alkaline phosphatase enzyme conjugated with streptavidin was performed in buffer medium and the optimised conditions tested in CRP-free human serum with a total dilution of 1:10. The C-reactive protein was captured from the media, using a sandwich immunoassay technique. The solid phase used for the realisation of the sandwich technique in solution was magnetic beads covered with streptavidin. The sandwich assay was performed with an RNA aptamer and a monoclonal antibody, both biotinylated. The optimised time of incubation was 10 minutes and the optimised concentration of the streptavidin-alkaline phosphatase conjugate 0.22U/ml. These conditions in buffer medium showed a detection limit of at least 0.1mg/l of CRP concentration with a mean of coefficient of variance equal to ±14.45%. For the same conditions in CRP-free human serum, the detection limit was at least 0.5mg/l of protein concentration and the coefficient of variance ±7.32%. The developed aptamer biosensor presented elevated accuracy, reproducibility, relative sensitivity due to some matrix effects and less consumed laboratory time when compared with traditional tests. For these reasons the new aptamer biosensor developed appears to be an excellent candidate to detect CRP in the diagnostic field.
Description
Dissertação de Mestrado, Engenharia Biológica, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2009
Keywords
Proteínas Doenças coronárias Diagnóstico