Microtech 2011

Nano-ELISA using gold nanoparticle-DNA complex for the detection of pathogenic microbe

H.-S. Kim, B.-K. Oh
Sogang Univeristy, KR

Keywords: nano-ELISA, pathogen, biosensor, nanoparticle


In this study, we report on the rational design of a nano-complex that consists of gold nanoparticle (AuNP), antibody, oligonucleotides and horseradish peroxidase (HRP) for the detection of pathogen. The AuNP was functionalized with a polyclonal antibody that has an affinity for target pathogen. The DIG-labeled oligonucleotides were covalently attached on the AuNP. And then HRP labeled anti-DIG was bound to DIG labeled oligonucleotide on the AuNP. The binding of multiple HRP enzymes to the AuNP was used to amplify the signal of the target probe. Afterwards, magnetic microparticles (MMP) were functionalized with capture antibody and used in a sandwich reaction with the target pathogen. The MMPs containing the target pathogen were then bound to AuNPs via immune reaction. A magnetic field was used to effectively remove unbound AuNPs. For detection of pathogen, we exploited the reaction of HRP enzyme immobilized on Au probes with TMB that is a substrate of HRP, followed by stopping the reaction with 2M H2SO4. The resulting end products were analyzed by UV-vis spectroscopy. Using this method, we could detect down to 50 CFU/mL of target pathogen within a hour. This method is very simple and highly sensitive relative to established pathogen biosensors.

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