An atto-molar detectable colorimetric DNA biosensor based on enzyme amplification method

In this study, we report on the rational design of a nano-complex that consists of silica nanoparticles (SiNP), probe DNA and horseradish peroxidase (HRP) for the detection of target DNA. SiNPs were modified to probe DNA and streptavidin for the subsequent binding of biotinylated HRP enzymes. The localized binding of multiple HRP enzymes to the SiNP was used to amplify the signal of the target probe. Afterwards, magnetic microparticles (MMP) were functionalized with capture DNA and used in a sandwich reaction with the target DNA. The MMPs containing the target DNA were then bound to SiNPs via DNA hybridization. The interaction forces that resulted from the DNA hybridization between the functionalized MMP containing target DNA and SiNPs was use to separate unbound MMP; a magnetic field was used to effectively remove unbound MMP. For detection of DNA, we exploited the reaction of HRP enzyme immobilized on silica probes with tetramethylbenzidine (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 500 aM of target DNA a short time. This method is very simple and highly sensitive relative to established DNA biosensors and can be used to detect specific DNA markers associated with tumors, bacterial infections or other disease.