Circle-to-circle amplification to improve the sensitivity of a magnetic nanoparticle-based DNA detection protocol

University essay from Uppsala universitet/Molekylärbiologi

Abstract: Magnetic nanoparticles have great potential in the biomedical and diagnostics field. Due to their small size, the particles have a high surface-to-volume ratio which enables for biofunctionalisation with different molecular probes. This makes itpossible to target them against a wide variety of biomarkers. In this project, the aim was to develop a magnetic nanoparticle-based DNA detection method with respectto sensitivity by employing circle-to-circle amplification, which is an extension of rolling circle amplification, in order to increase the assay sensitivity. The method provides high specificity due to the use of padlock probes for amplification. The project included testing and optimising the protocol used for DNA amplification and detection with a synthetic target, which involved testing different padlock probes, incubation times and incubation temperatures. Lastly, the method was tested on a biological target. It has recently been shown that specific aggregation occurs between magnetic nanoparticles and DNA, which enables for a visual readout strategy sincethe aggregates are visible to the naked eye. Initial testing of the method yielded asensitivity of about 100 attomoles. The achieved sensitivity after the optimisation work was 1 attomole of both synthetic and biological DNA targets. This is an improvement compared to the 400 attomoles that has previously been reported with one round of rolling circle amplification. The results can be used in further development of the naked-eye DNA detection method towards the realisation of a commercially attractive bioanalytical device. 

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