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.

Improved analytical methods provide new opportunities for both biological research and medical applications. This thesis describes several novel molecular techniques for nucleic acid and protein analysis based on detection or sequencing of amplified single molecules (ASMs). ASMs were generated from padlock probe assay and proximity ligation assay (PLA) through a series of molecular processes. In Paper I, a simple colorimetric readout strategy for detection of ASMs generated from padlock probe assay was used for highly sensitive detection of RNA virus, showing the potential of using padlock probes in the point-of-care diagnostics. In Paper II, digital quantification of ASMs, which were generated from padlock probe assay and PLA through circle-to-circle amplification (C2CA), was used for rapid and sensitive detection of nucleic acids and proteins, aiming for applications in biodefense. In Paper III, digital quantification of ASMs that were generated from PLA without C2CA was shown to be able to improve the precision and sensitivity of PLA when compared to the conventional real-time PCR readout. In Paper IV, a non-optical approach for detection of ASMs generated from PLA was used for sensitive detection of bacterial spores. ASMs were detected through sensing oligonucleotide-functionalized magnetic nanobeads that were trapped within them. Finally, based on in situ sequencing of ASMs generated via padlock probe assay, a novel method that enabled sequencing of individual mRNA molecules in their natural context was established and presented in Paper V. Highly multiplex detection of mRNA molecules was also achieved based on in situ sequencing. In situ sequencing allows studies of mRNA molecules from different aspects that cannot be accessed by current in situ hybridization techniques, providing possibilities for discovery of new information from the complexity of transcriptome. Therefore, it has a great potential to become a useful tool for gene expression research and disease diagnostics.