This work explores several issues of importance for the development of a diagnostic method based on circle-to-circle amplification (C2CA) and oligonucleotide-functionalized magnetic nanoparticles. Firstly, the performance of the detection method was evaluated in terms of sensitivity and speed. Synthetic target sequences for Newcastle disease virus and Salmonella were used as model sequences. The sensitivity of the C2CA assay resulted in detection of 1 amol of starting DNA target with a total amplification time of 40 min for both target sequences. Secondly, the functionalization of the nanoparticles was evaluated in terms of robustness and stability. The functionalization was shown to be very robust, and the stability test showed that 92% of the oligos were still attached on the particle surface after three months of storage at 4 °C. Altogether, the results obtained in this study provide a strong foundation for the development of a quick and sensitive diagnostic assay.
Keywords: Brownian relaxation; DNA detection; circle-to-circle amplification; magnetic nanoparticles; nanoparticle functionalization; zoonotic diseases.