Rolling Circle Amplification on a Bead: Improving the Detection Time for a Magnetic Bioassay

Detection of pathogens has become increasingly important, especially in the face of outbreaks and epidemics all over the world. Nucleic acid detection techniques provide a solid base to detect and identify pathogens. In recent years, magnetic sensors and magnetic labels have become of more interest due to their simplicity of use, low cost, and versatility. In this work, we have used the isothermal DNA amplification technique of rolling circle amplification (RCA) in combination with oligo-functionalized magnetic nanoparticles. Detection of RCA products takes place through specific binding between magnetic nanoparticles and RCA products. Upon binding, the relaxation frequency of the nanoparticle changes. This change was measured using an AC susceptometer. We showcase that the RCA time can be reduced for a quicker assay when performing the RCA on the surface of micrometer-sized beads, which consequently increases the hydrodynamic volume of the RCA products. This, in turn, increases the Brownian relaxation frequency shift of the nanoparticles upon binding. We performed optimization work to determine the ideal quantity of micrometersized particles, oligo-functionalized nanoparticles, and the amplification time of the RCA. We show that the detection of 0.75 fmol of preamplification synthetic target is possible with only 20 min of amplification time. Finally, we showcase the high specificity of the assay, as the functionalized nanoparticles are unable to bind to amplified DNA that does not match their labels. Overall, this paves the way for a simple bioassay that can be used without expensive laboratory equipment for detection of pathogens in outbreak settings and clinics around the world.

Automated summary

Pathogen detection is of increasing importance, and nucleic acid detection techniques provide a reliable method to identify and detect pathogens. Magnetic sensors and magnetic labels have gained interest due to their simplicity, low cost, and versatility. In this study, we used the rolling circle amplification (RCA) technique combined with oligo-functionalized magnetic nanoparticles to detect RCA products by measuring the relaxation frequency change of the nanoparticles. By optimizing the use of micrometer-sized beads, we reduced the RCA time for faster detection and increased the detection sensitivity. Furthermore, our assay exhibited high specificity by only binding to amplified DNA that matched their labels. Overall, this research paves the way for a simple bioassay that can be used in outbreak settings and clinics worldwide without expensive laboratory equipment.