Enzyme-free magnetic diagnostics circuit for detection of SARS-CoV-2 RNA?

The detection of nucleic acids has gained enormous attention due to their potential as disease biomarkers. However, deoxy-/ribonucleic acids (DNA/RNA) ?detection often requires extensive and costly sample processing, including extraction and enzyme-based amplification. Magnetic assays based on magnetic ?nanoparticles (MNPs) and Magnetic Particle Spectroscopy (MPS) show great potential for mix-and-measure detection as the magnetic signal of MNPs does ?not interfere with the typical magnetic signal of biological background, making MNPs detectable even in opaque sample media. Moreover, portable MPS ?devices make magnetic assays adaptable for point-of-care applications. By realizing magnetic assays amplification- and enzyme-free, they will become ?irreplaceable for point-of-care diagnostics.?
Generally speaking, the binding of molecules to the surface of MNPs increases the hydrodynamic size of the particles, which in turn increases the Brownian ?relaxation time constant. The change in particle's relaxation can be readout using MPS. This thesis comprises the development of magnetic assays for the ?detection of pathogen specific RNA, which are based on target-induced declustering events of responsive magnetic clusters. The declustering-approach ?makes use of toehold-mediated DNA strand displacement reactions. Upon the addition of target, the clusters are dissociated into their building block MNPs, ?which reduces their initial hydrodynamic size and results in an increase of the MPS signal. By exploiting DNA nanotechnology methods and nanoswitches, a ?Magnetic signal Amplification Circuit (MAC) was developed to further amplify the magnetic signal, improving the assay sensitivity by a factor of four. In ?order to enable whole genome detection, the assay was evolved into the initiated MAC assay. It is demonstrated that the initiated MAC assay is capable of ?detecting the whole RNA genome of cell culture-grown Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) samples in a one-pot, ?amplification- and enzyme-free fashion at isothermal conditions (25 °C). For comparison, the extracted RNA elutions were analyzed by polymerase chain ?reaction and cycle thresholds of up to 26 were determined. Furthermore, it is shown how the initiated MAC can reliably discriminate between SARS-CoV-2 ?and other human coronaviruses. Finally, a cost analysis was performed to evaluate the resource efficiency of the approach. Conclusively, the declustering-?based MAC assays introduce a whole new approach for MPS-based magnetic assays, enhancing their competitiveness among other detection platforms.?