Endowing Femtomolar Sensitivity to Magnetic RNA Assays by Omega-Loop-Mediated Dual-Signal Amplification Cascades

Homogeneous magnetic bioassays for the detection of disease-specific nucleic acids are performed in a "mix & measure" fashion, yet nonenzymatic schemes have picomolar detection limits, which hamper their advancement toward real-world applications. Here, we demonstrate a magnetic omega-loop-mediated dual-signal amplification cascade (MODAC) that combines recycling and duplication of a nucleic acid target and amplifies the magnetic signal gain near-exponentially. We tweak the omega-loop duplex stability by length and sequence variation to achieve a good trade-off between cascade reactivity and leakage. We showcase the nonenzymatic detection of the N gene of SARS-CoV-2 RNA at 23 fM within 90 min at 25 °C. Our validation study with ddPCR demonstrates that the MODAC signal scales linearly with the RNA concentration at low target concentrations. The MODAC tests are performed in nonsterile laboratory settings using a portable magnetic particle spectrometer, highlighting its prospect for point-of-care applications.