AND-Logic Cascade Rolling Circle Amplification for Optomagnetic Detection of Dual Target SARS-CoV-2 Sequences

DNA logic operations are accurate and specific molecular strategies that are appreciated in target multiplexing and intelligent diagnostics. However, most of the reported DNA logic operation-based assays lack amplifiers prior to logic operation, resulting in detection limits at the subpicomolar to nanomolar level. Herein, a homogeneous and isothermal AND-logic cascade amplification strategy is demonstrated for optomagnetic biosensing of two different DNA inputs corresponding to a variant of concern sequence (containing spike L452R) and a highly conserved sequence from SARS-CoV-2. With an "amplifiers-before-operator" configuration, two input sequences are recognized by different padlock probes for amplification reactions, which generate amplicons used, respectively, as primers and templates for secondary amplification, achieving the AND-logic operation. Cascade amplification products can hybridize with detection probes grafted onto magnetic nanoparticles (MNPs), leading to hydrodynamic size increases and/or aggregation of MNPs. Real-time optomagnetic MNP analysis offers a detection limit of 8.6 fM with a dynamic detection range spanning more than 3 orders of magnitude. The accuracy, stability, and specificity of the system are validated by testing samples containing serum, salmon sperm, a single-nucleotide variant, and biases of the inputs. Clinical samples are tested with both quantitative reverse transcription-PCR and our approach, showing highly consistent measurement results.