Ferromagnetic Resonance Multiplexing of Orthogonal Magnetic Nanoparticle Agglutination for Simultaneous Analysis of Nucleic Acid Mutations and Nonspecific Interactions

Abstract Developing multiplexed platforms capable of simultaneously analyzing specific biomolecular interactions and nonspecific binding effects in a single reaction system holds significant potential for accurate disease diagnostics. Here, a ferromagnetic resonance (FMR) biosensor is presented that integrates orthogonal magnetic nanoparticle (MNP) agglutination with genetic algorithm (GA)‐based spectral deconvolution. Different Mycobacterium tuberculosis drug‐resistance mutations can induce orthogonal reactions including padlock probe ligation, rolling circle amplification, and MNP agglutination, producing homogeneous MNP assemblies. Meanwhile, heterogeneous MNP assemblies form due to nonspecific interactions. FMR spectrum of the MNP agglutination mixture is deconvoluted into three components, each representing the rifampicin‐resistant single‐nucleotide mutation rpoB 531T, the isoniazid‐resistant single‐nucleotide mutations katG 315C and inhA −15T, and the total nonspecific binding effect in the suspension. The method demonstrates detection limits as low as 50 fm for target sequences within 80 min, and is validated by wild‐type sequences, 5% serum samples, and clinical sputum samples. This triplex analysis not only enhances detection accuracy but also enables real‐time error correction, which is a feature unattainable in conventional duplex assays.