Single-Excitation, Orthogonally Encoded Quantum Dot Probes for Dual-Mode Multiplexed Biosensing

Abstract Simultaneous quantitative detection of multiple rheumatoid arthritis (RA) biomarkers is often constrained by the need for separate analytical platforms and complex instrumentation. Here, we describe a dual-mode multiplex immunoassay that integrates a lateral flow immunoassay (LFIA) with a magnetic bead immunoassay (MBIA) using spectrally resolved SiO2-encapsulated quantum dot (SiO2@QDs) probes. By combining SiO2@CdSe and carbon dot-based SiO2 nanoparticles, three spectrally distinct fluorescence signals (blue, green, and red) were generated under single-wavelength excitation (365 nm), enabling concurrent detection of anticyclic citrullinated peptide antibodies (anti-CCP), rheumatoid factor IgM (RF-IgM), and C-reactive protein (CRP). The LFIA module enables multiplex quantification within 10 min with minimal instrumentation, whereas the MBIA module extends analytical sensitivity and dynamic range through homogeneous immunoreactions and magnetic separation. The limits of detection of the MBIA sensor were 0.142 U/mL for anti-CCP, 0.057 IU/mL for RF-IgM, and 0.075 mg/L for CRP, with linear responses (R2 > 0.98), coefficients of variation below 8%, and minimal cross-reactivity. Analysis of clinical RA samples showed good agreement with commercial reference assays, as assessed by Passing–Bablok regression and Bland–Altman analysis. By integrating rapid screening and laboratory-grade quantification within a single fluorescence-encoded nanoprobe system, this dual-mode platform illustrates a modular strategy for multiplex autoimmune biomarker analysis, in which assay format and signal encoding are decoupled within a unified optical framework.