A Spatially Encoded Fluorescent Biosensor for Multiplexed and High-Throughput Detection of HFMD Viruses via Parallel...

Hand-foot-mouth disease (HFMD), mainly caused by Enterovirus 71 (EV71), Coxsackievirus A6 (CVA6), and Coxsackievirus A16 (CVA16), poses a serious threat to children due to its high transmissibility and potential to induce severe complications. Herein, a spatially encoded fluorescence biosensing platform that integrates a parallel entropy-driven circuit (EDC) with a combinatorial photonic crystal (cPC) array was reported for ultrasensitive, multiplex, and rapid detection of HFMD viruses. In this system, three parallel entropy-driven circuits were designed to specifically recognize the VP1 gene sequences of EV71, CVA6, and CVA16, generating distinct fluorescence signals (FAM, Cy3, and Cy5) upon target binding. Simultaneously, the cPC array, constructed from polystyrene nanospheres of three different diameters (220, 245, and 293 nm), not only acted as a spatial encoder that enabled the simultaneous discrimination of multiple signals through predefined physical zones but also served as a signal amplifier by matching its photonic band gaps to the emission wavelengths of the fluorophores. Under optimized conditions, the sensing array exhibited excellent linearity across broad dynamic ranges with the detection limits down to the femtomolar level (100.55 fM for EV71, 23.06 fM for CVA6, and 52.76 fM for CVA16). This assay also demonstrated high specificity against non-HFMD viruses and satisfactory recovery rates (94.52-102.68%) in spiked serum samples. This work reports the first application of integrating a combinatorial PC array with a parallel EDC for the detection of HFMD, offering a low-cost, user-friendly, and versatile strategy with promising potential for clinical diagnostics and public health surveillance.