Probes based on fluorescein derivatives for rapid detection of H3N2 virus spike proteins without separation

H3N2 influenza virus poses a persistent serious threat to human health every year. Currently available detection methods often involve multi-step complex procedures. Such as enzyme-linked immunosorbent assay (ELISA) requires centrifugation, incubation with multiple antibodies, and addition of enzyme-labeled reagents. Reverse transcription-polymerase chain reaction (RT-PCR) necessitates RNA extraction, reverse transcription, and thermal cycling for amplification. These time-consuming and labor-intensive protocols hinder timely response. In this work, fluorescein (FLC), 5-carboxyfluorescein (5-FAM) and fluorescein-5-thiosemicarbazide (5-FTSC) exhibited almost no fluorescence emission in pure DMSO and strong fluorescence emission in DMSO/H₂O with high water fraction. Based on this property, we presented a separation-free, rapid, and quantitative detection method using three compounds as “turn-on” fluorescent probes to detect H3 and N2 spike proteins in mixed solutions. Among them, the limit of detection of FLC probe to N2 was as low as 0.442 nmol/L. Simulations based on molecular docking indicated that the fluorescent probe was able to enter the hydrophobic cavity of the protein restricted by the surrounding amino acid residues, and that the intramolecular motion was reduced to achieve a fluorescence enhanced response. Finally, comparative experiments using real H3N2 virus samples showed that the fluorescence probe method not only achieved faster and more cost-effective detection than ELISA, but also produced more consistent results, indicating the higher reliability of detection results. The separation-free detection method will provide a valuable tool for early public health monitoring.