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method for the simultaneous detection of maduramicin( MAD) and salinomycin( SAL) in chicken meat, as shown in Figure 5c. In this study, BsMAb was obtained by hybridoma technique and combined with specific fluorescent probe( MAD-FITC / SAL-EDF) to establish an efficient detection system, which could be completed in only 10 min with high sensitivity and no cross-reaction. Molecular docking revealed the specific binding mechanism of BsMAb, and the recoveries of the actual samples were 88.7 – 113.7 %, which were in high agreement with the LC-MS / MS results. This study provides an efficient solution for the detection of antibiotic multiresidues in food and fully demonstrates the potential of FPIA technology.
FPIA has shown important application value in many fields with its advantages of high sensitivity, strong antiinterference ability and simple operation. In environmental monitoring, FPIA can efficiently detect pollutants in wastewater. In the field of food safety [ 70 ], this method can quickly screen erythromycin in milk and a variety of antibiotic residues in chicken. In addition, FPIA does not need complex separation steps, and is suitable for on-site rapid detection, providing a reliable tool for infectious disease diagnosis, drug screening and other research. With the progress of polarization optical elements and fluorescent probe technology, the application scope of this technology is still expanding.
2.4 Fluorescence resonance energy transfer
In 1948, Theodor Förster first proposed [ 71 ] the theory of fluorescence resonance energy transfer( FRET), which describes a non-radiative energy transfer phenomenon: when the distance between the excited state molecule( donor) and the ground state molecule( acceptor) is less than 10 nm, the donor can transfer energy to the acceptor through near-field coupling, which is often referred to as“ fluorescence resonance energy transfer” when both the donor and the acceptor are fluorophores. In the process, the donor molecule can return to the ground state through two pathways: emitting fluorescent photons or transferring the energy through FRET [ 72 ], as shown in Figure 6 [ 73 ]. The existence of FRET adds new pathways for energy dissipation in the donor, leading to a shortening of its excited state lifetime, a property that plays a key role in unravelling the mechanism of interactions between biomolecules. In 1967, the FRET theory was experimentally validated for the first time. Its unique distance dependence( 1 – 10 nm) makes it an ideal‘ optical ruler’ for molecular scale interactions and provides a breakthrough method for quantitative study of biomolecular interactions [ 74 ]. In the 1980s, FRET technology was successfully applied to protein conformation studies, and through the combination with other analytical techniques, it has driven the innovation of molecular detection methods [ 75, 76 ]. With the development of technology, FRET-based biosensors have gradually become a research hotspot. Their design centers on coupling the donor and acceptor fluorophores within a single, responsive molecular scaffold. This is typically achieved by genetically fusing fluorescent protein pairs( e. g., CFP / YFP) to a sensing domain that undergoes a target-induced conformational change, or by conjugating organic dyes to complementary
Fig. 6. Schematic diagram of fluorescence resonance energy transfer.
biomolecular recognition elements( e. g., antibodies, oligonucleotides). When the target analyte binds or the enzymatic reaction occurs, it alters the distance and / or orientation between the donor and acceptor. This change modulates the FRET efficiency, which is directly read out as a quantifiable shift in the fluorescence emission ratio of the acceptor to the donor, enabling sensitive and spatiotemporally resolved detection. This design not only achieves specific detection of molecular interactions, but also dynamically reflects changes in the microenvironment, providing an important tool for molecular mechanism research and biomedical applications.
FRET technology has made significant progress in the application of in vivo imaging, especially in tumor metabolism and drug delivery monitoring. Rudkouskaya et al. [ 77 ] developed a FRET receptor technology based on the dark bursting agent QC-1, combined with near-infrared Macroscopic Fluorescence Lifetime Förster Resonance Energy Transfer( MFLI-FRET), enabling multiple non-invasive imaging for simultaneous monitoring of tumour glucose metabolism and receptor-ligand binding in living mice, as shown in Figure 7a. This technique enables the simultaneous quantification of two independent biological processes in the same tumor: 1) intracellular drug delivery efficiency, measured by the FRET signal generated when transferrin( Tf) donors and dark-quencher acceptors( e. g., Tf-QC-1) bind in proximity to transferrin receptors( TfR) and undergo internalization; and 2) tumor glycolytic activity, assessed by the fluorescence intensity of a 2-deoxyglucose( 2-DG) probe that accumulates upon cellular uptake via glucose transporters( GLUT1). It was found that drug delivery efficiency was lower in highly metabolically active tumour regions, revealing a potential correlation between metabolic status and the efficacy of targeted therapies. Multiscale validation( in vitro microscopy and in vivo wide-field imaging) confirmed the stability of QC-1 as a FRET acceptor. The study realized the synchronous monitoring of drug targeted delivery and metabolic response in living animals for the first time, providing an important tool