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improved sensitivity to 2.42 ns / pH;( 3) maintained linear response in a simulated intracellular environment, which overcame signal fluctuations of its predecessor in complex environments; and( 4) cellular experiments confirmed its excellent 24-hour stability, whereas the previous generation probe suffered from signal attenuation in long-term experiments. Mechanistic analyses showed that histidine residues facilitated the escape of the probe into the cytoplasm to avoid lysosomal degradation through enhanced pH sensitivity and the“ proton sponge hypothesis”. This improvement makes the CdSe / ZnS-pH probe a more reliable tool for long-term pH monitoring in living cells, and provides a new method for cell metabolism research and drug screening.
FLIM Flow Cytometry enables high-throughput, highresolution analysis of single cells by combining FLIM with flow cytometry. While conventional flow cytometry relies on fluorescence intensity, which is susceptible to interference from concentration, scattering and other factors, FLIM technology takes advantage of the stability of fluorescence lifetimes to dramatically improve detection accuracy. The high-throughput FLIM system developed by Kanno et al. [ 52 ] employs a dual-intensity-modulated, continuous-wave beam array to acquire fluorescence lifetimes images at a rate of more than 10,000 cells / second, as shown in Figure 3d [ 53 ]. Crucially, this system maintains cell-level resolution, associating each lifetime measurement with an individual cellular event, unlike bulk measurements that average signals across populations. The system successfully distinguished subpopulations of rat glioma cells and captured anticancer drug-induced changes in cell nuclear dynamics, revealing heterogeneous features that are difficult to observe by conventional methods. In addition, the sensitivity of FLIM technology to environmental factors( such as pH, ion concentration) provides a new tool for drug screening and tumour microenvironment studies. The breakthrough of this technology opens up new avenues for cell function analysis and clinical diagnosis.
These research fully demonstrate the broad application prospect of FLIM technology in many biomedical research fields. With probe optimization and system integration( such as quantum dot probes, multimodal imaging, etc.), FLIM has been widely used in live cell dynamic observation, drug screening and clinical diagnosis. In the future, FLIM will play an increasingly important role in precision medicine, drug development and other fields.
2.3 Fluorescence polarization immunoassay
Fluorescence intensity is susceptible to external noise, affecting the sensitivity and reliability of the analysis, whereas rapid and specific diagnosis is essential for the treatment and prevention of infectious diseases [ 54, 55 ]. Compared with traditional fluorescence detection, Fluorescence Polarization Immunoassay( FPIA) has significant anti-interference advantages and is a highly sensitive analytical technique based on the rotational kinetic properties of fluorescently labelled molecules. Figure 4 shows a schematic diagram of the principle of Fluorescence Polarization [ 56 ]. The core principle of this technology lies in the fact that when a fluorescently labelled molecule is excited by linearly polarised light, the Polarization state of the emitted light is closely related to the rotational state of the molecule, and since the rate of rotational diffusion of a molecule is inversely proportional to its molecular weight, the change of the fluorescence Polarization signal directly reflectsthechangeinmolecularweightofthemoleculein the process of binding or dissociation [ 57, 58 ]. Specifically, when a small molecule fluorescent tracer binds to a large molecule( such as antibody), its rotational speed decreases, leading to an increase in the Polarization of the emitted light; conversely, a decrease in molecular weight during processes such as enzymatic cleavage will manifest itself as a decrease in Polarization [ 59 ]. The system employs a ratiometric measurement mode to eliminate common-mode noise such as light source fluctuations by detecting the ratio of fluorescence intensity in parallel and perpendicular directions( I k / I \). Especially when coupled with quench-free reporter probes, it not only reduces the cost of detection, but also effectively suppresses the interference of ambient light and electronic noise, and significantly improves the stability and reliability of the detection [ 60 ]. This quantitative relationship enables fluorescence Polarization techniques to monitor the dynamics of biomolecular interactions in real time, and the binding ratios can be extrapolated from the changes in the Polarization values. Based on the above advantages, fluorescence Polarization immunoassay has been widely used in various fields: in basic research for protein-ligand interaction analysis and enzyme kinetic studies [ 61 – 63 ]; in drug discovery and development for the primary screening stage of high-throughput screening [ 64 ]; and in clinical diagnostics for rapid detection of infectious disease markers [ 65 ]. In addition, the method has shown unique value in biosafety monitoring( such as toxin detection) [ 66 ], as it is particularly suited for rapid detection in the field due to the absence of a separation step. With advances in polarised optical components and fluorescent probe technology, the range of applications for this technique continues to expand.
In the field of environmental pollutant detection, FPIA has become an ideal choice for rapid screening because of its simple operation and no complex pre-treatment. Raysyan et al. [ 67 ] successfully optimized the FPIA method and detected diclofenac( DCF) in wastewater by designing novel fluorescent markers( including homologous and heterologous structures), as shown in Figure 5a. Research has shown that the FPIA system using DCF-Ahx-AMF markers exhibits excellent selectivity and sensitivity, with detection performance highly consistent with LC-MS / MS methods, and analysis time significantly shorter than traditional ELISA. This work further confirms the low cross reactivity of FPIA to DCF metabolites, highlighting its reliability in complex matrices such as wastewater, providing an important technological paradigm for efficient monitoring of small molecule pollutants in the environment, and promoting the application and development of FPIA in the field of environmental analysis.
FPIA also has significant advantages in the field of food safety detection. A FPIA-based homogeneous assay was developed by Duan et al. [ 68 ] for the rapid determination