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needs of consumer health protection, but also provide technical support for the sustainable development of agriculture and the assessment of compliance with international trade standards [ 13 ]. In the field of environmental monitoring, the new optical biosensor has successfully realized highthroughput and real-time monitoring of water pollutants( such as organophosphorus pesticides), and its detection performance is significantly better than the traditional enzyme inhibition method [ 14, 15 ].
As a cornerstone method in the field of optical biosensing, fluorescence detection technology occupies an irreplaceable position in life science research and clinical diagnosis due to its excellent sensitivity, excellent molecular specificity and diverse signal transduction mechanisms [ 16 ] Through the coordinated development of multi-dimensional detection strategies such as fluorescence labeling technology, fluorescence lifetime imaging( FLIM), fluorescence polarization immunoassay( FPIA) and fluorescence resonance energy transfer( FRET), the technology system has built biological detection, environmental detection and other platforms. Among them, the development of new fluorescent markers such as near-infrared fluorophores and ratiometric probes has significantly improved the selectivity and signal-to-noise ratio of detection [ 17, 18 ]; Fluorescence lifetime imaging technology effectively overcomes autofluorescence interference in complex biological samples by resolving nanosecond fluorescence lifetime differences [ 19, 20 ]; Fluorescence polarization technology uses the dynamic changes of molecular rotational relaxation time to provide a unique analytical perspective for the study of molecular interactions [ 21 ]; Owing to its exceptional sensitivity to nanoscale proximity( 1 – 10 nm), fluorescence resonance energy transfer serves as a powerful“ molecular ruler”. This capability makes it an indispensable technique for revealing protein-protein interactions and monitoring nucleic acid conformational changes in real time [ 22 ], and is in great demand in emerging fields such as genomics and proteomics [ 23 ]. These technical advantages enable fluorescence detection methods to achieve rapid, highthroughput, ultra-high sensitivity and specific detection, which perfectly fits the urgent needs of modern genomics and proteomics research for precise analytical tools [ 24 ]. Multi-color fluorescence detection enables the simultaneous detection of multiple biomarkers for a more comprehensive understanding of the condition, leading to more accurate medical diagnosis [ 25 ]. This multiple detection ability provides more comprehensive biological information for disease diagnosis. For example, through the simultaneous detection of multiple tumor markers, the accuracy and reliability of cancer diagnosis can be significantly improved [ 26 ]. However, fluorescence technology is still limited by problems such as photobleaching and autofluorescence interference, driving the exploration of novel probes and detection modes.
2 Fluorescence detection
Fluorophores are a class of chromophores that can specifically respond to photoexcitation and emit fluorescence, and their luminescence mechanism is shown in Figure 1 [ 27 ],
Fig. 1. Energy levels and radiative transition from the excited state( S 1 / S 2) to the ground state( S 0).
where electrons jump to the singlet excited state( S 1 or higher energy level) when the ground state molecule( singlet state S 0) absorbs photons of a specific wavelength. Due to the instability of the excited state, the molecule rapidly relaxes to the lowest vibrational energy level of S 1 through nonradiative relaxation( such as internal transitions), and then returns to the ground state through radiative leaps and releases photons with energies lower than that of the excitation light, resulting in a redshift of the emission wavelength, a phenomenon known as the Stokes shift. As photosensitive compounds, fluorescent dyes have characteristic absorption( excitation spectrum) and emission spectra determined by their electronic structure. Due to their ability to efficiently convert short-wavelength light energy into long-wavelength fluorescence, fluorescent dyes have been widely used as highly sensitive optical probes in the fields of biomarking, cellular imaging and tissue detection. Building on this foundation, this technology enables the quantitative, real-time observation of biomolecules with exceptional sensitivity, utilizing a suite of techniques such as fluorescent labeling, FLIM, FPIA, and FRET.
2.1
Fluorescent labeling technique
Fluorescent labeling technique is a method for qualitative and quantitative analysis of the measured components through physical adsorption or chemical coupling of fluorescent substances with the target molecules specifically combined to form a fluorescent labeling complex. The core component of biofluorescent probes is a class of compounds that can absorb light at specific wavelengths and emit fluorescence at longer wavelengths. This technique can be traced back to the antibody fluorescence labeling studies in the 1940s [ 28, 29 ], and has now developed into an important analytical tool with significant advantages such as fast detection speed [ 30 ], high sensitivity [ 31 ], low sample consumption and non-radioactivity [ 32 ], and has been widely used in the fields of protein localization and activity detection, biomolecular interaction studies, and in vivo dynamic process monitoring. To address the poor selectivity of natural fluorescent dyes, researchers have significantly