Construction Of Dual-emission Fluorescent Probes Based On Non-conjugated Fluorescent Molecules

Fluorescent probes have the advantages of high sensitivity,good selectivity and easy operation,and can perform in situ,real-time and nondestructive analysis of targets.They are widely used for environmental monitoring,food analysis,biochemical analysis,drug detection,disease diagnosis and bioimaging.However,the evaluation of target analytes using only a single emission wavelength can be affected by many factors unrelated to the analyte,including photobleaching of the fluorophore,light scattering due to the sample matrix,sources of excitation fluctuations,variations in the local concentration of the probe,and the influence of the microenvironment surrounding the probe.These factors can interfere with the quantification of analytes and reduce the reliability of the probe.Fluorescent probes with two or more emission wavelengths can account for environmental changes by calculating the ratio between fluorescence intensities at different wavelengths.This ratio measurement provides an effective internal reference and greatly improves the sensitivity of the probe.Although conjugated fluorescent probes are widely used in various fields due to their excellent fluorescence properties,they also have non-negligible disadvantages,such as poor water solubility,high toxicity,and high difficulty in synthesis.In contrast,non-conjugated fluorescent molecules have remarkable photostability,excellent solubility in water,low toxicity and good biocompatibility,etc.,and are widely used in cell imaging,ion probes,drug delivery and luminescence devices.In this paper,dual-emission fluorescent probes were constructed using non-conjugated fluorescent molecules and fluorescent dye/gold nanoclusters connected by covalent and simple physical mixing,respectively.To summarize,Non-conjugated fluorescent molecules were prepared from polycarboxylic acid and polyamine molecules by hydrothermal and chloride methods,and four dual-emission fluorescent probes were prepared in combination with fluorescein isothiocyanate（FITC）,rhodamine B（RhB）and gold nanoclusters（AuNCs）.The structures and fluorescence properties of these fluorescent substances were also systematically analyzed,and their applications in the field of ion detection were explored.The specific research contents of this paper are as follows:1.Fluorescent polymer dots were prepared as dual-emission fluorescent probes f-PDs by a one-step hydrothermal reaction in aqueous phase using citric acid（CA）and polyethyleneimine（PEI）and fluorescein isothiocyanate as raw materials.f-PDs were successfully synthesized by covalent bonding of non-conjugated polymer dots with fluorescein isothiocyanate,as demonstrated by structural characterization·at.Two·emission peaks in the fluorescence emission spectrum of f-PDs one originates from a non-conjugated fluorescent molecule and the other from isothiocyanate fluorescein.Meanwhile,f-PDs can rapidly and sensitively detect Cu²⁺and ClO^-in aqueous solution.The analytical principle is that Cu²⁺binds to the polyethyleneimine part of f-PDs to form a complex,while ClO^-oxidizes the hydroxyl group on the surface of f-PDs with detection limits of 6.7 nM and 14.0 nM,respectively.In this chapter,the non-conjugated fluorescent polymer dots are connected with fluorescein isothiocyanate by covalent bonding.The advantage is that the excitation wavelength of fluorescein isothiocyanate and non-conjugated polymer dots are similar,which provides the possibility of constructing dual-emission fluorescent probes;meanwhile,the introduction of non-conjugated polymer dots improves the water solubility of fluorescein isothiocyanate and expands the application conditions;furthermore,the construction of dual-emission fluorescent probes makes the color change of the aqueous solution can be captured by the naked eye.The disadvantage is that the conjugated structure of fluorescein isothiocyanate itself has poor water solubility,and there may be a risk of contamination during the preparation process,and the detection limit is not as high as desired.2.Three dual-emission fluorescent probes were constructed by physically mixing non-conjugated fluorescent molecules with fluorescent components.The first one is a dual-emission fluorescent probe C-PDA&AuNCsFluorescent small molecules were first synthesized by the chloride method using citric acid and 1,3-propanediamine（PDA）as raw materials,and then the fluorescent polymer dots C-PDA were prepared by continuing hydrothermal synthesis of the small molecules and mixing C-PDA with gold nanoclusters.The results of FT-IR and XPS tests showed the successful preparation of C-PDA.The dual-emission fluorescent probe was applied to ion detection and found to be able to achieve a multi-ion response.Compared with other ions,the dual-emission fluorescence probe showed a more obvious and sensitive response to ClO^-with a detection limit of 1.75 u M,and also a significant fluorescence burst to Hg²⁺with a detection limit of14.68 u M.The second one is CA-AEP&RhB dual-emission fluorescent probesCA-AEP was first synthesized with citric acid and N-aminoethylpiperazine and constructed by simple mixing with rhodamine B.CA-AFP&RhB can be used as sensitive sensors for the detection of Hg²⁺and ClO^-in aqueous phase with detection limits of 27.3 nM and 15.7 nM,respectively.The third one is CA-AEP&AuNCs dual-emission fluorescent probesIt was prepared by mixing the above CA-AEP with gold nanoclusters.The dual-emission fluorescent probe was applied to ion detection and found to be able to respond to Hg²⁺,Cu²⁺and ClO^-.The ratio of the two emission peaks of its fluorescence spectrum showed a linear relationship with the concentration of ClO^-,and the detection limit was 21.6 nM.The responses to Hg²⁺and Cu²⁺were nonlinear and could be detected qualitatively.In this chapter,three dual-emission fluorescent probes,C-PDA&AuNCs,CA-AEP&RhB and CA-AEP&AuNCs,were constructed using a simple hybrid approach,which has the advantages of convenience and does not change the intrinsic properties of the original chromophores.The non-conjugated fluorescent molecules used in this paper have similar excitation wavelengths with rhodamine B and gold nanoclusters,and the positions of the emission peaks do not interfere with each other,which also provides the possibility of constructing dual-emission fluorescent probes.By comparison,it was found that the construction of dual-emission fluorescent probes improved the detection sensitivity and the color change could be observed with the naked eye,which has the potential to be applied in the field of bioimaging.The drawback is that the application is limited and no more in-depth study has been done in the field of bioimaging.In conclusion,in this paper,a variety of non-conjugated fluorescent molecules were first prepared,while the excitation wavelengths between fluorescent molecules and fluorescent components are similar,and the emission peaks do not interfere with each other and produce fluorescence bursts for specific ions,which provides the possibility of constructing dual-emission fluorescent probes.The fluorescent molecules were then chemically or physically combined with fluorescein fluorescein isothiocyanate,rhodamine B and gold nanoclusters to obtain several dual-emission fluorescent probes,which have excellent applications in the field of ion detection.