Design,Synthesis And Application Of A Novel Formaldehyde Fluorescent Probe-Dual Emission Detection And Imaging Studies In Food And In Vivo

Formaldehyde(FA)was a highly toxic gas.When inhaled in high concentrations,FA could cause damage to the human nervous system,immune system and respiratory system.However,FA had the functions of antiseptic,sterilization,and preservation,and could be used in the fields of food and environment.Illegal addition or use of FA in food caused FA residues to exceed the standard.Once the human body consumes food containing excessive FA,it caused some diseases.Therefore,the development of low-toxicity,high-sensitivity and high-selectivity detection methods to detect FA in food and the environment was of great significance to food safety and human health.In this paper,the current FA fluorescent probes had the problems of ultraviolet-visible excitation,single signal output and small stokes shift,and propose methods to reduce these problems by using two-photon ratio fluorescent probes for detection and imaging analysis.The two-photon ratio fluorescent probe was excited by two photons of near infrared light(NIR)and had two mutually corrected fluorescence emission signals,which could reduce photobleaching,tissue autofluorescence interference,tissue penetration depth in detection and imaging analysis Shallow(<100 μm)and other restrictions could also reduce the interference caused by instrument efficiency,probe molecule concentration and environmental factors.The specific contents of this thesis were as follows:1)Based on the fluorescence resonance energy transfer(FRET)mechanism,using naphthalene derivatives as the energy transfer donor,naphthalimide derivatives as the energy transfer acceptor.The two-photon ratio FA fluorescent probes CmNp-CHO and NpFA with two different structures were designed.In this structure,the hydrazine group was not only the active site of the selective chemical response of FA,but also the quenching group of the acceptor fluorescence.its structure was characterized by mass spectroscopy,hydrogen spectroscopy(1H NMR)and carbon spectroscopy(13C NMR);2)Secondly,the spectral properties of the two probes(CmNp-CHO and NpFA)and FA response were studied with an ultraviolet spectrophotometer and a fluorescence spectrophotometer,and their selectivity and optimal pH were investigated.After the two probes were respectively incubated with FA,the results showed that the probes had specific chemical responses to FA,and their fluorescence emission had a typical ratio emission characteristic.The fluorescence emission signal in the blue band gradually decreased and the new fluorescence signal gradually appeared in the yellow band.The solution changed from blue fluorescence to yellow fluorescence.It could be seen from the fluorescence spectrum that the two probes had large Stokes shifts of 165 nm and 175 nm respectively,and the distance between the two emission peaks were 124 nm and 140 nm respectively,which was very beneficial for ratio imaging.It could reduce or eliminate the false signal by the mutual interference of light between the two channels during imaging analysis.More importantly,with the gradual increase of FA concentration,the ratio between the fluorescence intensity of the probe in the yellow band and the intensity of the blue band increased by 4 times(I550/I426)and 5 times(I550/I410)respectively.When the concentration of FA was(0-1.0)μM,the probes CmNp-CHO and NpFA both had a good linear detection range,with lower detection limits of 8.3 nM and 5.8 nM,respectively;3)Finally,the detection of formaldehyde content in food by CmNp-CHO and NpFA and the fluorescence ratio imaging analysis of formaldehyde in biological models were investigated.Comparing the determination of FA concentration in real food samples with the national standard method for determination of formaldehyde in aquatic products,the relative deviations of FA detection by CmNp-CHO and NpFA were<4%and<2%,respectively.Moreover,the two-photon laser confocal microscope(TPM)was used to analyze the fluorescence imaging of FA in tissues and zebrafish.It was found that both probes had good tissue penetration and staining capabilities.There was no light interaction between the two channels and interference to obtain high-resolution blue or yellow ratio imaging(blue fluorescent signal and yellow fluorescent signal).Here,we predict that the detection of formaldehyde in food and organisms with two probes will help food safety and biomedicine detection research.