Synthesis Of Novel Organic Small Molecular Fluorescent Probes And Their Application In Imaging Of Metal Ions In Plants

Heavy metal ion pollutants in water and soil seriously affect the growth and development of crops,resulting in crop yield reduction or even extinction.At the same time,heavy metal ions will accumulate in grain through biological enrichment,causing food safety problems and endangering human health.Therefore,real-time and rapid detection of heavy metal ion content in farmland environment and crops is of vital importance.Compared with traditional detection methods that rely on large instruments and equipment,fluorescence spectrometry is highly sensitive,simple to operate,and has high visualization degree.It can detect heavy metal ion pollutants quickly and accurately,and has potential applications in the visual tracer in biological tissues.Therefore,it has attracted much attention in the past decades.Although fluorescent probes have been widely used in environmental and food detection,the limitations of imaging in plants are still great,mainly due to the complex microenvironment of plant tissues,especially leaves,strong background interference of chlorophyll and other pigments,poor penetration of fiber tissues and other factors.Therefore,the development of fluorescent probes with high selectivity,strong penetration and excellent anti-interference ability is an effective way to achieve high resolution metal ion detection of plant body.In this paper,we developed three single-photon and two-photon fluorescence probes,and confirmed the advantages of two-photon fluorescence probes in plant imaging through in vitro experiments and plant imaging.The specific research contents are as follows:（1）Firstly,we constructed heavy metal ion enhanced single photon fluorescence probe P1 using commercial 7-nitrobenz-2-oxazo-1,3-diazole（NBD）as fluorophore and 8-hydroxyquinoline（8-HQ）as recognition group.The simple synthesis of the probe provides a method for the rapid construction of fluorescent probe for the detection of me tal ions.The probe can realize the rapid detection of metal ions in vitro and provide a wa y for the detection of heavy metal ions in plants.（2）Next,in order to solve the problem of metal ion selectivity of the fluorescent probe,sulfur atom（S）with stronger affinity to mercury ion(Hg²⁺)was introduced into the recognition group of the probe according to the soft-hard acid-base theory,and 8-mertoquinoline（8-SQ）was substituted for 8-hydroxyquinoline to synthesize the single photon fluorescent probe P2.As expected,in vitro experiments,probe P2 only selectively recognizes Hg²⁺,but has a weak response to other metal ions including Ag⁺ions.Modification of the recognition group solves the selectivity problem well.P2 also achieves imaging in plant roots.It provides a method for the detection of heavy metal ions in plants.However,the resolution of single photon fluorescence probe was low in plant imaging,and it could not penetrate the leaf directly.（3）Considering that the two-photon fluorescence probe can be excited by near infrared light with long wavelength and strong penetrating ability and anti-interference ability,we take fluorene with large two-photon cross section as fluorophobe,introduce benzothiophene to improve electron conduction ability,improve water solubility of dioligonucleotide（ethylene glycol）chain,and use DPA as metal ion recognition group.A two-photon fluorescence probe LJTP1 was developed.In vitro tests show that the probe has good selectivity for Cu²⁺and S^2-,with a dual fluorescence"On-Off-On"response.The detection limits of Cu²⁺and S^2-were 0.775 and 0.486μM,respectively.In addition,probe LJTP1 can be used for non-invasive fluorescence imaging detection of Cu²⁺and S^2-in Arabidopsis leaf and root tissues under two-photon excitation at 810nm.At the same time,the cellular substructure of Arabidopsis thaliana,such as resting center and stomata,as well as morphological changes under heavy metal ion stress can be shown in high resolution.This work provides a new idea for the development of fluorescent probes for plant imaging.