Time-gated And Upconversion Multiplexed Detection Fluorescence Probes:Synthesis,Application

Optical imaging has become an important technique for biomedical research because of its non-invasive,real-time and high-amplification properties.At present,it has been proved that various kinds of luminescent materials can be used for optical bioimaging such as fluorescent protein^[1],organic dyes^[2,3],metal complexes^[4],gold nanorods^[5]semiconductor quantum dots?QD?^[6,7].The ideal luminescent materials for bioimaging should have excellent luminous efficiency,high physical and chemical stability,low toxicity and so on.Upconversion refers to the absorption of two or more low-energy photons,the long-wave excition into short-wave radiation,compared with the traditional organic fluorescent dyes and quantum dots,upconversion nanoparticles?UCNPs?with sharp adjustable emission peak,long fluorescent lifetime,high chemical stability,low cytotoxicity,large anti-Stokes shift,deep tissue penetration,good biocompatibility,low background fluorescence and so on,so that they have potential applications for living imaging,cell labeling,drug delivery,data storage,sensors,resonance energy transfer?FRET?,biosensing,photodynamic therapy.Timed-gated fluorescence analysis is a special fluorescence assay developed on the basis of fluorescence analysis.Rare earth ions with unique fluorescence properties(such as Eu³⁺,Tb³⁺,Sm³⁺and so on)and its chelate as a tracer to mark the antigen or antibody as a high sensitivity,long fluorescence life,low background fluorescence interference fluorescence analysis method,in clinical detection,nucleic acid hybridization analysis and biological imaging analysis which has been widely used.In this paper,an upconversion time-gated multi-channel fluorescent probe was designed and fabricated.On the basis of fully characterizing the fluorescence properties of the fluorescent probe,the application method of fluorescence probe in time-gated biochemical analysis was established.In this paper,a 808 nm excited NaYF₄:Yb³⁺,Nd³⁺,Er³⁺upconversion nanoparticles and a four-carbitan-?-diketone ligand BHHBCB with chlorosulfonyl-reactive groups were designed and synthesized as a time-gated and upconvertion multichannel fluorescent probe.Herein NaYF₄:Yb³⁺,Nd³⁺,Er³⁺upconversion nanoparticles,APTES-BHHBCB-Eu³⁺complexare covalently bonded in silanization reagent 3-aminopropyltriethoxysilane?APTES?,the UV-light,near-infrared light excitation and long fluorescent luminescence life of the core-shell fluorescent probe can be prepared in the microemulsion system.The fluorescent probes were incubated with transferrin?TF?and folic acid?FA?for time-gated and upconversion microscopic imaging of HeLa cell respectively,and demonstrated that the application of specific identification of TF-transferrin receptor?TFR?and FA-folate receptor?FR?on surface of the cancer cell can be implemented by time-gated and upconversion fluorescent imaging.