Construction Of Rare Earth Upconversion Nano-Probes And Time-Resolved Imaging Application Of Virus Tracking

Viruses are one of the most infectious pathogens that cause human diseases,and it is crucial to study the virus infection mechanisms.Viral visualization tracking is the most intuitive method to explore the virus infection mechanism,and many fluorescent probes have been applied to viral labeling for virus tracking research.However,traditional probes such as fluorescent proteins,organic dyes and quantum dots are mostly used for in vitro research.Due to the absorption and scattering of excitation light by biological tissues,the in vivo imaging effect is not ideal when used.Rare earth doped upconversion nanoparticles（UCNP）have the advantages of long fluorescence lifetime,good photostability,deep penetration depth,and low background fluorescence interference.Based on this,we intended to construct a biological probe based on UCNP,and use the probe to label the virus and trace the labeled virus in vitro and in vivo.The detailed work contents are as follow:1.Construction of rare earth doped upconversion nano-fluorescent probes and labeling of viruses.We synthesized three different UCNPsβ-Na Gd F4:10%Yb,1%Er,Na YF:Gd,Yb,Er@Na YF₄ and Cubic-Na Yb F₄:Er@4Na YF₄.The fluorescence lifetimes of the three UCNPs were 422μs,128μs,and 652μs respectively,which were significantly longer than traditional fluorescent probes（ns level）.Compared with traditional organic dye fluorescent probes,it was found that the photostability of UCNP was obviously better,and its luminescence intensity did not change significantly after12hours of continuous illumination.Different labeling strategies were used to label the envelope,capsid and nucleic acid of the virus by UCNP,and the labeling efficiency of different components of the virus with UCNP could reach more than 90%,without significant effect on the virus activity.The above results indicated that UCNPHad excellent optical properties as a fluorescent probe and could be used for virus labeling,providing a new strategy for virus labeling.2.Rare earth doped upconversion nano-fluorescent probe labeled virus for in vitro tracking.Taking influenza virus（IAV）as an example,UCNP was used to label the envelope of IAV,UCNP labeled IAV was co-cultured with canine kidney cells（MDCK）,and the trajectory of the virus was observed under a confocal microscope.The results proved that IAV entered the cell through endocytosis and moved from the cell membrane to the nucleus.In order to explore the intracellular transport mechanism of influenza virus,specific fluorescent probes were used to label the lysosome and microtubule structures of MDCK cells to study the interaction of IAV with them.The results showed that IAV would enter the host lysosome after entering the host cell,and released the nucleic acid by means of the lysosome.In addition,virus movement along microtubules could be observed in MDCK cells.And when microtubule inhibitors were used to inhibite the microtubule function of MDCK cells,the virus particles stopped moving in the cells,which proved that the transport of IAV in host cells was microtubule-dependent.This chapter showed that UCNP could be used for long-term virus tracking in vitro,laying the foundation for subsequent in vivo applications.3.In vivo tracking of rare earth doped upconversion nano-fluorescent probes labeled virus.In this chapter,IAV was double-labeled by UCNP and Cy5,and the distribution of the labeled IAV after infecting mice through the respiratory tract for 6hours was observed.The results showed that compared with the traditional fluorescent probe Cy5,the UCNP-labeled virus could provide imaging results with lower background fluorescence.In addition,according to the characteristics of long fluorescence lifetime of UCNPs,time-gated imaging technique was used to reduce the background fluorescence signal during the imaging process,increasing the signal-to-noise ratio by nearly 3 times.The mice were infected with pre-labeled influenza virus,or in situ labeling method were used to track the distribution of IAV in mice.At the same time,time-resolved imaging technology was used to simultaneously Image the distribution of the two viruses at the same infection site.This chapter showed that the labeling of viruses by UCNP could not only be applied to in vitro research,but also suitable for In vivo virus tracking research,which provided a general technical method for in vivo virus infection mechanism research.4.The application of rare earth doped upconversion nano-fluorescent probes in other microbial labeling.In addition to viruses,we tried to use UCNP to label other microorganisms.We delivered the UCNP-labeled luciferase-encoding engineered bacteria into mice by gavage.By collecting and analyzing the chemiluminescence signal and UCNP fluorescence signal,we found that UCNP could provide low-background fluorescence imaging results of engineered bacteria in the intestine,and showed more clearly distribution of engineered bacteria.In addition,a breast cancer tumor model was constructed,UNCP was used to label the engineered bacteria,and the anaerobic targeting of the engineered bacteria was verified by imaging the tumor site.In addition,UCNPs with different fluorescence lifetimes was modified PD-L1 antibody with and was used to label optogenetic engineering bacteria that secreted TNFα,and time-resolved imaging was performed at the tumor site 24hours after intravenous injection.It showed that both the PD-L1 antibody and the engineered bacteria reached the tumor site.The above shows that UCNP could be applied to the labeling of microorganisms and was a universal fluorescent probe,which broadened the application range of rare earth upconversion nano-fluorescent probes.