BSA Nanospheres Based Nanoprobe For Super-resolution Imaging

In recent years,the application of super-resolution microscopy in biological imaging has attracted the attention of many researchers.As One of the super-resolution microscopy,single-molecule localization microscopy(SMLM)is of great interest because of its excellent spatial resolution.However,there is a necessary condition for the fluorophore,which is used for SMLM imaging.That is,the random switching of the fluorescent signal between the fluorescent("on")and dark("off")states(called "blinking").Fluorescent blinking behavior is critical to improving image quality.Therefore,it is urgent for researchers to explore new fluorescent probes with excellent fluorescencent blinking properties.In this paper,based on bovine serum albumin(BSA)nanospheres,a super blinking composite nanoprobe that can be used for SMLM imaging is proposed.First,in the preparation of BSA nanospheres,we prepared a kind of BSA composite nanospheres with excellent fluorescent blinking properties by incorporation of Alexa Flour dyes,which can be called BSA@Alexa594(or BSA@Alexa647).This composite BSA nanospheres obviously improved the fluorsecent blinking properties of Alexa Fluor dyes.Escapially for BSA@Alexa594,it performed high blinking frequency,long blinking time and many “ on-off ” circles Secondly,BSA nanospheres,as a kind of protein nanospheres,are rich in amino and carboxyl groups.In the existence of natural amino and carboxyl groups,modifying targeting ligands(such as antibodies)on the surface of BSA nanospheres can be very easy,because there is no need of additional functional group modification.Therefore,we first modified the BSA@Alexa594 with Anti-HER2 antibody to prapare BSA@Alexa594-Anti HER2 probe,and successfully applied it to target breast cancer cells.Then,under a confocal laser scanning microscope,the BSA@Alexa594-Anti HER2 probe was observed to enter the tumor cells by HER2 receptor-mediated endocytosis,and its excellent targeting properties to breast cancer cells was verified.At the same time,SMLM imaging of probes mediated by receptor-mediated endocytosis was performed using SMLM microscopy.The detailed distribution of endocytosed probes in the cells was further observed within the diffraction limit(<200 nm).Fianlly The position of two probes whose diatance are less than 100 nm was successfully distinguished.Meanwhile,this paper also expand the potential of BSA@Alexa594-Anti HER2 probe used for the SMLM imaging of tumor cell-derived exosomes.The prepared BSA@Alexa594-Anti HER2 probe labeled the exosomes through the HER2 protein on the surface of SKBR3 cell exosome membrane.Through the SMLM imaging,we found that the super blinking BSA nanoprobe can be used for exosomes,whose size is under 200 nm.In summary,the main work of this paper is preparing a kind of super-resolution imaging probe with excellent fluorescent blinking behavior based on BSA nanospheres,and praparing various bioprobes with specificity by modifying the surface of nanospheres with antibodies.Finally,those BSA nanospheres bioprobes were used in the super-resolution imaging of breast cancer cells,and tumor-derived exosomes.