Detection Of Immunoassay Proteins Using DNA-PAINT

Tumor immunotherapy is a new anti-cancer approach.Detection of proteins such as PD-L1 and CTLA-4,which are important immune checkpoint molecules,is attracting more and more attention as they play key roles in immunotherapy.However,due to the tiny size of these proteins,they cannot be visualized and studied at the single-molecule level by conventional optical microscopy.Optical super-resolution imaging technology breaks through the diffraction limit,making it possible to study proteins at the molecular level.In this paper,two types of breast cancer cells(MDA-MB-231 and MCF-7)were used as models for the optical super-resolution imaging technology of DNA-PAINT(DNA points accumulation for imaging in nanoscale topography).High-resolution imaging and quantitative detection of various tumor protein markers including PD-L1 and CTLA-4 have been achieved.The main findings include:The expression levels of PD-L1 protein on MDA-MB-231 and MCF-7 cell membranes and exosomes were verified by fluorescence imaging.First,exosomes were extracted from breast cancer cell culture medium by ultracentrifugation,and the expression of PD-L1 protein in breast cancer exosomes was verified by two-color fluorescence co-localization method.Subsequently,the distribution and expression level of PDL1 protein were analyzed by immunofluorescence.In the experiment,PD-L1 on the cell surface was labeled with PD-L1 antibody modified with Alexa Fluor 642,and widefield fluorescence images were obtained.By comparing the fluorescence intensity,it can be concluded that MDA-MB-231 cells overexpress PD-L1,and MCF-7 cells have a low expression level of PD-L1.However,this method can only obtain the relative expression levels of PD-L1 in two types of cells,and cannot accurately quantify PD-L1.Furthermore,single molecule localization microscopy(SMLM)was used to qualitatively and quantitatively analyze the biomarkers on the surface of breast cancer cells.High-precision spatial localization and quantification of PD-L1 on breast cancer cell membrane was accurately performed by DNA-PAINT and quantitative PAINT(q PAINT).The expression levels of PD-L1 in cells before and after gene silencing treatment were successfully detected by q PAINT.In addition,five biomarkers(PD-L1,CTLA-4,Ep CAM,EGFR,HER2)on the membrane of breast cancer cells were subjected to super-resolution imaging and quantification analysis using Exchange-PAINT and q PAINT.The quantitative results were analyzed by a machine learning algorithm,which enabled the classification of MDA-MB-231 and MCF-7 cells.This superresolution quantitative analysis method has great potential in cancer diagnosis and immunotherapy.