The Novel Method For In Situ Imaging Of HER2 Protein On Breast Cancer Cell Surface Based On Proximity Effect

Breast cancer has become the most prevalent cancer in the world and the number one threat to women’s health worldwide.The incidence of breast cancer is still on the rise.Among the biomarkers used in the diagnosis of breast cancer,human epidermal growth factor receptor 2(HER2)is widely considered as the most valuable biomarker,and it is also the most important site of targeted drugs.Recent studies have shown that HER2 overexpression exists in about 30% of breast cancer patients,which has become an important indicator for clinical evaluation of malignant degree of breast cancer,prognosis and recurrence,and an important gold standard in the detection of tumor markers of breast cancer.Drugs targeting HER2 protein have become an important strategy in the treatment of breast cancer,which benefits a large number of HER2 positive patients.When HER2 protein is overexpressed,HER2 will spontaneously form homodimer or form heterodimer with other family members,and continue to be activated to initiate downstream signaling,which will directly affect the efficacy and treatment regimen of HER2 targeted therapy.Therefore,the efficient detection of HER2 protein including the expression level and dimer status of HER2 has important clinical guiding value for the early diagnosis and targeted drug therapy of breast cancer.In this paper,the organic integration of the clinical laboratory diagnostics,biology,analytical chemistry,and nano materials.Based on nucleic acid aptamer recognition technology and proximity hybrid technology,we build a series of simple and quick to detect HER2 and their status of dimers biosensor by using breast cancer HER2 protein receptor for detection model.This research mainly includes the following two parts:1 DNAzyme catalyzed tyramide depositing reaction for in situ imaging of protein status on the cell surfaceEffective characterization of protein biomarkers status on the cell surface has important value in the diagnosis and treatment of diseases.Traditional immunohistochemistry can only assess the protein expression level rather than accurately reflect their interaction and oligomerization,resulting in inevitable problems for personalized therapy.Herein,we developed a novel DNAzyme-catalyzed tyramide depositing reaction for in situ amplified imaging of membrane protein status.By using human epidermal growth factor receptor 2(HER2)as model,the binding of HER2 proteins with specific aptamers induced the formation of activated hemin/G-quadruplex(G4)DNAzyme on the cell surface to catalyze the covalent deposition of fluorescent tyramide on the membrane proteins for fluorescence imaging.The DCTDR-based imaging can conveniently characterize total HER2 expression and HER2 dimerization on the breast cancer cell surface with the application of aptamer-G4 probes and proximity aptamer-split G4 probes,respectively.The designed strategy was successfully applied to quantitatively estimate total HER2 expression and HER2 homodimer on clinical breast cancer tissue sections with high specificity and accuracy.This strategy provides a simple,pragmatic and enzyme-free toolbox to conveniently and sensitively analyze protein status in clinical samples for improving clinical research,cancer diagnostics and personalized treatment.2 Light up multiple protein dimers on cell surface based on proximity-induced fluorescence activation of DNA-templated sliver nanoclustersEfficient and multiple analysis of receptor protein dimers is highly necessary,due to their important role in the occurrence and development of cancer.Herein,we report a turn-on strategy to visualize human epidermal growth factor receptor(HER)dimers on cell surfaces.By taking advantages of specific aptamer recognition and proximity-induced fluorescence activation of DNA-templated sliver nanoclusters(DNA/Ag NCs)by guanine(G)-rich sequence,we attached the two kind of DNA/Ag NCs sequence with different fluorescence properties to the corresponding HER aptamer to form aptamer-functionalized Ag NCs probes,and attached G-rich sequence to the corresponding HER aptamer as enhancer.In the presence of protein dimers,after aptamer specific recognition and binding,it will draw the dark Ag NCs probes close to the G-rich probes and then excite corresponding fluorescence.As a result,this approach has successfully realized imaging of HER2:HER2 homodimer and HER2:HER3 heterodimer at the same time,which was provided a new idea for the simultaneous detection of multiple HER2 dimers in situ.This Ag NCs-based light up strategy provides a potential tool for further investigation of protein dimerization on cell surface,which is more conducive to the mechanism research,accurate classification and treatment of cancer.In conclusion,in this study the novel protein detection and dimer analysis system established cleverly integrates the high affinity and specificity of nucleic acid aptamers,the mechanism of DNA nano-assembly triggered by proximity effect and the unique optical properties of nanomaterials.The constructed biosensor detection method is simple,rapid and highly sensitive without the involvement of any antibody or protease,which provides a new idea for the accurate detection of protein dimer disease biomarkers,as well as a new technical support for the accurate molecular diagnosis and treatment.