Construction Of A Novel Biosensor Based On Nucleic Acid Isothermal Amplification System For The Diagnosis Of Pancreatic Cancer

In recent years,the five-year relative survival rate of pancreatic cancer is less than 10 percent due to a lack of early symptoms and inadequate diagnostic techniques.Therefore,it is urgent to develop a highly sensitive detection strategy for pancreatic cancer.The isothermal amplification reaction of nucleic acid shows excellent advantages due to its high amplification efficiency and no need for accurate temperature control instruments.In this study,we designed two isothermal amplification techniques(hybridization chain reaction and rolling circle amplification)to detect pancreatic cancer cells or pancreatic cancer exosomes detection with the help of fluorescence or UV spectrophotometer for signal output,it has good application prospect in the detection of pancreatic cancer patients.The specific research contents are as follows:1.In this study,the fluorescence quenching and magnetic separation abilities of Fe₃O₄@DOP NPs,as well as the signal amplification of hybrid chain reaction(HCR),were combined to detect overexpressed MUC1 protein in pancreatic cancer cells.FAM fluorescence was modified with HCR substrate(H1 and H2)and the detection limit of three different pancreatic cancer cell lines was 21-41 cells/m L.The fluorescence intensity of pancreatic cancer cells was significantly higher than those of normal pancreatic cells(HPDE-C7)and hepatocarcinoma cells(Hep G-2),therefore,the strategy has good selectivity.Moreover,the HCR amplification system was used to identify the cancer cells on pancreatic tissue,and the results of our strategy in MUC1 protein detection was consistent with the results obtained by traditional immunohistochemistry(IHC)method,indicating that our strategy has potential practical application value in clinical application.2.In this study,we developed a simple,sensitive,and rapid exosome diagnostic method that facilitates early diagnosis and prognostic tracking of cancer.we take advantage of the facile adaptability of aptamers to design an exosome quantitative method,which converts an exosome capture event to nucleic acid detection.First,CD63 aptamer/C-DNA linked to magnetic beads acts as the capture probe.In the presence of exosomes,aptamers identify and combine with exosomes,releasing c-DNA probes as primers to initiate rolling circle amplification(RCA).we propose a strategy that utilizes the electrostatic attraction between RCA products and positively charged silver nanoparticles(Ag NPs)to induce aggregation of Ag NPs.Thus,the concentration of exosomes can be determined by UV-Vis spectra or naked eye of the Ag NPs aggregation.The detection limit of exosomes by the spectrophotometry was 4×10³ particles/m L.Furthermore,this method can also achieve sensitive detection of exosomes in the serum samples.3.Exosomes have attracted more and more attention in the early diagnosis of diseases due to their unique biological properties.However,traditional analysis methods(PCR or ELISA)can not distinguish between exosomes from different sources.Therefore,we developed a fluorescence biosensing platform based on upconversion nanoparticles(UCNPs)to detect the pancreatic cancer exosomes overexpressed epidermal growth factor receptor(EGFR)and epithelial cell adhesion molecule(Epcam).In this experiment,exosomes were captured by magnetic beads that modified with CD63 antibody,then the captured exosomes was specifically recognized by multiple DNA aptamers(EGFR/Epcam),followed by rolling circle amplification to generate localized fluorescent signals(UCNPs-DNA).In this strategy,we obtained the detection limit of exosomes as low as 3×10³particles/m L.In addition,the method can be used to determine the existence of exosomes by the aggregation of RCA products with the magnetic beads.