The Fluorescence Assay For The Detection Of MicroRNA-21 In Plasma Exosomes Based On Molybdenum Disulfide Nanosheets

Lung cancer is the main cause of cancer death in the world.Although some progresses have been made in the diagnosis and treatment of lung cancer,the overall5-year survival rate of lung cancer patients range from 10%to 15%,and the prognosis is poor.Therefore,there is an urgent need for new tumor markers with higher specificity to assist the diagnosis and treatment of lung cancer.At present,liquid biopsy is a new field of cancer research,which can provide effective information about genetic background in diagnosis.Exosomes including RNA,DNA and proteins have been proved to participate in the occurrence and development of cancer.Exosomes are spherical nano vesicles with the diameter of 40 nm～160 nm and the density of 1.13 g/m L～1.19 g/m L,which exist in most of bio-fluids.Additionally,the microRNA involved in exosomes is one of the most potential tumor markers.Therefore,to achieve the early,highly sensitive and non-invasive detection of lung cancer,this study aims to establish a new method to detect microRNA in exosomes.In this paper,MoS₂ nanosheets were combined with hybrid chain reaction and duplex-specific nuclease to establish the rapid detection system of microRNA.The proposed method was also applied to the detection of microRNA in plasma exosomes.The main work includes the following aspects:1.Detection of microRNA-21 based on the signal amplification of hybridization chain reaction and the signal quenching of molybdenum disulfide nanosheetsIn this experiment,a method for detecting microRNA-21 was established based on the excellent fluorescence quenching performance MoS₂nanosheets and signal amplification of the hybrid chain reaction.In the system,in the absence of target microRNA-21,the fluorescence of carboxyfluorescein（FAM）labeled H1 and H2（FAM-H1 and FAM-H2）were quenched by the MoS₂ nanosheets,and there was no hybrid chain reaction occurred.In the presence of target microRNA-21,the hybrid chain reaction of FAM-H1 and FAM-H2 was triggered to amplify the fluorescence signal.In this experiment,the concentration of MoS₂ nanosheets,the fluorescence quenching time,fluorescence recovery time and the ratio of FAM-H1:FAM-H2 were optimized.The standard curve for the detection of microRNA-21 was established under optimal conditions.The limit of detection was down to 20 pmol/L,and the linear range was from 0.05 nmol/L to 25.0 nmol/L.In addition,the low fluorescence intensity among the five different interference sequences microRNAs indicated that this new enzyme-free detection method had high specificity and shown good performance in distinguishing single base differences.The establishment of this method provides a new strategy for the detection of liquid biopsy,and has great potential application in the early diagnosis of lung cancer and the screening of other diseases.2.Detection of microRNA-21 based on the signal amplification of duplex-specific nuclease and the signal quenching of molybdenum disulfide nanosheetsDuplex-specific nuclease shows a strong preference cleavage for double strand DNA and DNA strands in DNA/RNA,but is not active for single strand DNA,RNA or double-stranded RNA.In this study,a rapid,highly sensitive and selective method for microRNA-21 detection was established by MoS₂ nanosheets and duplex-specific nuclease.In the range of 0.5 nmol/L～50.0 nmol/L,there was a linear relationship between the fluorescence intensity and the concentration of microRNA-21.The standard curve was y=1350.55x+1556.89,and the limit of detection was 5 pmol/L.In addition,the method had the ability to identify single base mismatch,which showd that the method has good specificity.This method takes on the high sensitivity and specificity,and could simultaneously detect multiple microRNAs.Therefore,it has great prospect for detecting microRNAs in biomedical research and clinical diagnosis.3.Extraction of plasma exosomes and detection of microRNA-21Exosomes in plasma were extracted by ultracentrifugation and characterized by nanoparticle tracking analysis,dynamic light scattering,western blot and transmission electron microscopy.Finally,the two established methods were used to detect the content of microRNA-21 in plasma exosomes.Experimental showed that the two established methods could efficiently and sensitively detect the content of microRNA-21 in plasma exosomes and could distinguish normal control from lung cancer patients.To sum up,the two analytical methods have the advantages of high sensitivity,good specificity and rapid detection of microRNA-21 in exosomes.They have potential application prospects highly sensitive and non-invasive screening of lung cancer.