Multiplex Detection Of Microrna Biomarkers For Gastric Cancer Diagnosis Based On Aggregation-Induced Emission Luminogens Encoded Microspheres

The incidence and mortality of gastric cancer rank the fifth and the third respectively,among all malignant tumors all over the world.The 5-year survival rate of advanced gastric cancer is less than 20%.Therefore,diagnosis and treatment at an early stage is the most critical factor in reducing the mortality of gastric cancer.In recent years,a large number of miRNAs related to the occurrence and development of gastric cancer have been screened from cell-free fluids such as plasma or urine.Given the challenges to detect miRNAs with low abundance and instability,and the progress of suspension array technology,the purpose of this article is to build a multiplex detection platform based on suspension array for simultaneous quantitative measurement of miRNAs.Firstly,AIEgens-encoded microspheres(HPSMBs)were prepared by incorporating an AIEgen(hexaphenylsilole,HPS)into polystyrene-comaleic anhydride(PSMA)microspheres through facile shirasu porous glass(SPG)membrane emulsification method.HPSMBs owned ideal properties such as high and uniform fluorescence signal,high dispersion,high uniformity and good environmental resistance.By changing the amount of HPS particles in microspheres,the fluorescence barcodes of HPSMBs could be strictly controlled.Next,miRNA multiplex detection protocol based on suspension array was proposed,and general design principles and methods for detection probes were also given.Based on above methods,three sets of detection probes were designed for three selected target miRNAs.The results of homology analysis and electrophoresis test showed that the designed detection probes had no similarities with the target miRNAs,and the hybridization conditions of our detection protocol were suitable for all of probes.Finally,based on the as-prepared HPSMBs and designed detection probes,a novel suspension array platform was established and optimized to achieve multiple detections of let-7b-5p,mi R-16-5p and mi R-19b-3p,which are associated with gastric cancer.Triplex detection assay showed high sensitivity with LOD of 0.43,0.76 and 0.60 n M for the three miRNAs respectively.The results of specificity tests based on three target miRNAs and two interference sequences let-7a-5p and mi R-23a-3p showed that these multiplex assays could achieve excellent specificity even to recognize single base mismatch.The approach could be extended to simultaneous detection of more target miRNAs by designing specific detection probes and increasing the number of fluorescent barcodes.We could foresee it holds great potential in the future laboratory research and clinical applications due to its flexibility,strong multiplexed ability and good detection performance.