Research On The Application Of Portable Device In MicroRNA-21 Detection

MicroRNAs（miRNAs）are a class of physiological and pathological status-related endogenous non-coding single-strandedRNA molecules,which can be used as biomarkers for diseases such as cancers.The point-of-care testing（POCT）of miRNAs has great potential in early diagnosis and process monitoring of diseases.MicroRNA-21（miRNA-21）is not only one of the most highly expressed miRNAs,but also plays an important role in the proliferation and apoptosis of many tumors,including lung,breast,stomach,prostate,colon,brain and esophageal cancer.In this thesis,fast and dual signal outputs detection for miRNA-21 was established by using both portable personal glucose meter（PGM）and fluorescence spectrometer.In such an assay protocol,a dual-functional hairpin structure was rationally designed to recognize miRNA-21 and serve as the carrier of the reporter adenosine monophosphate（AMP）.The hairpin structure can be specifically degraded by exonuclease T after hybridization with the target miRNA-21,releasing a large amount of AMP as the reporter.Then a smart signal conversion machinery composed of four enzymes and the corresponding substrates was employed to produce dual output signals through enzymatic cascade reactions.The machinery includes two parts:an adenosine triphosphate（ATP）generation system and a glucose consumption/NADPH production system.The produced AMP in the former step triggers the production of ATP,and subsequently the consumption of glucose and the production of NADPH,which are both proportional to the concentration of miRNA-21 and can be determined by PGM and fluorescence spectrometer,respectively.Besides,the build-in substrate-recycling mechanism achieves signal amplification of the cascade enzymatic reactions.First,the designed dual functional hairpin structure was optimized,and the subsequent experiments were carried out by using the optimized hairpin structure.The stability of the hairpin structure in the absence of miRNA-21 and the opening of the hairpin structure in the presence of miRNA-21 were proved by polyacrylamide gel electrophoresis.The principle was also confirmed by the fluorescence signal.After that,the biosensor was used to detect the miRNA-21 standard under the optimal experimental conditions.The results showed that a linear relationship can be found between the signal difference of PGM and the concentration of miRNA-21 in the range of 5～150 nmol·L^-1,and the detection limit is 3.65 nmol·L^-1.Meanwhile,two linear relationships can be found between the difference of the fluorescence intensity caused by the change of NADPH and the concentration of miRNA-21 in the range of0.04～1 nmol·L^-1 and 5～200 nmol·L^-1,respectively,and the detection limit is 0.03 nmol·L^-1.Under the same conditions,four non-target miRNAs,Cutsmart buffer solution and two single-base mismatched sequences（mismatched at the 6th and 19th nucleotide,respectively）were used to verify the specificity of the sensor,and the results showed that the hairpin sequence recognizes miRNA-21 with high specificity.Finally,this method has been successfully applied in the detection of miRNA-21-spiked serum samples,as well as the actual serum samples from cancer patients,indicating its good practicability and interferent-resistance.In summary,this thesis established fast and dual signal outputs detection for miRNA-21,which can not only be used in clinical lab for accurate determination,but also provides a POCT solution for cancer diagnosis and prognosis.