| The formation and further development of many tumors are related to the abnormal expression of micro RNAs(mi RNA),so mi RNAs have become a new type of cancer markers.Because mi RNA has the characteristics of short sequence,high sequence similarity,and low content in actual samples,it is particularly important to establish a detection method with high sensitivity and high selectivity.This thesis is based on the dual-interface biosensing platform for specific recognition-functional sensing,using anodized aluminum oxide nanochannels immobilized with oligonucleotide recognition probes as the recognition interface,and carbon nanofiber modified electrodes as the detection interface.The detection of the target mi RNA is converted to the electrochemical detection of the redox probe methylene blue that is transmitted across the channels.Based signal amplification strategies based on the G-quadruplex formation and the catalytic deposition in the nanochannels,two new methods for ultra-sensitive detection of Micro RNA-21(miR-21)have been established.The study not only can provide new ideas for electrochemical biosensing analysis,but also supply reference techniques for early diagnosis and curative effects monitoring of related diseases.The thesis is divided into three chapters,and the main research includes the following two aspects.1.Electrochemical biosensing for mi RNA based on the signal amplification strategy of the G-quadruplex formation in nanochannels.The recognition interface was prepared by immobilizing the DNA recognition probes complementary to miR-21 and rich in guanine(Guanine,G)in the nanochannels.When the target miR-21 hybridize with the DNA recognition probes,with the assist of double-strand specific nuclease,the number of Gquadruplex in the channels decreases with the increase of miR-21.The steric hindrance caused by the G-quadruplex will affect the transmission of methylene blue across the nanochannels,a carbon nanofibers modified electrode is used to monitor the change in the concentration of methylene blue to realize the electrochemical detection of miR-21.Studies have shown that the specific recognition-functional sensing dual-interface biosensing platform can detect miR-21 in a linear range of 1 a M to 10 p M,with a detection limit of 0.5a M.This method has the advantages of electrochemical label-free,low detection limit and high specificity.It can be used for the determination of miR-21 in serum.2.Electrochemical biosensing based on G-quadruplex DNAzyme catalyzed deposition in nanochannels.The DNA recognition probe 1,which is partially complementary to miR-21,is immobilized in the nanochannels to form the recognition interface,and a porous carbon nanofibers modified electrode is used as the detection interface.In the analysis,the target miR-21 forms a DNA-RNA double strand after co-incubation with DNA recognition probe2,while the unhybridized G-rich sequence forms a G-quadruplex DNAzyme with hemin.Gquadruplex DNAzyme can catalyze the oxidation of 4-chloro-1-naphthol and thus formation of insoluble precipitate,which increases the steric hindrance in the channels.Then,the methylene blue transports crossing the nanochannels is greatly influenced.The porous carbon nanofibers modified electrode can sensitively monitor the concentration change of methylene blue,thereby realize highly sensitive electrochemical detection of miR-21.Studies have shown that the biosensing platform displays a linear range of 100 a M to 1 n M for the detection of miR-21,and a detection limit of 40 a M.This method is also label-free.What’s more,the method avoids the use of expensive double-strand specific nucleases,and has been successfully used for the determination of miR-21 in serum and tumor cells. |
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