A Novel Biosensing Strategy For Microrna-21 Detection In Type 2 Diabetes Mellitus

Purpose:As a biomarker,miRNA is playing an important role in early diagnosis of cancers,regulation of gene expression,screening of metabolic disorders,etc.,and concerned by more and more scientific research workers.At present,the general detection techniques for miRNAs are Northern blotting,microarray and qRT-PCR.However,these methods suffer from some inherent limitations,such as low throughput,poor sensitivity,time and labor consuming,which improve the cost and difficulty of detection.According to the present situation of miRNA detection,in this study,a new detection method for miRNA is developed,which has the advantages of rapid,highly sensitivity,low-cost and provides a new potential way for early diagnosis of cancers,disease screening and gene expression regulation of clinical work.MiRNA-21 is chosen as a detection target to lay a foundation for the convenient diagnosis of type 2 diabetes and to provide a new research for the rapid and low-cost detection of biomarkers of other diseasesMethods:MiRNA-21 is the biomarker of various metabolic disorders and cancers.It is up-regulated in the serum and urine of patients with type 2 diabetes,fatty liver and colon cancer.We developed a simple,low-cost and highly sensitive electrochemical biosensor for the detection of miRNA-21.In the homogeneous system,the target miRNA-21 hybridizes with the designed multifunctional strand probe in the premise of abundant nucleic acid substrates,KF exo-and template strand.As a result,primer extension is processed to form a heteroduplex to provide a precondition for polymerization of T7 promoter.Afterwards,highly effective catalytic transcription is realized under the T7 pol.Following the transcription,a large amount of single-stranded RNA products can be formed and hybridize with the biotinylated detection probes,resulting biotinylated double strains.Finally,the biotinylated double strains hybridize with pre-immobilized capture sequence on the surface of the gold electrodes to realize the signal signify,detection and achieve the quantitative determination of miRNA-21.Results:The developed strategy achieves the quantitative detection of miRNA-21 with a LOD of 0.6 fM and a linear range of 1 fM to 10 nM.In similar sequences of miRNAs,this strategy exhibits excellent specificity in discriminating the differences of single nucleotide.In real samples detection,it is applied to miRNA-21 detection in total RNA extracted from HepG2 cells.Conclusion:This work combines homogeneous system with T7 pol assisted high transcription amplification efficiency and KF exo-to develop a HTITA electrochemical biosensing strategy for miRNA-21 detection,which can avoid heterogeneous polymerization and other troublesome procedures.This biosensing strategy has the advantages of rapid,highly sensitivity and low-cost.Because of the versatility of the design of the multifunctional probe,the detection of other target can be achieved by the change of a small number of sequences on the multifunctional probe.This simple and sensitive electrochemical biosensing method might be a potential alternative tool for the quantitative analysis of miRNAs in physiological fluids,biomedical research and point-of-care testing in the future.