Target Recycling Amplification Strategy For MicroRNA Detection In Electrochemiluminescence

MicroRNAs?miRNAs?have turned out to be important regulators of biomarkers for early disease diagnosis.Due to the intrinsic characteristics of miRNAs,such as highly homologous sequences,shorten sequence and low abundance in cells,investigators are constantly exploring ways to achieve sensitive and selective miRNAs detection.Electrochemiluminescence?ECL?assays is a new analytical technology which combines electrochemical with high controllability and chemiluminescence technologies highly sensitive,showing great potential applications value in the fields of environment,food,life analysis and so on.Recently,ECL has combined with a variety of analytical strategies and grown vigorously in the direction of economy,greenness,and sensitivity.It particular,nucleic acid amplification strategy play an important role for its development,such as the target recycle amplification strategy,which can N times reuse the input target to improve the detection sensitivity.Thus,it is suitable for the detection of microRNA.This article combine target recycle amplification strategy with ECL analysis technology to achieve highly sensitive microRNA detection to meet the needs of life analysis.This work mainly including the following three tasks:1.Based on the microRNA induced catalyzed hairpin assembly and self-catalyzed carbon dots anode-ECL composite to construct signal-off ECL biosensorRecently,to improve the sensitivity of microRNA detection methods,researchers are committed to introducing nucleic acid amplification strategy into analysis technology.Among these amplification strategies,catalyzed hairpin assembly?CHA?was a simple,enzymatic and economical amplification strategy based on two hairpin DNA structures.Usually,it acted as carrier to introduce signal probe and applies in the construction of signal-on or signal-off ECL biosensors.Nevertheless,the background signal of signal-on ECL biosensor was high,which was not suitable for low concentration microRNA detection.In this work,CHA was induced by microRNA with abundant quenchers introduced on the electrode surface to construct a signal-off ECL biosensor.First,the self-catalyzed carbon dots complex with an anodic ECL signal was synthesized to obtain an enhanced ECL signal.In addition,amino-modified H1 could be immobilized on the complex.Simultaneously,dopamine is labeled on H2.After CHA,a large amount of dopamine could be in the electrode surface to achieve sensitive detection of microRNA.By combining highly efficient CHA technology with high-performance self-catalyzed carbon dots complexes,the detection range of biosensor changed from 0.1 fmol·L^-1 to 100 pmol·L^-1 with the detection limit of 0.03fmol·L^-1.2.K-junction structure mediated exponential signal amplification strategy for microRNA detection in electrochemiluminescence biosensorAs we know,exponential signal amplification strategies showed higher efficiency than common signal amplification strategies,while exponential signal amplification strategies always assisted by multiple biological enzymes,which increased the cost,limited experimental range and made operation complicated.Herein,we had designed the exponential signal amplification strategy mediated by novel K-junction structure with one enzyme assisted and applied in ECL biosensor to achieve sensitively microRNA detection.It was noteworthy that the recognition domain for target microRNA was constructed with the formation of K-junction structure.When target microRNA sensed,paired domains of substrate DNA were completely digested from5'-terminus,accompanied by the release of target microRNA and unpaired DNA fragment of substrate DNA called trigger DNA.Afterwards,the released microRNA and trigger DNA were recycled over and over with the digestion of K-junction structure linear and exponential grade respectively.As a result,numerous uninhibited reporter DNAs were left on the electrode lonely to capture hemin with hemin/G-quadruplex yielded which could enhance ECL emission significantly due to its catalysis for the luminol-H₂O₂.As expected,this method exhibited excellent specificity and high sensitivity for microRNA detection from down to 0.033 fmol·L^-1.3.An ultrasensitive electrochemiluminescent strategy based on highly-efficient luminol immobilization approach and exponential strand displacement reaction to monitor microRNA expression in cellAlthough the experimental operation and the cost had simplified by one enzyme assisted exponential signal amplification strategy in some degree,the special operation of biological enzyme was still a problem.To overcome this problem,a cruciform DNA structure was designed to mediate exponential strand displacement reaction?SDR?.And an ultrasensitive electrochemiluminescence?ECL?biosensor was constructed for microRNA-21?miRNA-21?detection based on the exponential SDR.The signal probe,which was synthesized utilizing the?-?stacking interaction between PTCA and luminol,could be opened by miRNA-21 resulting in the disaggregation of cruciform DNA structure to mediate exponential SDR for target recycling amplification.On the basis of highly effective and stable luminol immobilization and exponential SDR,this biosensor achieved excellent sensitivity with wide linear range from 10 amol·L^-1 to 100 pmol·L^-1and detection limit was 2 amol·L^-1.Moreover,this ECL biosensor was applied to estimate the expression level of miRNA-21 and pharmacodynamics of matrine in human breast cancer cells?MCF-7 cells?.