Chemiluminescence Resonance Energy Transfer-Based Multistage Nucleic Acid Amplification Circuit For MiRNA Detection

The abnormal expression of miRNA is closely related to tumorigenesis and development,making it a promising tumor marker with a wide range of clinical applications.Chemiluminescence（CL）is a highly attractive method for miRNA detection due to its lack of external excitation light source,autofluorescence,and anti-photobleaching properties.Chemiluminescence resonance energy transfer（CRET）is a method that transfers energy from a CL donor to an appropriate acceptor,which extends the wavelength of the luminescence signal.This process shows great potential in biochemical analysis and biomedicine.However,CRET sensing methods have limitations such as low sensitivity and short luminescence time,which restrict their application in cell imaging.Therefore,we constructed a CRET-based multi-stage nucleic acid amplification circuit for sensitive detection of miRNAs and cellular imaging.The main work can be divided into two parts:The first part demonstrates the basic principle and feasibility of the cascaded nucleic acid amplification circuit based on chemiluminescent resonance energy transfer.It consists of catalytic hairpin assembly（CHA）,hybridization chain reaction（HCR）,and hemin/G-quadruplex DNAzyme.The target triggers CHA reaction,and the hybridization product further triggers HCR to form a large amount of nanowires containing G-quadruplex.The G-quadruplex conjugates with hemin to form the hemin/G-quadruplex DNAzyme,which possesses horseradish peroxidase-mimicking catalytic activity.Then DNAzyme catalyzes H₂O₂-mediated luminol oxidation and simultaneously transferring energy released from the chemical reaction to the adjacent energy acceptor photosensitizer dihydroporphyrin e6（Ce6）,generating luminescent signals and single oxygen.This CRET-based multi-stage nucleic acid circuit enables sensitive detection of the target with a detection limit of 184.17 p M.The second part is the miRNA detection and cellular imaging application of the chemiluminescent resonance energy transfer-based nucleic acid amplification circuit.By introducing a hairpin that specifically recognizes miRNA-21 into the constructed nucleic acid amplification circuit,miRNA-21 specifically opens hairpin H7,exposes the sequence that triggers the subsequent CHD reaction,and generates an amplified luminescence signal,enabling precise detection of the tumor marker miRNA-21 with a detection limit of 174.49p M.Compared to other interfering nucleic acids,our system demonstrated excellent specificity for miRNA-21 detection.A reactive oxygen species（ROS）probe was used to indicate miRNA-21activated singlet oxygen signal for intracellular miRNA-21 imaging.Through imaging of miRNA-21 in three different cells,MCF-10A,He La,and MCF-7,it demonstrated that the CHD system is capable of accurately distinguishing different cells by miRNA-21 expression levels.In summary,the constructed multi-stage nucleic acid amplification circuit based on chemiluminescence resonance energy transfer successfully achieves intracellular miRNA-21 imaging with the assistance of ROS probes,which provides a powerful tool for early diagnosis and prognosis of diseases.