Self-Enanced Dual-Ligand MOF Binding Framework Nucleic Acid To Construct Electrochemiluminescence Biosensor

Electrochemiluminescence（ECL）refers to the process of high energy electron transfer reaction on the electrode and the formation of excited states,including electrochemical and chemiluminescent reaction processes.This process combines the controllability of electrochemical analysis and sensitivity of luminescence analysis,with high sensitivity,simple operation,strong controllability,fast analysis,and other advantages.ECL biosensors based on biomolecular recognition could convert biometric activity into quantifiable ECL signals to analyze and measure the number of targets.At present,it is a hot research topic to utilize the high programmability and biocompatibility of framework nucleic acids to design the topology of biosensor interfaces to enhance the sensitivity and specificity of target detection.However,the construction of ECL biosensors still faces the following problems:（1）the performance of novel ECL materials is not excellent enough and the stability is poor.Also,it is difficult to adjust the luminescence performance of the materials by rational design.（2）Using programmability of the framework nucleic acid for signal output to meet the requirements of low detection limits still needs to be studied in depth.Based on this,a dual-ligand self-enhanced metal-organic framework（d-MOF）was designed in this thesis.Owing to the synergistic promotion of structure and components,the prepared d-MOF has excellent ECL intensity and stability.Meanwhile,in combination with the easily programmable framework nucleic acid structure,efficient biosensors are constructed using direct or indirect target recognition transduction strategies to achieve sensitive detection of biomarkers.The research work of this paper is mainly divided into two parts as follows:1.Ordered heterogeneity in dual-ligand MOF combined with DNA triangular prism as signal switch to construct microRNA-141 ECL biosensor.The emission behaviors of polycyclic aromatic hydrocarbons（PHAs）are controlled by the spatial arrangement state.It remains a challenge to obtain high-performance ECL materials by controlling the ordered arrangement of ECL molecules in micro/nano space.In this work,we modulate the ordered assembly of luminescent ligands and coreactant ligands through the rigid scaffold of MOF leading to enhanced ECL performance due to the improvement of both chemiluminescence efficiency and radical reaction efficiency.First,the intramolecular vibration and rotation of ECL chromophores are limited due to the flexibility of the ligands constrained by the framework thus improving the chemiluminescence reaction efficiency.Secondly,the introduction of coreactant ligands into the framework shortens the electron transfer distance improving the radical reaction efficiency.Specifically,a d-MOF with an ECL chromophore ligand（1,1,2,2-tetra（4-carboxylbiphenyl）ethylene,H₄TCBPE）and a coreactant ligand（1,4-diazabicyclo[2.2.2]octane,D₂H₂）was designed and prepared.The emission from TCBPE in framework matrix was enhanced 5.14 times in comparison to H₄TCBPE microcrystals.Considering the unique ECL performance of d-MOF,the ECL biosensor using the triangular prism DNA walker was developed with microRNA-141 as targets.This ECL biosensor achieved a low detection limit of 22.9 a M within a wide linear range from 100a M to 100 p M.Generally,the implementation of a synergistic strategy,which combines advantages of both the highly ordered assembly of ECL ligands and self-enhanced ECL effect,enriches the fundamentals and applications of ECL systems.2.DNA tetrahedron-based specific sequence ds DNA generator for activating CRISPR/Cas12a to construct APE1 ECL biosensor.ECL biosensors possess the advantages of high sensitivity,excellent selectivity,and reproducibility.However,there is still a need to improve the efficiency of transduction and amplification processes in biometric events to enhance biosensor performance.In this work,a DNA tetrahedral framework nucleic acid as a scaffold for the specific sequence double-stranded DNA（ds DNA）generator was designed with a ds DNA probe containing a depurinic/apyrimidinic endonuclease 1（APE1）active site at each of the two vertices of the DNA tetrahedron.By cleavage the AP active site in the presence of the APE1,the binding sequence of the primer in primer exchange reaction is exposed,and a large amount of specific ds DNA that could activate the CPISPR/Cas12a system is amplified in situ through strand replacement and strand migration processes.The signal switch at the GCE surface enables the recovery of the ECL signal.We prepared novel self-promoted ECL micro-nano-materials（d-MOF@Pd NPs）by in situ synthesis of palladium nanoparticles on the surface of d-MOF and constructed an ECL sensing platform.The ECL biosensor combines the advantages of a ds DNA generator-mediated target conversion process and an efficient CRISPR/Cas12a activation process,which achieved sensitive detection of APE1 with low detection limit(2.06×10^-11 U/μL)and wide linear range(1×10^-10 to 1×10^-5 U/μL).