CRISPR/Cas12a Mediated T-junction DNA Cyclic Amplication Electrochemiluminescence Biosensor

Bacterial pathogens are commonly found in soil,the ocean,and the human gut.Humans carry more than 150 species of bacteria.Some of these bacteria can have profound effects on people and animals and may cause different infectious diseases.16S r DNA is the most useful marker and the most commonly used molecular clock in the study of bacterial classification system.It exists in all organisms and is highly conserved in structure and function.Therefore,it is very important to realize the sensitive detection of 16S r DNA.Electrochemiluminescence technology has attracted wide attention in the scientific research field because of its advantages such as high sensitivity,low detection limit,wide linear range,fast response speed,low background signal and strong stability.In order to enhance electrochemiluminescence signals,a ternary ECL system is introduced,which refers to the system involving luminescent reagents,co-reactive reagents and co-reactive accelerators.The co-reactive accelerators have no direct influence on the luminescent body,but can improve the performance of the co-reactive materials.In order to solve the defects of multiple interference factors and poor stability in the detection process,the biological amplification method was used to improve the concentration of nucleic acid and the CRISPR/Cas system to cooperate with the rapid signal conversion.In this paper,ECL as a biological analysis means,through the preparation of new luminescent materials and different co-reaction accelerators to enhance the ECL intensity,and combined with different nucleic acid amplification strategies,the establishment of different kinds of ECL biosensors,to achieve the rapid sensitive detection of trace targets.This paper specifically carried out the following aspects of work:（1）The programmable T-shaped structure assisted CRISPR/Cas12a electrochemical luminescence biosensor to detect Sa-16S r DNA.In this strategy,an ECL biosensor for quantitative analysis of Sa-16S r DNA based on the advantages of porphyrin Zr-MOF（PCN-224）/Cd S QDs composites and SDA-assisted CRISPR/Cas amplification strategy was proposed.In the presence of Sa-16S r DNA,DFP 1 and DFP 2 bound specifically and hybridized with each other to produce defective T-shaped structures.The T-structure had high specificity and stability.A large number of substantial single stranded DNA products were exported through cascaded SDA amplification,thus realizing effective signal amplification.In addition,PCN-224 was prepared as co-reaction accelerators to promote the conversion of S₂O₈^2-and SO₄^*-,thus enhancing the reaction with Cd S QDs and amplifying the ECL emission signal.The CRISPR/Cas12a system with high dynamic catalytic efficiency could realize non-specific trans-cleavage of hp DNA and avoid false identification results.Therefore,the new Sa-16S r DNA assay provided a promising platform for clinical detection of other biomolecules.（2）3D DNA nanomatines assisted CRISPR/Cas12a combined with electrochemical luminescence biosensors to detect Ec-16S r DNA.In this strategy,an ECL biosensor for quantitative analysis of Ec-16S r DNA was proposed based on SDA and 3D DNA nanomachines combined with assisted CRISPR/Cas amplification strategy.A large amount of SP 1 was exported through the cascade amplification of SDA.Triggered by SP 1,the polymerization based on KF polymerase and the shear of Nt.Bbv CI shear enzyme,hp DNA and track DNA were reversed and simultaneously rolled to complete the operation of 3D DNA roller.The release of rich SP 2,thus achieving exponential signal amplification.In addition,a new ternary(Sn S₂ QDs/S₂O₈^2-/Mn O₂ NFs)ECL system had been developed.The Mn O₂ NFs with a large specific surface area was loaded with a large number of Sn S₂ QDs and acted as a co-reaction accelerator to promote the conversion of S₂O₈^2-and SO₄^*-,thus amplified the ECL emission signal.The CRISPR/Cas12a system with high dynamic catalytic efficiency could realize non-specific trans-cleavage of hp DNA and avoid false identification results.Therefore,the proposed strategy achieved efficient detection of DNA in complex substrates.