Construction And Application Of Label-free Fluorescent Biosensor Based On CRISPR/Cas And Primer Exchange Reaction Signal Amplification

Objective:Various biological molecules,such as proteins,amino acids,and small molecular metabolites,exist in the human body.They have special physiological functions and play a crucial role in life activities.Therefore,accurately monitoring the concentration and distribution of these biomolecules in the body is of great significance for studying various physiological and pathological changes in the human body and for early diagnosis of diseases.However,due to the low content of relevant biomarkers in the early stages of major diseases such as malignant tumors,it is necessary to use highly sensitive and selective analytical methods for the detection of these biomolecules.Therefore,two labelfree fluorescent probes have been constructed based on CRISPR/Cas technology and primer exchange reaction strategies for rapid and highly sensitive detection of disease related markers.1.In the first part,a CRISPR/Cas12 a biosensor was built with G4 MB containing a hairpin structure with a split-G-quadruplex,we developed a label-free fluorescent biosensor for highly specific detection of circulating tumor DNA(ctDNA)activity.2.In the second part,based on the primer exchange reaction(PER)and the specific dye Thioflavin T(ThT),we constructed a label-free fluorescent biosensor for the amplification detection of Apurinic/apyrimidinic endonuclease 1(APE1).Methods:1.G4 MB containing a split-G-quadruplex is combined with CRISPR/Cas12 a to build a sensor system.The stem-loop structure of G4 MB can keep the split G-quadruplex at the end to come close to form complete G-quadruplex.In the absence of ctDNA,the CRISPR/Cas12 a system is inactivated and the G4 MB cannot be cleaved.Therefore,ThT can specifically bind with the G-quadruplex structure of G4 MB and significant fluorescence enhancement is observed.In the presence of ctDNA,ctDNA can hybridize with crRNA and form the“Cas12a/crRNA/activator”ternary complex,resulting in the activation of trans-cleavage activity of Cas12a.Because ss DNA is the most susceptible substrate of Cas12a,the single-stranded loop structure of G4MB probe is rapidly cleaved first.As a result,the G4 structure is disassembled and the fluorescence intensity is decreased obviously.2.The hairpin probe is labeled with an AP site near the 5 ’end for easy identification of the target APE1,while the 3’ end is modified with an inverted d T to avoid nonspecific amplification.There is no G4 structure generation in APE1,so there is basically no background fluorescence.The hairpin structure marked with an AP site is cracking when the target APE1 is existed.Part of the sequence of the 5 ’end fracture of the hairpin becomes a primer of the primer exchange reaction(PER).Next,the primer extended in the influence of BST polymerase.After the sequence is extended to stop,the 3 ’end modification of the hairpin is dotted dt to avoid non-specific amplification.In the end,the extended ss DNA(PER product)dropped from the hair clip under the competition of the molecule,and combined with the specific dye ThT to generate a significant fluorescent signal.Results:1.The transliteration activity of Cas12 a is activated and the G4 MB is cracked,so that the specific dye ThT cannot be combined with the G4 structure,causing the fluorescent signal to significantly reduce.So as to achieve specific detection of ctDNA activity.2.As ss DNA(PER products)fell from the hair clip under the competition of the molecular interchange,a significant fluorescent signal was produced with the dye ThT,thereby achieving a high-sensitivity measurement of the target APE1.Conclusion:1.In the first part,by constructing the G4 MB sensor system,the ctDNA can be sensitive and rapidly detected,and the detection limit can reach 2 pM.This solution implements label-free testing,avoiding complex modifications and reducing costs.Finally,the biosensor was applied to the detection of ctDNA in the human blood sample,and the results were satisfactory.2.In the second part,the sensor has high sensitivity through multiple transitions of primers,specific binding of dye ThT,and the zero-background design of the hairpin structure,and its detection limit can reach 0.008 mU/mL.At the same time,APE1 recovery experiments were performed in serum,and the results were satisfactory.