Construction Of Aptamer-CRISPR/Cas12a-based Biosensor And Its Application In Biomarker Detection

Rapid and accurate early diagnosis is of great significance to the prevention and treatment of clinical medical diseases.Biomarkers,represented by proteins,nucleic acids,small molecules,have been applied in the field of early disease diagnosis and drug development as targets of drug development and measurable indicators of disease pathogenesis and prognosis.CRISPR/Cas is an adaptive immune system used in bacteria and archaea to defend against viral infection,which can guide Cas proteins to target specific nucleic acid sequences mediated by guide RNA(g RNA).Cas12a(Cpf1)is an endonuclease from the class 2 type V-A CRISPR/Cas system.It not only cleaves target DNA like many other CRISPR/Cas systems,but also cleaves any non-target ss DNA near the target DNA.This property shows great application prospect in nucleic acid related disease diagnosis.However,due to the lack of a general signal transduction strategy,the system is low genarality,low sensitivity and complex operation for non-nucleic acid targets,which limits its clinical application.Aptamers are single stranded DNA or RNA molecules that can bind to target molecules with high selectivity and affinity.Currently,with the rapid development of biotechnology,aptamers for different biomarkers have been screened.In this paper,aptamer-linked CRISPR/Cas12 a based immunoassays(denoted as ALCIA)was developed by using the recognition function of nucleic acid aptamer and the trans-cleavage activity of CRISPR/Cas12 a system.In addition,gold nanoparticles were introduced on the basis of ALCIA to design a fluorescence immunoassay method based on spherical nucleic acid nanoprobe for signal amplification.(1)ALCIA and Nano-ALCIA fluorescent biosensors were constructed to detect platelet growth factor(PDGF)-BB.Firstly,the stability of aptamer-CRISPR/Cas12 a was investigated by fluorescence spectroscopy.Then,the feasibility,sensitivity and specificity of ALCIA and Nano-ALCIA for PDGF-BB detection were investigated.Among them,the limit of detection(LOD)of ALCIA for PDGF-BB detection reached1.57 p M,the LOD of Nano-ALCIA fluorescence sensor is as low as 0.55 f M.(2)The ALCIA colorimetric biosensor(denoted as Opti-ALCIA)was constructed to detect small molecular ATP.The feasibility,sensitivity and specificity of Opti-ALCIA for ATP detection were investigated by naked eye observation and ultraviolet spectroscopic technology.The results show that our method can detect ATP quickly and conveniently,and LOD is as low as 0.14 p M.Then,we further explore its application in the detection of the three analytes of protein,bacteria and virus,and constructe detection kits for the three analytes.Through the analysis of the orthogonality and receiver operating characteristic(ROC)curve of the three kits,it is found that the three kits have strong orthogonality and diagnostic accuracy.(3)The ALCIA electrochemical biosensor(denoted as E-ALCIA)was constructed to detect human hepatocellular carcinoma(Hep G2)cells.The feasibility,sensitivity and specificity of E-ALCIA for detection of Hep G2 cells were investigated through electrochemical workstation.The results show that our method can be used to detect Hep G2 cells quickly and sensitively,LOD is 20 cells.What's more,we can be used to detect different cancer cells by replacing aptamers that target specific cancer cell biomarkers.