A Versatile Biosensing Platform Coupling CRISPR-Cas12a And Aptamers For Detection Of Diverse Analytes

The development of sensitive,rapid,stable,cheap and universal detection method is of great significance in clinical diagnosis,scientific research and environmental monitoring.However,large analytical instruments do not always meet the increased detection requirements due to the high cost and cumbersome operation.Small and convenient biosensors for point-of-care testing provide a promising solution.Therefore,new biological tools must be broadly deployed to develop a general bio sensing platform,and this endeavor should accelerate the construction of diverse biosensors.CRISPR(clustered regularly interspaced short palindromic repeats)nucleic acid diagnostic technology is widely used in pathogen detection,virus detection,genotyping,cancer mutation detection and single-nucleotide polymorphism identification,with advantages of low cost,good portability and sensitivity etc.However,the CRISPR nucleic acid diagnostic technology cannot be applied to the detection of analytes other than nucleic acids,which limits the application of this technology.In order to overcome this limitation and extend the CRISPR-Cas system(CRISPR-associated proteins system)to a wider range of analyte detection,we combined the trans-cleavage amplification capability of Cas12a with the broad analyte recognition capability of aptamers to create a novel versatile biosensing platform coupling Cas12a and aptamers for detection of diverse analytes.We demonstrated that the biosensors developed by this platform can be used to detect the tumor marker alpha fetoprotein with the detection limits of 0.07 fM.CRISPR based detection was successfully extended to protein detection by our platform.Diverse aptamers can be selected for sensing detection by SELEX technology.In order to streamline the construction of biosensors for diverse analytes,a plug-and-play modular design strategy is created to enable versatile detection of other analytes with only replacement of aptamer.A cocaine detection biosensor was constructed by means of a plug-and-play method.And achieved the detection limit of 0.34 μM.The low sensing efficiency and detection sensitivity are still big challenges for ultrasensitive detection(e.g.,antigen).Here,we created a PRC-free multiple trigger dsDNAs tandem activator for stable and rapid signal amplification which is directly generated by Cas12a.Compared with the SARS-CoV-2 nucleocapsid protein detection biosensor constructed by the single copy activator,the biosensor constructed by the multiple copies tandem activator shows a higher sensitivity.The detection limit reached 3.48 fM,and the detection sensitivity increased 9.4 folds.For most of the aptamers,the affinity towards its corresponding target is usually at nM level(i.e.,dissociation constant,KD),and it’s often not an easy work to further increase the binding affinity by SELEX(Systematic Evolution of Ligands by Exponential Enrichment).However,as we know,the KD of aptamer directly determines the sensing efficiency and then affect the detection sensitivity.Therefore,innovating the aptamer-based sensing module to more sensitively response antigen,which is independent of the affinity between aptamer and antigen,will have general interest for all the aptamer-based detection system.In this study,by using a Y-shape DNA,two aptamers A48 and A61 recognizing different epitopes of the antigen,have been designed to sense the Np.And a significant synergistic sensing effect has been observed,namely,the binding of A48 to Np will facilities the binding of A61 and Np,and vice versa.By combining the multiple trigger dsDNA and synergistic sensing strategy,the LOD for Np detection reached 0.17 fM.Lastly,to demonstrate the merits and potential of our method on clinical diagnosis,the portable fluorometer was used as the fluorescence output device.To this end,we achieved single virus level detection of inactivated SARS-CoV-2 in less than 21 minutes.As a result,for further expending its detection scopes of Cas12a,we developed a novel biosensing platform by coupling the collateral ssDNA cleavage activity of Cas12a with the excellent affinity of aptamers for a broad range of target analytes.Focusing on the signal amplification and sensing efficiency issues in the CRISPR-Cas12a and aptamer-based detection paradigm,we developed a PCR-free multiple trigger dsDNAs tandem-based signal amplification for high-performance CRISPR-Cas-based detection,and design a dual aptamer synergistic sensing strategy for highly efficient sensing.By integration of these strategies and finally realized the ultrasensitive,fast,and high-performance analytes detection.