Study On CRISPR-Cas12a Biosensor Regulated By Magnetic Assisted Y-dna Probe And Its Application In The Detection Of Schistosoma Japonicum Eggs

Objectives:Schistosomiasis is an acute and chronic parasitic disease caused by Schistosoma,which poses a serious threat to human health.The existing detection methods for Schistosoma mainly include parasitology detection,immunological detection and nucleic acid detection.These traditional detection methods play an important role in the diagnosis and surveillance of schistosomiasis.However,there are also some shortcomings,such as relying on large-scale instruments,requiring professionals to operate,time-consuming,low specificity and so on,which greatly limits the scope of application.Most CRISPR-Cas12a systems have high sensitivity and specificity,in the meanwhile nucleic acid aptamers have the characteristics of high specificity,high stability and no immunogenicity.In this study,combining nucleic acid aptamer with CRISPR-Cas12a system,a magnetic-assisted Y-DNA-regulated CRISPR-Cas12a biosensor was designed for sensitive detection of Schistosoma japonicum eggs.Methods:In this study,monodisperse Fe₃O₄ microspheres were synthesized by solvothermal reaction,and gold nanoparticles and polyethyleneimine were modified on the surface of Fe₃O₄ by ultrasound to obtain gold-coated magnetic nanoparticles（Au MNPs）.Secondly,a Y-type DNA probe（Y-DNA）was designed based on the egg aptamer of Schistosoma japonicum.Y-DNA is composed of fixed DNA,activator DNA and captured DNA.Finally,Y-DNA was immobilized on magnetic nanoparticles by S-Au bond to construct Au MNPs@Y-DNA nanoprobe.The probe dissociated after recognizing the target Schistosoma japonicum eggs,and the released Activator could regulate the CRISPR-Cas12a to cut the ss DNA-FQ fluorescence reporter molecules and produce significant fluorescence signals,thus realizing the sensitive and rapid detection of Schistosoma japonicum eggs.Results:The characterization results of a series of materials showed that Au NPs were modified on the surface of Fe₃O₄ microspheres and Au MNPs were successfully prepared.A relatively stable Y-DNA probe was successfully constructed,and Y-DNA would dissociate when the target appeared.Y-DNA was modified on the surface of Au MNPs,and Au MNPs@Y-DNA nanoprobe was successfully prepared.The ratio of cr RNA to Cas12a,ss DNA-FQ,reaction temperature,p H,reaction time and Au MNPs concentration in the detection system were optimized and designed.We verified the feasibility and performance of the detection system,and detected Schistosoma japonicum eggs.The results showed that the detection system could detect eggs with great sensitivity and specificity,and could detect the eggs of actual samples.Conclusions:Au MNPs was successfully synthesized based on solvothermal reaction and ultrasound,and Y-DNA was modified on the surface of Au MNPs by S-Au bond to prepare Au MNPs@Y-DNA nanoprobe successfully.The magnetically assisted Y-DNA-regulated Au MNPs@Y-DNA biosensor constructed by the combination of Au MNPs@Y-DNA nanoprobe and CRISPR-Cas12a could detect individual eggs sensitively and could detect the eggs of real samples.The biosensor has certain practical application potential in the diagnosis of schistosomiasis.In addition,the Y-DNA probe is programmable.Theoretically,the systems can be used to detect a variety of non-nucleic acid targets and has a broad application prospect.