Immune And DNA Repair Enzyme Analysis Based On Nanoparticle Tracking Analysis Technology And CRISPR/Cas12a System

Sensitive detection of protein and enzyme biomarkers is the most dependent means for diagnosis and prognosis of diseases in clinical practice at present.Therefore,it is of great significance to develop sensitive,accurate,rapid and simple detection methods for protein and enzyme biomarkers.Based on the advantages of Nanoparticle Tracking Analysis（NTA）technology to visually detect the particle size and concentration information of nanoparticles,as well as the specific targeting ability and the efficient trans digestion activity of CRISPR/Cas12a system,two new methods of immunoassay and enzymatic activity analysis were established in this paper.Specific research contents are as follows:In the second chapter,a new immunoassay method based on NTA was developed to detect prostate specific antigen（PSA）sensitively.In this design,gold nanoparticles（GNPs）were used as a nano-probe for quantitative determination of PSA concentration.The surface of GNPs is functionalized with PSA-specific monoclonal antibody（mAb1）for PSA recognition and capture.The sandwich immune complex is formed by the specific reaction of mAbl,PSA and another biotinylated PSA specific monoclonal antibody（mAb2-biotin）.After further adding streptavidin-functionalized magnetic beads（STV-MBs）to the system,GNPs-mAbl/PSA/mAb2-biotin sandwich immune complex structure will bind to STV-MBs.Under the action of magnetic field,the sandwich structure bound to PSA will fix and separate the functional GNPs,so that the number of GNPs in the system decreases with the increase of PSA.NTA is a new technology that can visually analyze the particle size and concentration of nanoparticles in homogeneous solution.In this paper,the unique advantages of NTA technology were used to directly and real-time monitor the changes of the number of nanoparticles in the system,so as to achieve quantitative analysis of PSA.The immunoassay method based on NTA proposed in this paper has simple steps,rapid reaction and does not depend on nanomaterials with specific optical and electrical properties.It is applicable to a wide range and avoids the interference of other conditions such as enzyme labeling,thus providing a new idea for clinical biochemical analysis.In the third chapter,a new method for the analysis of immune and DNA repair enzyme activity based on CRISPR/Casl2a was discussed.In order to further improve the sensitivity of immunoanalysis,this chapter first attempted to combine the self-pruning ability of CRISPR/Cas12a with the probes of RNA nanofflowers（RNFs）to construct a new signal amplification strategy for immunoanalysis.A sandwich immune reaction was carried out on the surface of STV-MBs using a reaction pattern similar to that in Chapter 2,and RNFs rich in crRNA repeat units were used as sensing probes.After immune reaction,RNFs on the surface of STV-MBs were proportional to the antigen content.Cas12a can self-pruning and assemble crRNA units in RNFs,thus identifying double-stranded DNA（dsDNA）activation sequence to initiate"trans-cleavage activity",cleavage single-stranded DNA reporter molecule（FQ）,generate fluorescence signal,and achieve quantitative analysis of PSA.In the above studies,we found that Casl2a-crRNA often needed to bind the double-stranded specific sequence with the protospacer-adjacent motif（PAM）to activate its trans-cleavage activity,while the sequence without PAM could not bind to Cas12a-crRNA.To solve this problem,we attempted to construct a "self-made Loop"mechanism in the double-stranded structure without PAM to activate the trans-cleavage activity of Cas12a-crRNA and successfully applied it to the analysis of uracil DNA glycation enzyme（UDG）activity.In this design,the common dsDNA structure was replaced by Hairpin DNA（H）to avoid unhybridized single strand,in which four uracil（U）bases were designed to replace PAM sequence at the non-target near-ring end of H.Under the effect of UDG,the U base in the Hairpin probe was removed to produce purine/aprimidine（AP）sites.Thus,a Loop structure is formed,which enables Cas12a-crRNA to interact through the Loop region,promote the binding with the target terminal sequence of H,and activate the trans-cleavage activity of Casl2a.In this system,the binding mechanism of Casl2a-crRNA and double-stranded target was preliminarily explored,and a simple and sensitive analysis of UDG activity was also realized.In this paper,we firstly established a new immunoassay strategy based on the absolute counting function of nanoparticles by using NTA technology.This strategy is generally suitable for a variety of nanoparticles probes and has the generality of detecting different disease markers,which expands the application range of NTA in clinical medicine field.On the other hand,the CRISPR/Cas12a system was used in this paper to construct a new method for the analysis of immune and enzyme activity,which not only broadened the detection range of biomarkers other than nucleic acids by Cas12a,but also broadened the thinking for exploring the binding principle of Cas12a-crRNA and target nucleic acids.