Study On Electrochemical Detection Method Of Bladder Cancer Assisted By CRISPR/Cas System

Bladder cancer is the most common malignant tumor in the urinary system.Due to the need for frequent monitoring and treatment,bladder cancer has been treated costly.Early,residual and recurrent bladder cancer is difficult to be detected by existing clinical diagnostic methods.Studies have found that a variety of urine biomarkers are closely related to the occurrence and development of bladder cancer.Therefore,the development of detection methods of urine biomarkers is helpful for the diagnosis and treatment of bladder cancer.Urine biomarkers of bladder cancer include exfoliated bladder cancer cells,cell-free nucleic acid molecules,exosomes,and apolipoproteins so on.In this paper,CRISPR/Cas system and electrochemical technology were combined to propose electrochemical detection methods for urine biomarkers of bladder cancer,which is expected to be applied to early screening of bladder cancer.The main research contents are as follows:1.Electrochemical evaluation of bladder cancer development via cellular interface supported CRISPR/Cas trans-cleavageEvaluating bladder cancer development is of great importance for clinic treatment and therapy.It has been known that the amounts of sialic acids on tumor cell membrane surface are closely associated with the degree of cancerization of the cell.So,in this work,cellular interface supported CRISPR/Cas trans-cleavage has been explored for electrochemical simultaneous detection of two types of sialic acids,i.e.,N-glycolylneuraminic acid(Neu5Gc)and N-acetylneuraminic acid(Neu5Ac).Specifically,Pb S quantum dot labeled DNA modified by Neu5 Gc antibody is prepared to specifically recognize Neu5 Gc on the cell surface,followed by the binding of Neu5 Ac through our fabricated Cd S quantum dot labeled DNA modified by Sambucus nigra agglutinin.Subsequently,the activated Cas12 a indiscriminately cleaves DNA,resulting in the release of Pb S and Cd S quantum dots,both of which can be simultaneously detected by anodic stripping voltammetry.Consequently,Neu5 Gc and Neu5 Ac on cell surface can be quantitatively analyzed with the lowest detection limits of 1.12 cells/m L and 1.25 cells/m L,respectively.On this basis,a ratiometric electrochemical method can be constructed for kinetic study of the expression and hydrolysis of Neu5 Gc and Neu5 Ac on cell surface,which can be further used as a tool to study bladder cancer cells at different development stages.Our method to evaluate tumor development is simple and easy to be operated and it can be potentially applied for the detection of tumor occurrence and development in the future.2.Study on electrochemical detection method of circular RNA in bladder cancer based on tetrahedron supported CRISPR/Cas13 a systemAs a diagnostic biomarker,the detection of circular RNA(circ RNA)is vital for the early screening of bladder cancer.Usually,the low abundance of circ RNA in clinic urine samples results in the necessarily complicated extraction before detection.In this work,a tetrahedron supported CRISPR/Cas13 a system has been explored for direct electrochemical detection of circ RNA in urine from bladder cancer.CRISPR/Cas13 a system has been reasonably designed to recognize the characteristic back-splice junction site of circ RNA,so as to activate its trans-cleavage ability.The activated CRISPR/Cas13 a can cleave uracil bases composed of DNA tetrahedron immobilized on the surface of gold electrode,resulting in the breakage of DNA tetrahedron and the release of electrochemical active molecule methylene blue.By virtue of highly catalytic efficiency of CRISPR/Cas13 a and rigid structure of tetrahedron,the developed electrochemical biosensor can directly detect circ RNA in 25 μL urine sample with the lowest detection limit of 0.089 f M and linear detection range from 10 f M to 50 n M.The detection process can be completed within 10 min,and the complicated extraction process is avoided.It is expected to be applied to on-site detection.