Nanopore Counter With Nucleic Acid Double-strand Discrimination Accurately Detects Biomarker

Cancer is a major disease that threaten human life in the world,and lots of people still die of cancer today.Because of the limitations of cancer diagnosis and treatment,its death rate has not decreased with the progress of technology.Therefore,early diagnosis of cancer is the key to successful treatment.Biomarker detection is one of the most effective methods to obtain biological information and has been used in research and development for many years.Biomarker-based cancer diagnosis can significantly improve early diagnosis and subsequent treatment.Biosensors play an important role in biomarker detection because of their simplicity,portability and real-time analysis.In addition,glass nanopores have been widely used as a single molecule sensing technology due to their simplicity and lack of labels,which can detect nucleic acid,protein and small molecules and other biomarkers.The detection of nucleic acid double strand is easy to detect by glass nanopore,and this method is easy to count and is a relatively simple and attractive method.Therefore,in this paper,double-stranded nucleic acid was used as the signal transduction carrier of the glass nanopore counter.First,DNA methylation was evaluated by combining the dual endonucleases digestion strategy with polymerase chain reaction(PCR).The second is to detect cfDNA in combination with the designed dumbbell double stranded nucleic acid structure and CRISPR-Cas12 system.DNA methylation is considered as a vital epigenetic biomarker in the diagnosis of various diseases,such as cancer.It plays a key role in the regulation of gene expression,but its abnormality can inhibit gene expression,which can lead to cancer and other problems.Therefore,DNA methylation analysis is very crucial for early disease diagnosis.In job 1,dual endonucleases used can selectively digest unmethylated DNA more thoroughly,leaving only methylated DNA intact and triggering PCR to produce large numbers of PCR amplicons that were fixed-length,and then these PCR amplicons were detected by glass nanoporbs.Finally,by simply calculating the event rate of translocation signal,the concentration range of methylated DNA was determined to be 1 a M to 0.1 n M,the detection limit was as low as 0.61 a M,and the level of 0.01 % DNA methylation was successfully identified.This highly sensitive evaluation of DNA methylation using glass nanopore is a low-cost and reliable method for DNA methylation analysis.cfDNA,cell-free DNA,which can play an important role as a biomarker in disease prediction and cancer treatment,is an early disease diagnosis strategy.In job 2,a dumbbell nucleic acid double-stranded structure was successfully designed,which consists of two ds DNA of different lengths(amplified by PCR)and a small fragment of ss DNA.Then,cfDNA was used as the target DNA to activate Cas12,and the CRISPR-Cas12 system was used to recognize and arbitrarily cut ss DNA,so that the dumbbell nucleic acid double-stranded structure was cleaved into two original ds DNA segments.Finally,the translocation signal changes of dumbbell nucleic acid double-stranded structure were detected by glass nanopore,thereby indirectly measuring the level of cfDNA.The detection range was between 10 f M and 1 n M,the detection limit was 0.24 p M,and the linear range was 1 ～ 10 p M.The detection of cfDNA using a glass nanopore counter combined with the CRISPR-Cas12 system is simple and fast,and this method can be used in early cancer diagnosis and other applications.