Construction Of Glass Nanopore Sensors With DNA Cubes As Signal Transduction Carrier

Nanopore technology is a kind of sensing technology with single-molecule analysis capability.Its basic working principle is that when the analyte passes through a nano-sized pore under the influence of an electric field,its concentration,charge and characteristics can be determined by reading the frequency,peak shape or pore charge change of the resulting resistance pulse signal.Due to its high sensitivity,label-free and low cost,nanopore technology has broad application prospects in gene sequencing,biosensing and biomedical fields.Nanopores are mainly classified into three types: biological nanopores,solid-state nanopores,and hybrid nanopores.Among them,solid-state nanopores have been proven to be a powerful platform for label-free single-molecule analysis due to their good mechanical stability and pore size flexibility.However,compared with protein nanopores,solid-state nanopores have lower resolution,which poses two major challenges in the production of biomarker sensors with strong signal-to-noise ratio and good selectivity:(1)Due to the difficulty in producing smaller pore sizes(<10 nm),interaction force between the nanopore and smaller chemical or biological target analytes passing through the pore is weak,making it difficult to produce recognizable translocation signals.(2)In practical sample testing,interference from abundant protein impurities(bovine serum albumin,recombinant albumin)in the sample or buffer would greatly degrades the noise level of nanopores,thereby submerging the useful translocation signal.Therefore,designing and producing solid-state nanopore biosensors with good translocation signals is the urgent scientific problem to be solved at present.This study found that DNA cubes can produce very strong resistance pulse signals when passing through glass nanopores,and their ultra-high signal-to-noise ratio(SNR>50:1)enables them to maintain clear and readable translocation signals in buffers containing protein impurities.Based on this,this study proposes an indirect detection technology between analytes based on DNA cubes as signal transducers,and carries out a series of studies on DNA cubes,applying them to the detection of specific biomarkers.In Chapter two,this study screened and studied various signal transducers,and ultimatel determined DNA cubes as the signal transducers for this study.At the same time,we further optimized the buffer system,synthesis concentration and storage time of DNA cubes.In order to achieve efficient transmission performance of DNA cubes,this study also determined the optimal pore size of glass nanopores that matches their size.The results showed that changing the composition elements of DNA cubes can significantly change the number of translocation events.Finally,this study determined a synthesis concentration of 100 n M for DNA cubes,a storage time of 6 hours,and an optimal pore size of 11-12 nm for glass nanopores.In Chapter three,the author combined DNA cubes with polymerase chain reaction(PCR)and clustered regularly interspaced short palindromic repeats-associated protein(CRISPR-Cas)to develop a detection strategy for hepatitis B virus(HBV)based on the cleavage of DNA cube elements.HBV is a virus that causes hepatitis B infection so the detection of its content in the human body is of great significance to clinical medicine.This chapter found that when the elements E and F of DNA cube compositions were cleaved,the translocation signal event rate of DNA cubes decreased most sharply.Then,combined with PCR and CRISPR-Cas12 a,the detection of HBV was realized,and the detection limit reached 5 a M.In Chapter four,this study further explored the characteristics of DNA cubes,and by introducing the blockers of DNA cube elements,it realized the transition of the response mechanism of the target analyte from the “off” to the “on”,improving the deficiencies in the strategy in chapter three.The 2019 coronavirus disease(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)is a pandemic disaster that continues to occur globally.Achieving rapid and efficient detection of SARS-Co V-2 is of great significance.The signal strategy improved in this chapter: the element closed chain can combine with the composition elements of DNA cubes,thereby destroying the strict folding spatial structure of DNA cubes,making the resistance pulse signal change sharply.Combined with CRISPRCas12 a and PCR,glass nanopores were used to detect the E gene of SARS-Co V-2.This chapter also optimized the number,location and length of the blockers and the detection limit of the E gene of SARS-Co V-2 reached 20 a M.Finally,the glass nanopore sensing strategy based on DNA cubes as signal transducers was applied to the ultra-sensitive detection of HBV and the E gene of SARS-Co V-2,which reduced the detection limit and provided profound inspiration for future research.