Design And Application Of Intracellular Biosensor Based On DNA Nanostructure

Based on its programmable base pairing,DNA has become a promising biomaterial for assembling various nanostructures.It also has other significant properties,including stability,outstanding biocompatibility,and can be further applied by modifying functional groups.Over the past few decades,researchers have established various design rules and assembly techniques to improve the stability and complexity of DNA nanostructures.Detection of cancer-related biomarkers is important in identifying patients with different clinical stages and developing adaptive therapeutic strategies.Due to its unique advantages,DNA nanostructures can be designed as universal units to form biosensors for the detection of tumor biomarkers.In order to improve the sensitivity of detection of tumor markers,biosensors based on DNA nanostructures was constructed in this paper.The works are as follows:1.A novel multicolor tetrahedral DNA nanostructure-based nanoprobe with embedded DNAzymes（TDNzymes）was presented which could enter the cells easily and achieve the simultaneous and quantitative detection of intracellular physiologically-related Cu²⁺and Zn²⁺.Two types of DNAzymes specific for Cu²⁺and Zn²⁺were encoded in the DNA tetrahedral scaffold by thermal annealing.The formation of well-ordered TDNzymes’nanostructure brought the fluorophore and quencher close to each other,therefore the fluorescence was quenched.In the existence of Cu²⁺and Zn²⁺,the structure of TDNzymes would be specifically cleaved and the corresponding fluorophore would be released.The recovered fluorescence in living cells could be observed by confocal microscope and quantitative analyzed by flow cytometry with low-nanomolar sensitivities for both metal ions.The proposed method offers new opportunities for quantitative detection of multiple targets in living cells.2.We develop an amplification strategy for microRNA-21（miRNA-21）imaging in living cells with a MoS₂-based three branched catalyzed hairpin assembly（TB-CHA）probe.MoS₂ nanosheet serves as the nanocarrier and excellent fluorescence quencher,which can transfer fluorescent metastable hairpin DNA into the cells easily with a nondestructive manner and significantly reduce background signals.TB-CHA probe containing three types of well-designed DNA molecular beacons with modification of Cy3 in the terminal.In the presence of miRNA-21,catalyzed hairpin assembly（CHA）reaction would be triggered and a“Y”shaped three branched duplex nanostructure would be formed,which would release from the surface of MoS₂ nanosheet due to the reduced affinity between DNA duplex and MoS₂ nanosheet.The multisite fluorescence modification and the circular reaction of TB-CHA probes allows a significant fluorescence recovery in live-cell microenvironment.Ultrasensitive detection of miRNA-21 is achieved with a detection limit of 75.6 aM,which is^five orders of magnitude lower than that of the simple strand displacement-based strategy（detection limit:8.5 pM）.The method offers great opportunities for ultrasensitive live-cell detection of miRNAs and helps the deeper understanding of the physiological functions of miRNAs in cancer research and life processes.