| Since the central dogma was discovered by F.H.C.Crick,deoxyribonucleic acid(DNA)carries all the genetic information of organisms necessary for transcription into ribonucleic acid(RNA)and translation into proteins.It is an essential biological macromolecule for the growth and reproduction of organisms and the maintenance of normal life activities.It has become a recognized fact in the scientific community that DNA plays a very important role in organisms.DNA nanostructures are complex DNA structures formed by folding,bending,winding,etc.Typical DNA nanostructures,including quadruplex,DNA origami,DNA nanospheres,etc.,are widely used in biosensors,medical diagnosis,and environmental detection.Isothermal nucleic acid amplification technology is a technology that can carry out nucleic acid amplification without thermal circulatory system.Compared with conventional PCR,which requires strict high temperature denaturation,annealing,extension and other steps to simulate in vitro DNA molecular amplification,isothermal amplification technology uses a constant temperature and has a wider application space.At present,DNA nanostructures combined with isothermal nucleic acid amplification technology can increase the sensitivity of biosensors and greatly shorten the signal response time.Therefore,the combination of the two has a good application prospect in clinical detection and diagnosis,food safety testing and environmental sanitation.In this paper,we mainly use a variety of DNA nanostructures,combined with catalytic hairpin self-assembly(CHA),hybridization chain reaction(HCR),rolling circle amplification(RCA)and other temperature amplification techniques.At the same time,the cleavage ability of endonuclease is used to detect the target and simultaneously perform gene therapy to construct a biosensing platform for detection and treatment.The main research contents of this paper are summarized as follows :In the first part,a glutathione(GSH)-responsive detection of intracellular micro RNA155(mi RNA155)and functional DNA nanoflowers for gene therapy were constructed.Repeated DNA sequences were formed by rolling circle amplification(RCA),including MUC1 aptamer sequence,sequence binding to 10-23 DNAzyme and complementary sequence of fluorescent hairpin H.Hairpin H and M(blocked 10-23 DNAzyme)were added to form a spherical DNA nanostructure,which was incubated with MCF-7 cells.Through the targeting of aptamers,DNA nanostructures were endocytosed into cells by cells.In the presence of high expression of GSH in tumor cells,GSH cleaved the disulfide bond in M and released blocked 10-23 DNAzyme,thereby cutting VEGFR-2 m RNA,achieving the purpose of gene silencing and promoting tumor cell apoptosis.At the same time,the binding site of mi RNA155 was exposed.HCR reaction occurred between mi RNA155 and nanostructures,which squeezed the fluorescent hairpin H and restored the fluorescence resonance energy transfer(FRET)to achieve the purpose of detection.This protocol can induce 25% apoptosis of MCF-7 cells in actual sample detection.The constructed biosensor can simultaneously detect and treat tumor cells,which is expected to provide a new method for tumor detection and treatment.In the second part,a biosensor platform based on DNA Walker with Au NRs particles for visual detection of glutathione was constructed.Using the characteristic that glutathione can specifically cleave disulfide bonds(S-S),the DNA Walker chain is released through the cleavage of S-S.DNA Walker can walk continuously along Au NRs,and finally form a large amount of G-quadruplex / hemin DNAzyme.In the presence of hydrogen peroxide,luminol emits light to achieve ultra-sensitive detection.The linear equation of biosensor detection was y = 210.814 lg C-52.828.R2 = 0.997.The detection range is 10 nm-500 μM,and the minimum detection limit is 0.15 n M.The constructed biosensor platform can be used for the specific detection of glutathione and is expected to be applied to cancer diagnosis. |
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