Construction And Application Of Novel Fluorescent Sensors Based On DNA Nanomaterials

With the uninterrupted growth and improvement of life science technology,human beings have gradually deepened their understanding to it,but meanwhile they are facing various challenges from the nature and themselves,such as envir onmental deterioration and disease outbreaks.Therefore,the development of fast and sensitive biosensors is of great significance to the development of environment monitoring and disease screening.In particular,due to the novel coronavirus(SARS-COV-2)rapid spread,COVID-19(Corona Virus Disease)has exploded all over the world,making humans deeply aware of the importance of rapid and accurate early detection to assist early intervention of the disease and prevent the spread of the virus.Fluorescence biosensors have become one of the most widely used biosensors due to their non-destructive operation mode,sensitive signals and fast response.Traditional fluorescent biosensors use enzymes,antibodies,or small organic molecules as recognition units.Because of the difficulty of preparation and poor versatility of them,researchers have been working to develop simpler and more universal fluorescent sensors.The emergence of DNA nanomaterials has greatly expanded the application of fluorescence biosensors.DNA as carrier of genetic information,are also regarded as building blocks for nanomaterials.Due to the advantages of good programmability,thermodynamic stability,biocompatibility,as well as excellent functions such as molecular recognition,encapsulation and signal amplification,DNA nanomaterials are widely used in the design of fluorescence biosensors.At present,fluorescent sensors based on DNA nanomaterials still have a lot of room for improvement in biosensing applications due to the fact that the DNA molecules themselves are susceptible to nuclease degradation and the lack of construction strategies.This research paper makes full use of the advantages and functions of DNA nanomaterials to construct a more stable,accurate and sensitive fluores cence biosensor,which is expected to provide a new strategy for the development of biosensors.The specific research content is as follows:(1)In Chapter 2,in order to solve the problems of traditional functional nucleic acid probes being susceptible to nuclease degradation,non-specific binding to proteins,and poor cell uptake ability,we take advantage of the programmability and encapsulation function of DNA nanostructures to encapsulate the functional nucleic acid probe(L-histidine DNAzyme in this case)in the cavity of the DNA nanocage to construct the DNA nanocage probe.Compared with traditional DNAzyme fluorescent probes,DNA nanocage probes not only improve the stability of DNAzyme and protect it from nuclease degradation and non-specific adsorption of proteins.More importantly,by adjusting the cavity of the DNA nanocage,we found that the protective effect of the DNA nanocage on the encapsulated probe is size-selective.Besides,DNA nanocage probes also exhibit excellent cell self-transport ability and biocompatibility.Therefore,the encapsulation strategy of DNA nanocage pr ovides a simpler,versatile and efficient method for the application of functional nucleic acid probes in the fields of biomedical research and clinical diagnosis.(2)In Chapter 3,based on the size dependence of DNA nanocage,we further construct a DNA molecular sieve for biosensing to achieve size-selective identification of targets.The system not only protects the encapsulated nucleic acid probes,but also successfully realizes size-selective discrimination between mature micro RNA(mi RNA)and precursor pre-micro RNA(pre-mi RNA)in living cells,avoiding the interference of pre-mi RNA to mi RNA and further improving the accuracy of functional nucleic acid probe detection.Therefore,DNA molecular sieve provides a simple,universal,and controllable strategy for size selective identification in biomedical research and clinical diagnosis.(3)In Chapter 4,in order to further improve the sensitivity of DNA molecular sieve,we combined DNA molecular sieve with CRISPR/Cas12a(Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a)fluorescence detection system,and developed a size-selective DNA nanocage-based activatable CRISPR/Cas12a fluorescence biosensor for sensitive and accura te detection of mature mi RNA.The DNA molecular sieve inhibits the cleavage acti vity of CRISPR/Cas12a through spatial isolation,and realizes the size-selective activation of the trans-cleavage activity of CRISPR/Cas12a by mi RNA and pre-mi RNA.In addition,the highefficiency trans-cleavage activity of CRISPR/Cas12a can further improv e the sensitivity of mi RNA detection.Therefore,this strategy is expected to realize the accurate analysis of mi RNA in the fields of biological science and medical diagnosis.(4)In Chapter 5,in order to solve the problems of poor stability of traditional CRISPR/Cas12a signal reporters and un-adjustable detection range,we used DNA functionalized gold nanoparticle to construct spherical nucleic acid signal reporters and explored the potential of them as CRISPR/Cas12a signal reporter s.This chapter studied the relationship between the CRISPR /Cas12 a system and spherical nucleic acid,proved that the spherical nucleic acid signal reporter is more stable than the traditional signal rep orter,and realized the regulation of the detection range of the CRISPR/Cas12a s ystem and the improvement of detection sensitivity.Therefore,this work is expected to open up new ideas for the construction of fluorescent biosens or based on CRISPR/Cas12a.