Construction Of Aptamer-based Nanoprobes And Investigation Of Their Bioanalytical Applications

The main content of bioanalytical chemistry is to obtain the chemical composition,amount,structure and function of bioactive substances in biological systems.The complicated structure and similar chemical characteristics of bioactive substances have put forward very high demands for the analytical methods.Nanoprobe is a kind of ultramicro biosensor that is made up of atoms or molecules,suggesting that nanoparobe is suitable for the analysis of bioactive substances at the molecular level.Generally,nanoprobe is composed of recognition unit and signaling unit.The recognition unit not only directly determines the selectivity of the nanoprobe,but also significantly affects the sensitivity of the nanoprobe.Therefore,the design of recognition molecules is highly important in the construction of nanoprobes.The usually encountered molecular recognition pairs in biological systems are antibody/antigen,enzyme/substrate and so on.Researchers have also developed various organic molecules with recognition function.However,these recognition molecules face considerable challenges in bioanalytical chemistry,such as limited kinds of targets,poor biocompatibility and poor solubility.Aptamers(single-stranded DNA or RNA molecules)are a newly emerged class of functional molecules that can bind to their targets with high selectivity and strong binding affinity,which provides a promising platform for the construction of nanoprobes.Generally,aptamers specifically bind to their targets by folding into unique secondary or tertiary structures that accommodate the molecular structure of the target.Moreover,the dissociation constants of aptamer/target complex are usually in the range of picomolar to nanomolar.Compared to antibodies or organic molecules,aptamers display many distinct advantages,such as design flexibility,a wide range of available targets,easy chemical synthesis/modification,and good biochemical stability.In the past decades,aptamers have been largely employed in the construction of nanoprobes for biosensing,bioimaging and cancer therapy.With the rapid development of nucleic acid research and nanotechnology,aptamers are being used for construction of nanoprobes with special structures and functions.These nanoprobes hold good potential in the analysis of bioactive substances in complicated biological systems.In this work,we designed several highly selective and sensitive nanoprobes through combining aptamers with the excellent optical and magnetic properties of inorganic nanomaterials.We explored the applications of these nanoprobes in the analysis of bioactive substances.The main contents of this dissertation are as follows:(1)We have designed a nanoprobe for protein activity regulation through combining proximity-dependent surface hybridization and the localized surface plasmon resonance of gold nanorods.Two thrombin binding aptamers bind to thrombin simultaneously to form a closed-loop structure,and the activity of thrombin is efficiently inhibited.Subsequent near infrared(NIR)light stimulation liberates the trapped thrombin in the closed-loop structure,resulting in the recovery of thrombin activity.(2)We have constructed an aptamer-guided upconversion nanoprobe to overcome the background fluorescence interference in the analysis of bioactive substances.The upconversion nanoprobe can not only specifically recognize the target in biological samples,but also efficiently avoid the background fluorescence.High sensitivity has been obtained in the detection of lysozyme in fingerprints with the upconversion nanoprobe.(3)We have synthesized NIR emitting persistent luminescence nanoparticles with tunable size and persistent luminescence.Aptamer-guided persistent luminescence nanoprobe is further constructed and its bioimaging application is investigated.The nanoprobe can specifically target the tumor in mice,and it can efficiently eliminate tissue autofluorescence interference as well.Superior sensitivity and resolution have been achieved in mice bioimaging.(4)We have prepared one-dimensional persistent luminescence nanorods with tunable length and persistent luminescence.Aptamer-guided persistent luminescence nanoprobe is further constructed for biosensing.The persistent luminescence nanoprobe can not only recognize serum lysozyme with high selectivity,but it can completely avoid serum autofluorescence as well.Good selectivity and sensitivity have been achieve in lysozyme detection in serum samples.(5)We have combined the denaturation property of aptamer with magnetic separation for the construction of enrichment nanoprobes.Cyclic separation and enrichment of analytes with different dimensions have been realized with the magnetic nanoprobes.