Novel Analytical Platform And Protein Delivery Based On Nanomaterials

In recent years,fluorescent biosensors have attracted extensive attention,and been widely used in clinical diagnosis,pharmaceutical development and environm ental protection.At the same time,utilizing fluorescent nanomaterials and novel analytical technologies has also achieved rapid development.Among them,fluorescent carbon nanoparticles?CNPs?,with good biocompatibility,easy preparation,and controlled light emission provide new ideas and means for the development of novel sensors.By rationally changing composition and surface functional groups of CNPs,researchers have prepared many new CNPs,studied their luminescence properties,and constructed different CNPs-based sensors to broaden the application of fluorescent nanomaterials.By using new nanomaterials to build sensor platforms,researchers have continuously developed fluorescent sensors with lower background signals and stronger detection signals.On the one hand,due to its excellent electronic properties,structural characteristics and mechanical properties,novel two-dimensional nanomaterials have attracted widespread attention in the field of constructing new types of sensors.Researching new two-dimensional nanomaterials for the construction of sensing platforms can improve the ability to analyze and identify objects,reduce detection limits,and increase selectivity.This helps to further apply analytical methods and detection systems to live analysis and diagnose.However,there has been little research reported on Ti₃C₂ nanosheets-based fluorescent sensors.Herein,we presented a proof-of-concept demonstrating the application of Ti₃C₂ nanosheets as a novel fluorescence-based enzyme activity detection platform.On the other hand,protein drugs have made great progress in the field of medicine,but protein membranes have poor membrane permeability.There are still great challenges in how to transport proteins into cells.Based on the excellent specific surface area and transport function of nanomaterials,we have designed a highly efficient and large-scale transporter strategy.The main research content is as follows:?1?Highly photoluminescent nitrogen-doped carbon nanoparticles?N-CNPs?were prepared by a simple and green route employing sodium alginate as carbon source and tryptophan as both of nitrogen source and functional monomer.The as-synthesized N-CNPs exhibited excellent water solubility and biocompatibility with a fluorescence quantum yield of 47.9%.The fluorescence of the N-CNPs was intensively suppressed by the addition of ascorbic acid?AA?.The mechanism of the fluorescence suppression was investigated,and the synergistic action of inner filter effect?IFE?and static quenching effect?SQE?contributed to the intensive fluorescence suppression,which was different from those reported for the traditional redox based fluorescent probes.Owing to the spatial effect and hydrogen bond between AA and the groups on the N-CNP surface,excellent sensitivity and selectivity for AA detecting was obtained in a wide linear relationship from 0.2?M to 150?M with a detection limit as low as 50 nM?signal-to-noise ratio of 3?.The proposed sensing systems also represented excellent sensitivity and selectivity for AA analysis in human biological fluids,providing a valuable platform for AA sensing in clinic diagnostics and drug screening.?2?In this work,N-CNPs modulated turn-on fluorescent probes were developed for rapid and selective detection of histidine.The fluorescence of N-CNPs can be selectively quenched by Cu?II?ions with high efficiency,and restored after addition of histidine,owing to the competitive binding of Cu?II?ions and histidine that removes Cu?II?ions from N-CNPs surface.Under the optimal conditions,a linear relationship between the increased fluorescence intensity of N-CNP/Cu?II?ion conjugates and the concentration of histidine was established in the range from 0.5 to 60?M.The detection limit was as low as 150 nM?signal-to-noise ratio of 3?.In addition,the as-prepared N-CNP/Cu?II?ion nanoprobes showed excellent biocompatibility and were applied for a histidine imaging assay in living cells,which presented great potential in bio-labeling assays and clinical diagnostic applications.?3?As one of emerging inorganic graphene analogues,two-dimensional titanium carbide?Ti₃C₂?nanosheets have attracted extensive attention in recent years due to their remarkable structural and electronic properties.Herein,a sensitive and selective nanoprobe for fluorescent probing the phospholipase D activity was developed based on ultrathin Ti₃C₂ nanosheets-mediated fluorescence quenching effect.Ultrathin Ti₃C₂nanosheets with¹.3 nm in thickness were synthesized from bulk Ti ₃AlC₂ powder by a two-step exfoliation procedure,and further modified by natural phospholipid that doped with rhodamine B-labeled phospholipid?RhB-PL-Ti₃C₂?.The close proximity between RhB and Ti₃C₂ leads to efficient fluorescence quenching?>95%?of RhB by energy transfer.Phospholipase D-catalyzed lipolysis of the phosphodiester bond in RhB-PL results in RhB moving away from the surface of Ti₃C₂ nanosheets and subsequent fluorescence recovery,providing a fluorescent“switch-on”assay for the phospholipase D activity.The proposed nanoprobe was successfully applied to quantitative determination of phospholipase D activity with a low limit of detection(0.10 U L^-1),and for its inhibition measurement.Moreover,in situ monitoring and imaging the activity of phospholipase D in living cells were achieved using this biocompatible nanoprobe.These results reveal that Ti₃C₂ nanosheets-based probes exhibit great potential in fluorometric assays and clinical diagnostic applications.?4?Efficient protein delivery into target cells is highly desirable for protein therapeutics.Current protein delivery methods commonly suffer from low protein loading capacity,poor cell-type specificity,and invisible protein release.Herein we report a protein@inorganic nano-dumpling?ND?system as a novel intracellular protein delivery platform.Structurally similar to Chinese traditional food dumpling,ND consists of a pre-assembled protein core formed by metal-ion-directed self-assembly of protein cargos that are fused to histidine-rich green fluorescent protein(H₃₉GFP),which is further wrapped by manganese dioxide?MnO₂?nano-shell via in situ biomineralization.The unique protein@inorganic structure endows NDs with an unprecedentedly high loading capacity?>70 wt.%?and enhanced stability of proteins.NDs use surface-tailored folic acid to target and internalize into cancer cells specifically,and the protein release is bistimuli-responsive triggered by reductive and acidic intracellular microenvironments.Moreover,the MnO₂ nano-shell is an efficient fluorescence quencher of inner fused GFPs and a“turn-on”magnetic resonance imaging?MRI?contrast agent via triggered-release of Mn²⁺ions,which enables dual-activatable fluorescence/MRI bimodal imaging of protein release.For functional protein delivery,the NDs is highly potent and specific to deliver cytotoxic protein ribonuclease A?RNase A?into cultured target cells and tumor site in a xenografted mouse tumor model.Our approach may open up new opportunities to advance pr otein-based cancer therapeutics.