Research And Application Of The Novel Organic Electrochemiluminescence Luminophores And DNA Walkers In Biosensors

The electrochemiluminescence(ECL)biosensor with good stability,good selectivity,fast response,high sensitivity,fast analysis speed and simple operation have been used in the the analysis of biological systems with complex components and low concentrations.The improvement of recognition capability to the target objects has always been the research topic in the ECL biosensing field.There are two main ways to construct highly sensitive ECL biosensors to realize the sensitive recognition of target,(1)synthesizing the new ECL luminophores,which could improve the ECL efficiency of luminophores and further improve the ECL intensity and stability of the ECL biosensor,(2)improving the recognition capability to the low abundance target of the ECL biosensor by DNA nanomachines.In view of this,this paper mainly used precipitation method,hydrothermal method and sol-gel method to synthesize the new ECL luminophores with high efficiency,combined with the amplification function of DNA nanomachines and the advantages of good biocompatibility to construct a variety of ECL sensors to achieve sensitive recognition of the target,providing a new vision for the application of ECL analysis.The research work of this paper mainly includes the following parts:(1)Construction of microRNA ECL biosensors based on the assembling ordered flower-shaped DNA walkersDNA walkers are a kind of nanoscale molecular device formed by swing arm strands and predetermined track strands,which can move autonomously along the preset orbit and further output the signals during the walking process.In general,the swing arm strands and the DNA tracks were assembled on the gold nanoparticles randomly.The uneven distribution of the swing arm strands and the DNA tracks will lead to the lower recognition efficiency of DNA walkers for target.In this study,the DNA flower nanomachines(DFNs)walkers were construct with the reasonably to detect microRNA let-7a.The DFNs walkers has the characteristics of simple synthesis,low cost and controllable size,which could realize the orderly distribution of the swing arm strands and the DNA tracks,reduce the uneven distribution and cross reaction of the swing arm strands and the DNA tracks on the electrode surface,and improve the recognition ability of the target.The assembling ordered DFNs walkers based ECL biosensor was developed for the detection of microRNA let-7a ranging from 10 fmol/L to 100 nmol/L with the detection limit of 4.5 fmol/L.In conclusion,this work proposed a novel flower-shaped DNA walker,which provided a new idea for designing quantitative and ordered DNA walkers.(2)Construction of microRNA ECL biosensors based on DNA structure transition-inducedaffinityswitchcombinedwith9,10-Diphenylanthracene-regulated perylene micrococrystal as ECL luminophorePlane perylene and its derivates as organic ECL luminophores have received great attentions for ECL bioanalysis due to their high fluorescence quantum efficiency,relative low cost and modifiable structure.However,Pe as a typical plane molecule was susceptible to aggregation induced quenching(ACQ)effect via inter-molecular?-?stacking,resulting in the low ECL efficiency.In this work,we developed a facile method to minimize the aggregation-caused quenching effect(ACQ)effect and improve the ECL efficiency of Pe molecule by preparing 9,10-Diphenylanthracene(DPA)-doped perylene(Pe)micrococrystals(DPA@Pe MCs)to regulate the?-?stacking.We developed an effective recognition strategy using the target-trigerred DNA structure transition as affinity switch for microRNA 21 detection based on DPA@Pe MCs as a novel ECL luminophore,which showed an excellent linear response from 10 amol/L to1 pmol/L with the detection limit down to 4.14 amol/L.In conclusion,this work reduced ACQ by changing the spatial arrangement of Pe molecules in microcrystals and achieved the strong ECL emission of DPA@Pe MCs,which provided a way for the further application of planar PAHs.(3)Construction of microRNA ECL biosensors based on the swing arm location-controllable DNA walkerIn this work,we developed a swinging arm location-controllable DNA walker based on the DNA tetrahedral nanostructures(DTNs)due to the DTNs with the intrinsic nature of mechanical rigidity and well-controlled orientation,in which the swing arm strands and track strands were fixed on the vertices and three edges of DTNs respectively.It avoided the invalid collision caused by the uncontrollable position of the swing arm strands and track strands and improved the reaction efficiency.In addition,an economic and facile strategy was developed through the doping of the nonplanar molecule with propeller?shaped structure tetrakis(4-aminophenyl)ethene(TAPE)into the planar molecular Pe microcrystals via?-?stacking to generate the TAPE-Pe microcrystals(TAPE-Pe MCs),which achieved a significantly amplified ECL in aqueous solution.As a proof-of-concept,the TAPE-Pe MCs based-ECL sensing platform was developed in combination with a swinging arm location-controllable DNA walker to detect microRNA let-7a ranging from 10 fmol/L to 100 nmol/L with the detection limit of 4.92 fmol/L.In conclusion,this work proposed an efficient DTNS walker,which opened a new chapter for designing DNA walkers with controllable relative position.(4)Construction of microRNA ECL biosensors based on the DNA cube walker combined with the novel perylene-titanium oxide clusters as ECL luminophorePerylene and its derivates were the novel organic electrochemiluminescence(ECL)luminophores with high fluorescence quantum efficiency and structural controllability.However,Perylene and its derivates were was susceptible to aggregation induced quenching(ACQ)effect because of their own structure,resulting in resulting in the low ECL efficiency,which limited the practical applications of perylene and its derivates in biological analysis.In this work,the pore confinement-enhanced perylene titanium-oxide clusters(PT-Ti_nO_m)as ECL luminophore were synthesized by sol-gel method.In addition,in this study,we designed an efficient DNA walker with DNA cube as a scaffold combined with the self-promoting PT-Ti_nO_m as ECL platform for microRNA let-7a detection.The ECL biosensor has the following advantages,the DNA cube based walker with DNA cube as the scaffold,which could realize the quantitative and orderly arrangement of swing arm strands and track strands,and reduce the non-specific adsorption caused by the direct introduction of swing arm strands to the electrode surface,improving the recognition efficiency for the microRNA let-7a detection.In addition,the self-promoting PT-Ti_nO_m luminophore could limit the luminophore into a small space,avoiding the aggregation of luminophore and improving the emission efficiency.In conclusion,this work not only opened up a new way to improve the ECL efficiency of planar organic luminophore,but also provided a new idea for the construction of multi-functional DNA walkers.(5)Construction of ECL biosensors based on the novel metal-organic framework(MOF)material as ECL luminescence for detection of H2O2 secreting from cancer cellsOrganic luminescent crystals of polycyclic aromatic hydrocarbons(PAHs)with the non-coplanar construction were synthesized by the reprecipitation method,which showed a strong ECL response due to the aggregation induced enhancement(AIE)effect and greatly improved the stability of PAHs organic luminescent crystals in the aqueous phase.Because their dense molecular stacking caused the increase of inner filter effect and hindered the internal ECL luminophores from being electrochemically activated.In this work,a novel and efficient metal-organic framework(MOF)ECL luminophore(Tcpe-MOF)was synthesized by a simple solvothermal method using 1,1,2,2-tetrachlorophenol(4-carboxylic benzene)ethylene(Tcpe)organic luminophore as ligand.Compared with the Tcpe aggregates prepared by reprecipitation method,the Tcpe ligands were fixed on the rigid metal frame,which limited the free rotation of molecules and reduced the internal filtration effect,realizing the enhanced ECL response and the higher ECL efficiency.We utilized the advantages of the Tcpe-MOF ECL luminophore to construct an ECL biosensor,achieving the sensitive detection of hydrogen peroxide(H₂O₂)and further used for the detection of H₂O₂ secreted from the living cells.Therefore,this work not only prepared a novel and efficient ECL luminophore,but also applied the novel ECL luminophore based biosensor to cell analysis.This work provided a new way to improve the ECL efficiency of AIE type ECL luminophore.