The New Electrochemiluminescence Sensing Method Based On Efficient Metal Complexes And Novel Nucleic Acid Amplification Strategy

Electrochemiluminescence(ECL)is a self-emission of light from electrochemically excited luminophores through a series of redox reactions,and its intrinsic properties based on dual electrochemical/photophysical nature make it a powerful and attractive method across diverse fields.ECL is superior to photoluminescence(PL)in many aspects and has become a reliable analytical tool in biomedical,biochemical analysis,food industry and environmental science due to its advantages of near-zero background response,wide dynamic range,high sensitivity,simple device,low cost,good spatial and temporal resolution.In recent years,ECL has been widely used in biosensing systems,mainly including the detection of trace biomarkers such as small biological molecules,proteins,RNA and DNA fragments,and applied to the early diagnosis of diseases.However,with the rapid development of modern nanomaterials and the deepening of biological research by researchers,more stringent requirements have been put forward for the analytical performance of ECL sensing platform,such as sensitivity and specificity.Therefore,this paper mainly explores how to achieve the performance improvement of ECL biosensor from two aspects.On the one hand,a novel ECL reaction system with excellent luminescence characteristics is developed to overcome the defects of traditional ECL luminophores.On the other hand,a creative nucleic acid amplification strategy is designed to achieve high-efficiency conversion of targets,thereby providing new ideas and methods for the construction of highly sensitive and highly selective ECL biosensors,which can be further applied to the quantitative detection of biomarkers.And it is expected to promote its potential applications in the field of luminescent molecular devices and disease diagnosis.The research work is mainly divided into the following parts:1.Construction of an ultrasensitive electrochemiluminescence biosensor for the detection of multiple microRNAs based on a novel nucleic acid amplification strategy of dual circuit catalyzed hairpin assemblyTraditional ECL biosensors are usually only used for single-component detection,but there are false positive or false negative phenomenon.Although multi-component detection can improve the accuracy and efficiency of disease diagnosis,there are crossreactions between different ECL luminophores,which is not conducive to the precise treatment of diseases.Simultaneously,the catalyzed hairpin assembly(CHA)has the shortcoming of insufficient amplification efficiency in conventional nucleic acid amplification strategy.How to further improve its amplification efficiency is also an urgent problem to be solved.Therefore,here,a novel nucleic acid amplification strategy for double-hairpin DNA-induced dual circuit catalyzed hairpin assembly(DC-CHA)was cleverly designed to fabricate ECL biosensor for dual-target(microRNA-21 and microRNA-155)ultrasensitive detection.Compared with the traditional CHA,the superiority of this novel DC-CHA strategy was that using a special double-hairpin DNA to induce two CHA process effectively,which not only realized the dual-target detection,but also achieved ultrahigh detection sensitivity for the dual circuit amplification of targets.On the one hand,microRNA-21 triggered the first CHA circuit amplification so that a large number of H2 labeled with N-(4-Aminobutyl)-N-ethylisoluminol(ABEI)was immobilized,which could greatly improve the ECL response to achieve the sensitive detection of microRNA-21 due to the energy transfer between ABEI and Ru(bpy)32+.On the other hand,microRNA-155 could trigger the second CHA amplification process,which resulted in the immobilization of the abundant H3 labeled with dopamine(DA)and further a sharply deceased ECL signal due to the distinct double quenching effect of DA for both Ru(bpy)32+ and ABEI,reaching the sensitive detection of microRNA-155.Thus,the proposed biosensor with simple operation presented excellent performance for microRNA-21 and microRNA-155 with the low detection limits of 14.8 amol/L and 5.3 amol/L,respectively.Furthermore,this ECL biosensor could be applied to monitor the expression level of microRNA-21 and microRNA-155 in human breast cancer cells(MCF-7).In general,the novel strategy establishes a simple and effective ECL method for multi-component detection,which can provide more comprehensive reference information for the early diagnosis of the disease and improve its diagnostic efficiency.2.Target-induced 3D DNA network structure as a novel signal amplifier for ultrasensitive electrochemiluminescence detection of microRNAsIt is well known that tripropylamine(TPrA),as a typical coreactant for the luminophore Ru(bpy)32+,has been widely used in the field of ECL.However,TPrA has certain defects such as corrosivity,toxicity and volatility,and the long electron transport distance between TPrA in solution and Ru(bpy)32+on the electrode leads to the decrease of ECL emission efficiency.In addition,the CHA amplification strategy only produces one output DNA per cycle,resulting in insufficient amplification efficiency.Target conversion is typically carried out by enzymatic cleavage or ionic cleavage,but the conversion efficiency is insufficient due to the low cleavage efficiency and needs to be further improved.In view of this,here,target-induced three-dimensional DNA network structure(T-3D Net)by CHA was proposed as a novel signal amplifier to fabricate an ultrasensitive ECL biosensor for microRNA detection.Although conventional CHA could produce only one output DNA in each target cycle,the proposed strategy could produce multiple output DNA by using DNA-functionalized magnetic beads(MBs)and gold nanoparticles(AuNPs)to form T-3D Net.Then,the T-3D Net with high loading capacity could be completely collapsed by dissolving AuNPs to efficiently convert trace microRNA-21 into a large amount of output DNA.Furthermore,boron nitride quantum dots(BNQDs),as an environmentally friendly coreactant,the self-enhanced ECL emitter BNQDs/Ru/PtNPs/Nafion provided a strong initial ECL response owing to the short electron transfer distance between Ru(bpy)32+and BNQDs(signal-on).Next,the DNA duplex probes labeled with ABEI and DA(S1-ABEI/S2-DA)were further immobilized on the nanocomposite to reduce the background signal due to the double quenching effect of DA for both ABEI and Ru(bpy)32+(signal-off).In the presence of the output DNA with enzyme-assisted self-recycling,S2-D A detached from the electrode surface and S1-ABEI restored the hairpin structure to promote effective resonance energy transfer(RET)between ABEI and Ru(bpy)32+,thus greatly improving the final ECL response(signalsuper on).Thus,the ECL biosensor demonstrated superior performance for ultrasensitive detection of microRNA-21 with low detection limit(0.33 amol/L)and was successfully applied to monitor the expression of microRNA-21 in human cancer cell lysates.This strategy provided an ultrasensitive way for the detection of biomolecules,and revealed an effective avenue for diseases diagnosis.3.Highly efficient aggregation-induced electrochemiluminescence of Al(Ⅲ)-Cbatpy metal-organic gels obtained by ultrarapid self-assembly for a biosensing applicationAggregation-induced electrochemiluminescence(AIECL)have attracted extensive interest due to the significant increase in ECL response by restricting free intramolecular rotation and torsion,but traditional AIECL luminophores suffer from limited ECL efficiency,high cost and complex synthetic steps,which dramatically limit their wide application in many fields.To solve the above problems,herein,a novel Al(Ⅲ)-Cbatpy metal-organic gels(Al(Ⅲ)-Cbatpy-MOGs)with nanofiber morphology by ultrarapid coordination of Al3+and 4’-carboxylic acid-2,2’:6’,2"-terpyridine(Cbatpy)is developed,which demonstrates excellent AIECL enhancement behavior far beyond the reported ECL supramolecular gels.In view of the strong affinity of N and O atoms in Cbatpy toward Al3+,Al(Ⅲ)-Cbatpy-MOGs with high viscosity and stability can be assembled in one step within about 15 seconds,easily conquering the main predicaments of current AIECL luminophores:complicated synthesis steps and poor film formation.Impressively,the ECL efficiency of Al(Ⅲ)-Cbatpy-MOGs with super emission is about 20 times higher than that of individual Cbatpy molecules,which is attributed to the aggregation of the organic ligand Cbatpy restricting intramolecular rotation and torsion to reduce nonradiative relaxation.Furthermore,compared with traditional metal complexes,Al(Ⅲ)Cbatpy-MOGs shows the benefits of remarkable biocompatibility and low cost without the involvement of any organic solvents,noble metals and rare metals.As proof,a "signaloff" sensing platform based on Al(Ⅲ)-Cbatpy-MOGs/S2O82-system was constructed for sensitive detection of DA with a low detection limit of 0.34 nmol/L.This strategy provides a novel method to prepare cheap metal-organic gels as a highly efficient AIECL luminophore,promising as luminescent molecular devices and biosensors for clinical diagnostic applications.4.Al(Ⅲ)-Cbatpy-MOGs as an AIECL luminophore combined with rigid triplex DNA walker as an efficient signal magnifier for ultrasensitive DNA assayAt present,the reported single or duplex DNA walkers often suffer from the entanglement and lodging of leg DNA,which seriously limits the walking efficiency and destroys the walking trajectory of the walkers,thus affecting the analytical performance of the constructed sensing platform.In this work,based on the aforementioned Al(Ⅲ)Cbatpy-MOGs with high ECL luminescent efficiency and excellent biocompatibility as an AIECL luminophore,a sensing platform for rapid and ultrasensitive detection of Flu A virus biomarker DNA(fDNA)was constructed in the aid of rigid triplex DNA walker(T-DNA walker)as a high-speed and hyper-efficiency signal magnifier.In the presence of the cheap metal ion Al3+,the ligand Cbatpy can assemble Al(Ⅲ)-Cbatpy-MOGs with 3D nanofiber morphology in only one step within about 15 seconds,which demonstrates high ECL efficiency,excellent biocompatibility and outstanding stability.Notably,on the basis of loop-mediated isothermal amplification-generated hydrogen ions(LAMP-H+),the target-induced pH-responsive rigid T-DNA walker overcomes the limitations of conventional single or duplex DNA walkers in walking trajectory and efficiency due to the entanglement and lodging of leg DNA,exhibiting high stability,controllability and walking efficiency.Therefore,Al(Ⅲ)-Cbatpy-MOGs with excellent AIECL performance combined with CG-C+T-DNA nanomachines with high walking efficiency and stability,the proposed“on-off" ECL biosensor displayed a low detection limit down to 23 ag/μL for target fDNA.And the strategy provided a useful platform for rapid and sensitive monitoring of biomolecules,considerably broadening its potential applications in luminescent molecular devices,clinical diagnosis and sensing analysis.