Construction Of Biosensor Based On High Efficiency Electrochemiluminophores For Detection Of SARS-CoV-2 Nucleocapsid Protein And MiRNA

Electrochemiluminescence（ECL）is a chemiluminescence phenomenon initiated by electrochemical reactions at the interface of electrodes.As a clever combination of electrochemical and chemiluminescence analysis technology,ECL technology possesses the advantages of strong controllability,rapid response,simple operation,high sensitivity and accuracy,and has attracted wide attention in basic research and commercial applications.Electrochemiluminophores with excellent ECL performance is the key to realize sensitive ECL analysis.Iridium（Ⅲ）complexes and porphyrins have been applied in ECL biosensing analysis as electrochemiluminophores due to their excellent photoelectric properties.However,the two electrochemiluminophores mentioned above have the problem of low ECL efficiency in aqueous solution environment,which further limits their application in ECL field.Therefore,the development of efficient and simple strategies to improve the ECL efficiency of iridium（Ⅲ）complexes and porphyrins is of great value to expand their ECL analytical applications.Novel Coronavirus pneumonia（COVID-19）is a highly contagious viral infection caused by severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）.Nucleocapsid protein（ncov NP）provides a possibility for the diagnosis of SARS-CoV-2 due to its high stability and abundance.Micro RNA（miRNA）is a class of short,single-stranded and non-coding RNA（18-25 nucleotides）.Its abnormal expression level is closely related to the occurrence of cancer and cardiovascular diseases.Therefore,it is essential to realize ultrasensitive detection of ncov NP and miRNA for intervention therapy of related diseases.Among the technologies used for ncov NP and miRNA analysis,ECL sensing technology has attracted much attention due to its advantages of simplicity and high sensitivity.Based on the above observations,this paper aims to improve the ECL efficiency of iridium（Ⅲ）complex and porphyrin nanomaterials by means of morphology regulation,interfacial charge modulation and the introduction of porous complexes,and construct ECL biosensors with excellent performance to achieve the ultrasensitive analysis of ncov NP and miRNA.The main work is as follows:（1）An ECL biosensor based on morphology controlled iridium complex nanomaterials for SARS-CoV-2 nucleocapsid protein detectionThe morphology of electrochemiluminophores is closely related to ECL properties,thus the control of their morphology is greatly essential for the construction and application of ECL sensor system.In this paper,tris（2-phenylpyridine）iridium（Ⅲ）（Ir（ppy）₃）nanomaterials were prepared by a facile nanoprecipitation method using poly（styrene-co-maleicanhydride）（PSMA）as a functional reagent.The morphology of iridium complex nanomaterials was regulated by modulating the volume ratio of solutions PSMA to Ir（ppy）₃,and the effect of morphology on ECL performance of iridium complex nanomaterials was systematically investigated.Iridium complex nanoparticles（Ir NPs）with excellent ECL performance were selected as admirable electrochemiluminophores to construct biosensors to detect ncov NP.Ir NPs were modified on the electrode surface,which served as the matrix to stepwise capture primary antibody,antigen of SARS-CoV-2 nucleocapsid protein（ncov NP）and secondary antibody bioconjugate coupled with dual quenchers and detection antibody.Taking advantage of the significant quenching effect of noradrenaline（NA）and gold nanoparticles（Au NPs）in the secondary antibody bioconjugate on ECL emission from Ir NPs,the developed ECL biosensor realized the sensitive detection of ncov NP.The integration of morphology-controlled iridium complex nanomaterials and dual quenchers Au NPs-NA provides a promising ECL platform.（2）Porphyrins-mediated interfacial charge modulation to enhance aggregation-induced ECL of iridium complex for ultrasensitive bioassayIr（ppy）₃ has attracted much attention as ECL luminophore,but its inferior ECL efficiency(Φ_ECL)in the aqueous solutions has impeded its further application.Herein,meso-tetra（4-carboxyphenyl）porphine（TCPP）was developed as interfacial charge mediator to dramatically enhance aggregation-induced electrochemiluminescence（AIECL）of Ir（ppy）₃,thus improving itsΦ_ECL.As an interfacial charge mediator,TCPP can not only efficiently accelerate the electrooxidation of Ir（ppy）₃ through its high hole transport ability,but also inhibit the motions of Ir（ppy）₃ to elevate radiative transition efficiency in emission process through its rigid structure,thereby obtaining aggregation-induced emission luminogens（AIEgens）Ir（ppy）₃@TCPP with a significantly enhanced AIECL.Compared to monomer Ir（ppy）₃,Ir（ppy）₃@TCPP increasedΦ_ECL by 18 times.Ir（ppy）₃@TCPP as AIEgens combined with double nucleic acid amplification strategy to construct ECL biosensor for detecting miRNA-126.In the presence of target miRNA-126,strand-displacement amplification（SDA）reaction was triggered to output large amounts of ST1 and ST2.When the obtained ST1 and ST2 hybridized with S2 captured by Ir（ppy）₃@TCPP modified on the electrode,rolling-circle amplification（RCA）reaction was initiated,and S3 labeled with gold nanoparticles and glucose oxidase（GOx）was subsequently captured.GOx catalyzed substrate glucose to produce H₂O₂,which quenched the ECL signal of Ir（ppy）₃@TCPP/tripropylamine system,thus realizing the highly sensitive detection of miRNA-126.This work creatively exploited interfacial charge modulation as an effective way to enhance AIECL efficiency.The prepared Ir（ppy）₃@TCPP provided much attractive ECL system for bioanalysis.（3）Porous complex-mediated dual ECL of porphyrins for bioassayPorphyrins have been considered as promising electrochemiluminophore candidates,but their properties of aggregation-caused quenching（ACQ）lead to inferiorΦ_ECL.Furthermore,current application about porphyrins only limited to cathodic ECL emission.This paper utilized self-assembly of TCPP and cage-like porous complex（C₂H₅）₃NH[Co₄Cl（TC4A-SO₂）（INA）₄（Co Cl₂）]（SWU-1）to prepare TCPP@SWU-1nanocapsules（TCPP@SWU-1 NCs）.The introduction of cage-like porous complex SWU-1 not only effectively weakened ACQ effect of TCPP to improve electron-hole recombination efficiency,but also increased the stability of anion and cation radicals.Compared with TCPP aggregate,the anodic and cathodicΦ_ECL of designed TCPP@SWU-1 was significantly increased by 8.7-fold and 3.9-fold,respectively.Dual emission TCPP@SWU-1 as ECL luminophore was successfully applied to construct ECL biosensor for miRNA-126 ratio detection based on the quenching effect of BHQ2 on dual emission.Firstly,the glass carbon electrode（GCE）was modified with TCPP@SWU-1NCs to capture S3,and then S4 labeled with BHQ2 was introduced to quench the dual ECL signals.In the presence of target miRNA-126,target-driven non-enzymatic toehold-mediated strand displacement reaction（TSDR）was initiated to output large quantities of single stranded S1.By utilizing the hybridization reaction between S1 and S4 modified on the electrode surface,S4 labeled with BHQ2 was removed from the electrode surface,and the dual ECL signals were restored.The highly sensitive ratio detection of miRNA-126 was realized based on the relationship between the ratio of dual ECL signals and the concentration of the target.This work pioneered the use of cage-like porous complex SWU-1 to weaken the ACQ effect of TCPP and significantly enhance its dual ECL emission.Porous complex-mediated dual-emitting TCPP@SWU-1 NCs provided an ideal dual-emitting ECL luminophore,extending the application of TCPP in single-luminophore-based ECL ratio analysis.