Electrochemiluminescence System Based On Polymer Nanomaterials For Detecting COVID-19 Markers And MicroRNA

Electrochemiluminesence（ECL）combines the advantages of electrochemical and chemical luminescence technology,possessing high sensitivity,low background signal,and wide detection range.It has been widely used in the field of biological analysis.In ECL system,luminophores play important role.The enhancement of ECL intensity of luminophore is the key for improving the sensitivity of ECL detection.Polymers exhibit advantages including excellent photostability,easy functionalization and good biocompatibility,and have attracted attention in the field of ECL biosensing.However,their ECL efficiency is usually not high enough.The tactics of coreactants enrichment,coreactants mediation and resonance energy transfer can effectively enhance the ECL intensity of luminophores,which also provide inspiration to improve the ECL efficiency of polymers.In addition,ECL luminescent nanomaterials possessing near infrared（NIR）emission have aroused the interest of researchers in biosensing and imaging,and it is of great significance to develop novel NIR polymer nanomaterials in the ECL field.COVID-19 caused by severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）has endangered human health.SARS-CoV-2 nucleocapsid protein（ncov NP）can be served as one of the diagnostic markers of COVID-19 due to its abundant protein.SARS-CoV-2 Rd RP gene has high specificity,which can effectively distinguish SARS-CoV-2 from SARS-CoV,and is often used as a marker for the diagnosis of COVID-19.Therefore,sensitive detection of ncov NP and SARS-CoV-2Rd RP genes is of great significance for the detection of COVID-19.Micro RNA-21 is a marker for some tumors,and change of its content can reflect the differentiation status of tumors.Therefore,sensitive detection of micro RNA-21 is of great significance for protecting human health.Among various technologies analysis for ncov NP,SARS-CoV-2 Rd RP gene and micro RNA-21,ECL sensing technique has shown great application prospect owing to its advantages of high sensitivity and simple operation.This paper explored the application of coreactants enrichment and coreactants mediated enhancement strategies to improve the ECL efficiency of polymer nanomaterials.Meanwhile,energy transfer between polymer and dye was utilized to synthesize ECL luminophore with NIR emitting,thus constructing three kinds of biosensors based on single signal and double signal ratio strategies to realize sensitive detection of ncov NP,SARS-CoV-2 Rd RP gene and micro RNA-21.The research works are displayed as follows:（1）Hydrophobic localized enrichment of coreactants to enhance ECL of conjugated polymer for detecting SARS-CoV-2 nucleocapsid proteinsThe enrichment of coreactants is one of the keys to improve the sensitivity of ECL detection.This work developed a novel hydrophobic localized enrichment strategy of coreactants utilizing the inner hydrophobic cavity ofβ-cyclodextrin（β-CD）.Pt nanoparticles（Pt NPs）were grown in situ on the coordination sites for metal ions ofβ-CD to prepareβ-CD-Pt nanocomposites,which not only could highly efficiently enrich coreactant 3-（dibutylamino）propylamine（TDBA）through its hydrophobic cavity,but also could immobilize TDBA via Pt-N bond,thus significantly enhancing the ECL intensity of carboxy-functionalized poly[2,5-dioctyl-1,4-phenylene]（PDP）polymer nanoparticles（PNPs）.The ECL luminophores PDP PNPs coupled nanocomposites TDBA-β-CD-Pt to construct a biosensor for ncov NP determination.PDP PNPs were first modified to the surface of a glass carbon electrode,and then the primary antibody（Ab₁）,capture antigen and secondary antibody complexes（TDBA-β-CD-Pt@Ab₂）were sequentially captured to construct a sandwich immunobiosensor.The limit of detection was as low as 22 fg/m L for ncov NP.TDBA-β-CD-Pt nanocomposites shared with an inspiration in hydrophobic localized enrichment of coreactants for improving the sensitivity of ECL detection.The luminophores PDP PNPs integrated TDBA-β-CD-Pt nanocomposites to open up a promising and sensitive ECL platform,providing a new method for ncov NP detection.（2）Persulfate-mediated dual-emitting conjugated polymers for ECL ratio analysis of SARS-CoV-2 Rd Rp genePolymers have attracted attention as luminophores due to their excellent ECL properties.However,the current research and application of polymers mainly focus on anode emission,and ECL efficiency is not high enough,thus showing a limited application.This work exploited the persulfate-mediated dual-emission characteristics of poly[2,5-dioctyl-1,4-phenylene]polymer nanoparticles（PDP PNPs）.The two ECL emissions were collected synchronously at-2.0 V and+1.0 V with persulfate(S₂O₈^2-)as cathodic coreactant and 3-（dibutylamino）propylamine（TDBA）as anodic coreactant,respectively.Interestingly,S₂O₈^2-can simultaneously mediate the double emissions,significantly enhancing both cathode emission and anode emission.The dual-emission mechanism was explored carefully and enhancement mechanism of cathodic coreactant S₂O₈^2-to anodic emission was hypothesized to be attributed to SO4^?-radicals,which was produced from S₂O₈^2-during cathodic potential scanning and oxidized PDP PNPs to generate more cation radical,thus enhancing anodic emission of PDP PNPs.Moreover,the black hole quencher-2（BHQ2）was exploited as dual-function moderator to quench dual emissions of PDP PNPs synchronously.PDP PNPs coupled with BHQ2 to build ECL ratiometric system for detecting SARS-CoV-2 Rd Rp gene and its limit of detection was 25.1 a M.Persulfate-mediated double emissions provided a new way to improve the efficiency of ECL emission from polymers and expand their application.The clever integration of dual-emitting PDP PNPs and dual-regulating BHQ2 created a promising single-luminophore-based ratiometric ECL platform,developed an attractive ECL method for detecting SARS-CoV-2 Rd Rp gene.（3）Coreactants-free biosensor based on polymer-enhanced near-infrared ECL nanomaterial for micro RNA-21 detectionNear-infrared（NIR）luminescence has attracted attention in biological imaging and disease diagnosis due to its deep tissue penetration and high spatial and temporal resolution.Dyes have been widely used in biological imaging and fluorescent labeling due to their high sensitivity and high quantum yield.However,dyes have drawbacks in their application,such as easy aggregation induced quenching,lack of functional group modification,and easy signal interference of excitation light.In this work,fluorescent dye（IR-783）wasdopedwith poly[9,9-bis（3‘-（N,N-dimethylamino）propyl）-2,7-fluorene]-alt-2,7-（9,9 dioctylfluorene）]（PFN）to prepare near-infrared emitting polymer nanoparticles（IR-PFN）by nanoprecipitation method.The prepared IR-PFN nanocomposites exhibited strong ECL emission in the near-infrared region with an luminescence wavelength of 815 nm.The IR-PFN luminophores integrated with the spontaneous entropy driven chain replacement reaction（ESDRs）to construct the biosensor for micro RNA-21 detection,and the detection limit was 0.251f M.The enhanced near-infrared emission of IR-PFN by polyfluorene opens a new avenue for the design of NIR-ECL materials,which has broad application prospects in ECL biosensing and imaging.