Construction Of Electrochemiluminescence Biosensor Based On Metal Nanoclusters As Signal Probe And Coreaction Accelerator As Amplification Strategy

With the rapid development of bioanalysis chemistry,there is an increasing demand for biocompatibility and chemical property of signal probes in electrochemiluminescence?ECL?analysis methods.The new luminescent nanomaterials not only possess ECL performance,but exhibit good biocompatibility,large specific surface area,strong conductivity and chemical catalytic properties,opening new directions for the application of ECL analytical methods in biomedicine,clinical diagnosis and environmental assessment.Among them,metal nanoclusters as an ultrasmall nanoprobe stand out with good biocompatibility,excellent photostability and ease of labeling,and have received extensive attention and coverage.However,the development of ultrasensitive biosensing based on metal nanoclusters still faces the following two challenges:?1?How to break through the limitation that ultrasmall metal nanoclusters with uniform particle size are difficult to prepare,it is of critical importance to find a simple,rapid and high yield preparation method of metal nanoclusters;?2?How to reduce the luminous efficiency of metal nanoclusters is lower than traditional luminophor,such as Ru?bpy?₃²⁺and its derivatives or luminol,and it is challenging to seek an efficient and simple catalytic pathway to promote its ECL efficiency.Herein,this paper mainly prepares a series of metal nanoclusters via the high controllable electrodeposition method,furthermore,improves the ECL efficiency of metal nanoclusters through coreaction accelerator pathways.The signal probe combined with a variety of biologically amplification strategies,constructs an ECL biosensing platform for achievingultrasensitive detection of biomarkers,to open up a new path for the development of metal nanoclusters in biosensing and bioimaging.The research work is mainly divided into the following parts:1.A highly-efficient electrochemiluminescent Ag nanoclusters/TiO₂ nanomaterials as signal probe for ferrocene-driven light switch bioanalysisThe introduction of coreaction accelerator provides a new way to improve the luminescence efficiency of luminophor,but the new ECL promotion pathways is still placed in the start stage.In this work,the ECL of Ag NCs on the TiO₂ NFs surface could be significantly promoted in comparion with that of pure Ag NCs in solution,through combining the TiO₂ NFs as the co-reaction accelerator and dissolved O₂ as the intrinsic coreactant to in suit generate strong oxidizing intermediate radical OH^·.Further,this developed assay was constructed for the detection of amyloid-??A??based on Fc-labeled DNA as ECL quenching probe and immunoreaction-induced DNA nanostructure,realizing excellent sensitivity with linear range from 50 fg/mL to 500 ng/mL and limit of detection down to 32 fg/mL.As a result,the strategy opened up a new research direction for ultrasensitive ECL bioanalysis based metal NCs.2.In situ electrodeposited synthesis of electrochemiluminescent Ag nanoclusters as signal probe for ultrasensitive detection of cyclin-D1 from cancer cellsMetal nanoclusters?NCs?as a new type of ECL nanomaterials have attracted great attention but their applications are limited due to relative low luminescent efficiency and complex preparation process.Herein,an ultrasensitive ECL biosensor for the detection of cyclin-D1?CCND1?was designed by utilizing in situ electrogenerated Ag NCs as ECL emitters and Fe₃O₄-CeO₂ nanocomposites as coreaction accelerator.As a result,the assay for CCND1detection achieved excellent sensitivity with a linear range from 50 fg/mL to 50 ng/mL and limit of detection down to 28 fg/mL.Impressively,the efficiency of traditional Chinese medicines-sophorae toward MCF-7 cell was successfully investigated due to the overexpression of CCND1 relation to the growth and metastasis of MCF-7 human breast cancer cell.In general,the proposed strategy provided an effective method for anticancer drug screening,and expanded the application of metal NCs in ultrasensitive biodetection.3.Ternary electrochemiluminescence nanostructure of Au nanoclusters as highly efficient signal label for ultrasensitive detection of cancer biomarkerHerein,the Au nanoclusters?Au NCs?functionalized ternary nanostructure with significant ECL emission was first proposed to fabricate an ultrasensitive immunosensor for carcinoembryonic antigen?CEA?detection,which was established by combining bovine serum albumin?BSA?templated Au NCs as the luminophor,tris?3-aminoethyl?amine?TAEA?as the coreactant and Pd@CuO nanomaterial as the coreaction accelerator via covalent attachment in a nanostructure.Through the dual self-catalysis including intramolecular coreaction between TAEA and Au NCs,and intramolecular coreaction acceleration from Pd@CuO to TAEA,the Au NCs-TAEA-Pd@CuO achieved excellent ECL performance so that the detection limit of the immunosensor for measuring CEA antigen was down to 16 fg/mL in the absence of any additional signal amplification assay.Moreover,the method for preparing ECL nanostructure-based metal NCs should be devoted to the development of highly efficient ECL signal label,outlining a significant scheme toward biological testing and clinical diagnosis.4.Programmable modulation of copper naonoclusters electrochemiluminescence via DNA nanocranes for ultrasensitive detection of microRNAThe ECL efficiency of metal nanoclusters is limited as the tight molecular arrangement of the ECL probes,due to the inner filter effect.Herein,the DNA nanocrane with functionalized manipulator and fixed-size base offered a programmable approach to modulate the luminous efficiency of copper naonoclusters?Cu NCs?for achieving remarkable ECL enhancement,further the Cu NCs as signal label was constructed in biosensor for ultrasensitive detection of microRNA-155.Specially,the DNA nanocrane was first constructed by combining of binding-induced DNA assembly as manipulator and tetrahedral DNA nanostructure?TDN?as base,which harnessed a small quantity of specific target?miRNA-155?binding to trigger assembly of separate DNA components for producing numerous AT-rich double-stranded DNA?dsDNA?on the vertex of TDN.Upon the incubation of Cu²⁺on the AT-rich dsDNA,each DNA-stabilized Cu NCs probe could be in situ electrochemically generated on an individual TDN owing to the A-Cu²⁺-T bond.Thus,the generation of Cu NCs was highly regulated with AT-rich dsDNA as the template,and its lateral distance was tuned by the TDN size,which were two key factors to influence the luminous efficiency of Cu NCs.By coordinate modulation,the detection limit of the ultrasensitive biosensor for miRNA-155 down to 36 amol/L.5.Highly efficient and simultaneous cathodic and anodic electrochemiluminescence emission of Au nanoclusters for the detection of multiple biomarkersAs a controllable and sensitive analytical method,ECL provides a feasible platform for high-throughput detection of multiple biomarkers.Howevwe,traditional dual-biomarkers detection with dual-luminophors cannot eliminate the cross-reaction of different ECL probes,thus limiting popularization of the methodology.For the first time,simultaneous cathodic and anodic ECL emissions of Au nanoclusters?Au NCs?as highly efficient bipolar ECL probes were constructed for synchronous determination of carcinoembryonic antigen?CEA?and mucin 1?MUC1?based on different coreaction accelerator and coreactant.Under the potential cathodic scanning,TiO₂ nanosheets?TiO₂ NSs?as cathodic coreaction accelerator catalyzed the reduction of coreactant dissolved O₂ for promoting cathodic ECL emission of Au NCs,while,under the potential anodic scanning,Cu₂O@Cu nanoparticles?Cu₂O@Cu NPs?as anodic coreaction accelerator stimulated the oxidation of coreactant N,N-diethylethylenediamine?DEDA?for enhancing anodic ECL emission of Au NCs.Thus,Thus,the large difference of ECL peak potential?².7 V?of Au NCs-TiO₂ NSs/O₂ and Au NCs-Cu₂O@Cu NPs-DEDA provided an access for potential-resolution detection of dual biomarkers,resulting in great sensitivity and accuracy toward the detection of CEA down to 0.43 pg/mL and MUC1 down to5.8 fg/mL.As a proof of concept,the detection methodology containing only luminophor easily solved one main technical challenge of dual-biomarkers detection with cross reactions of dual-luminophors.