Construction Of Electrochemiluminescence Biosensor Based On Different Signal Amplification Strategies For MicroRNA And DNA Dection

Electrochemiluminescence（ECL）is a phenomenon that induces redox reactions to generate chemiluminescence by applying voltage.This method combines the advantages of electrochemical controllability and photochemical sensitivity,so it is widely employed in biological analysis.The traditional ECL system limits the luminous efficiency of the ECL system due to the low luminous efficiency of the luminophore itself and the low concentration of the coreactant,resulting in lower sensitivity.In recent years,the integration of different amplification strategies（such as nanomaterial amplification,nucleic acid amplification）into ECL biosensors is of great significance in improving its sensitivity and specificity.Inspired by this,this study proposed some different novel methods of sensitivity and specificity to construct an ECL biosensor for detecting the target based on different signal amplification strategies.The specific work are as follows:1.An ultrasensitive electrochemiluminescence biosensor for micro RNA detection based on luminol-functionalized Au NPs@ZnO nanomaterials as signal probe and dissolved O2 as coreactantThe luminol-dissolved oxygen system is an ECL luminescence system developed in recent years,which overcame the traditional biological destructiveness of using hydrogen peroxide as coreactant.However,due to the low concentration of solution oxygen in the solution,lower ECL luminescence was caused effectiveness.In this work,the ZnO nanostars with excellent catalytic performance were used as the coreaction accelerator of luminol-O₂system,ZnO nanostars could expedite the reduction of dissolved O₂,generating more reactive oxygen species（ROSs）to extremely promote electrochemiluminescence（ECL）luminous efficiency of luminol.Thus luminol-functionalized Au NPs@ZnO（L-Au NPs@ZnO）nanomaterials were employed as signal probe to fabricate sensing nano-platform for achieving significant ECL emission as“signal on”state.Moreover,upon the addition of a tiny minority of target mi RNA-21,massive ferrocene（Fc）could be immobilized on the sensing interface through hybridization chain reaction（HCR）triggered-DNA dendrimers self-assembly,in which Fc consumed dissolved O₂ for prominently quenching the ECL emission of signal probe and then reached a“signal off”state.As a result,the biosensor performed a good linearity in 100 a M-100 p M and a low limit of detection（LOD）down to18.6 a M.In general,this work utilized a new coreaction accelerator as an efficient amplification approach for ultrasensitively detecting target analyses,providing a promising approach in luminol-centric ECL bioanalysis fields.2.An electrochemiluminescence biosensor for ultrasensitive detection of micro RNA was constructed based on the enhancement of Au Ag NCs-S2O8^2-system with mixed valence Ce-MOF as coreaction acceleratorCompared with single metal nanoclusters,bimetallic nanoclusters have stronger luminous intensity.Gold-silver alloy nanoclusters showed higher ECL luminous efficiency and luminescence stability,but they still have the disadvantage of relying on large concentrations of coreactants.In this work,an electrochemiluminescence（ECL）biosensor was constructed for mi RNA-21 detection by using gold and siliver bimetallic nanocluster（Au Ag NCs）as luminophore,mixed valence Ce-MOF（MVCM）nanocomposite as coreaction accelerator.Specifically,MVCM could promote the reduction of peroxydisulfate(S₂O₈^2-)to generate more SO₄^·-and expedite electron transfer,thus significantly improving the luminous efficiency of the Au Ag NCs-S₂O₈^2-system.Furthermore,once a tiny amount of mi RNA-21（target）was introduced,the enzyme-asitant strand displacement reaction（SDR）could be triggered,generating plentiful secondary target（ST）,which facilited the sensitivity of the proposed biosensor.Moreover,Fc-labled DNA（H2）as signal quenching probe was designed for restraing the ECL signals of nanocomposite,further realizing the ultrasensitive detection of target.The results performed that proposed biosensor had a detection limit of31.6 amol/L,and also realized the detection of mi RNA-21 in cells.In conclusion,this method proposed a novel coreaction accelerator as an amplification method,which opened up a new method for clinical application of biology.3.Double hairpin DNAs recognition induced a novel cascade amplification for highly specific and ultrasensitive electrochemiluminescence detection of DNAGenerally,nucleic acid amplification strategies are often used to improve detection sensitivity.Simutaneously,it is extremely important to improve the specific recognition of targets and the ability to recognize single nucleotide polymorphism,which helps to enhance the ability to diagnose diseases.Herein,a novel DNA cascade amplification,including double hairpin DNAs recognition-triggered single target recycling（D-STR）and concatenated DNA structure-controlled rolling circle amplification（C-RCA）,was developed as signal amplifier to construct a highly specific and ultrasensitive electrochemiluminescence（ECL）biosensor for human immunodeficiency virus（HIV）DNA fragments detection,which not only revealed tremendous potential in avoiding false positive signals,but also obviously promoted the amplification efficiency simultaneously compared to conventional single recognition of single target.Once the target DNA triggered the rolling circle amplification（RCA）,the obtained RCA products could be anchored on Pt-modified glassy carbon electrode（GCE）via Pt-N bond,capturing massive ruthenium（Ru）-labeled ss DNA as ECL signal tag to generate remarkable ECL emission.As a result,the proposed biosensor showed highly specific and ultrasensitive detection of target with detection limit down to 27.0 a M,which gives great impetus to the development of novel specific biosensor for practical bioanalysis and diagnostic technologies.