Construction Of Electrochemiluminescence Biosensors For Sensitive Detection Of MicroRNA Based On Transition Metals And Transition Metal Oxides As Coreaction Accelerators

Electrochemiluminescence（ECL）analytical technology is a neotype analytical method that combines the electrochemical technology and chemiluminescence analytical technology.ECL not only owns the controllability of electrochemical technology and the sensitivity of luminescence analytical technology,but also possesses the merits of broad dynamic range,low background signal and simple operation,which has therefore become a hotspot in the research field of analytical chemistry.In the field of bioanalysis,ECL biosensor,a combination of ECL analytical technology and biosensing technology,has been widely used in the analysis of microRNAs（miRNAs）,a type of tumor markers.Unfortunately,the expression level of miRNAs in cancer cells is extremely low.As a result,in practical application,it’s inevitable to adopt some novel and efficient amplification strategies to improve the sensitivity of miRNA detection.On the one hand,in some binary ECL system,such as nanoclusters/persulphate system,quantum dots/persulphate system and organic luminophores/dissolved oxygen system,because the reaction activity of luminophore or coreactant is so weak that the ECL intensity of the system is unable to meet the requirement of analysis.Whereas,by introducing coreaction accelerator to expedite the decomposition process of the coreactant to intermediate radicals,the ECL signal of the system can be significantly enhanced,so as to improve the detection sensitivity.On the other hand,in order to improve the detection sensitivity of miRNA,highly efficient nucleic acid amplification strategies are often needed to convert the target miRNA signal to massive nucleic acid signal output,hence to improve the detection sensitivity,simplify the experimental operation and shorten the analytical time.Therefore,taking luminol/dissolved oxygen system as the research object,this paper was aimed to improve the sensitivity of ECL biosensor for miRNA detection from two aspects,searching for transition metals and transition metal oxides materials with excellent properties,good biocompatibility and low costs as coreaction accelerators,and designing highly efficient nucleic acid amplification strategy.The research work of this paper mainly included the following aspects.1.Ternary electrochemiluminescence system based on multivalent manganese oxides microflowers（MnO_x MFs）as a coreaction accelerator for the construction of biosensor in sensitive miRNA detectionDissolved oxygen is a type of safe,mild and endogenous coreactant.However,the low reaction activity of dissolved oxygen limits its application in ECL bioanalysis.The multivalent manganese oxides microflowers（MnO_x MFs）contained three kinds of manganese oxides with different valence states（MnO/Mn₂O₃/γ-MnO₂）,which could effectively boost the generation of reactive oxygen species（ROS,including OH·and O₂·^-,etc.）from dissolved oxygen.In this work,we employed MnO_x MFs as a coreaction accelerator of luminol/dissolved oxygen and a carrier for the luminophore for shortening the electron transfer distance in ECL reaction and reducing the energy loss.Therefore,the initial ECL signal of the system was remarkably enhanced and showed as a“signal on”state.Then,target miRNA-21 triggered hybridization chain reaction（HCR）and formed dendritic DNA structures to immobilize amounts of ECL quencher doxorubicin-ferrocenecarboxylic acid（Dox-Fc COOH）,for distinctly quenching the ECL signal of the biosensor and turning it into a“signal off”state.Therefore,an ultrasensitive miRNA biosensor was proposed for the detection of a tumor marker,miRNA-21.The detection linear range of the biosensor was 100 amol/L～100 pmol/L,and the limit of detection was 10.72 amol/L.Furthermore,the fabricated biosensor was applied to the detection of miRNA-21 in tumor cell（MCF-7）lysate,and it also exhibited good linear relationship,indicating that the biosensor possessed promising prospect in clinic diagnosis.2.A novel luminol/dissolved oxygen/Fe@Fe₂O₃ nanowires ternary ECL system combined with highly efficient strand displacement amplification strategy for sensitive detection of miRNAStrand displacement amplification（SDA）strategy is a kind of classical nucleic acid amplification strategy.However,in traditional SDA,each complete polymerization and nicking cycle can only produce a single strand of DNA,therefore the amplification efficiency is remained to be further improved.In this work,Fe@Fe₂O₃ nanowires were used as a coreaction accelerator in luminol/dissolve oxygen system,which could decompose the dissolved oxygen to active intermediate radicals for enhancement of the ECL intensity from the system.Meanwhile,an SDA cascading with a strand displacement reaction（SDR）was designed to further amplify the output signal of SDA.In short,the secondary target（ST）strands produced through SDA reacted with a T structure formed by three DNA strands to undergo a SDR so as to replace two DNA strands S1 and S2 that were labelled with ferrocene（Fc）at both ends for quenching the ECL signal of the biosensor.Therefore,every time one ST strand was produced,it was equivalent that the target signal was amplificated by four times.Employed for miRNA detection,the constructed biosensor exhibited satisfactory selectivity and stability with a linear range of 100 amol/L～100 pmol/L,and the calculated detection limit was 50.2 amol/L.3.An ultrasensitive biosensor based on ternary electrochemiluminescence system using copper nanospheres as a coreaction accelerator with a target-recycled double-output strand displacement amplification strategy for the detection of miRNAFor traditional SDA reaction,after hybridization,the target is combined with the template DNA and hasn’t been released throughout.Therefore,the utilization efficiency of target is restrained.Herein,we designed a target-driven catalyzed hairpin assembly（CHA）reaction,in which the product of CHA was used as the substrate of SDA.Hence,this reaction system could not only achieve double output,doubling the output efficiency on the basis of traditional SDA,but also realize the recycling of target miRNA,improving the amplification efficiency of SDA distinctly.Besides,for the output mimic target（MT）strand,it was designed to undergo a non-linear hybridization chain reaction（HCR）with another single-stranded DNA（ss DNA）to generate hyperbranched DNA dendrimer,which was used for embedding a mass of luminescent substance,the compound of doxorubicin and luminol（Dox-Lum）.What’s more,in the presence of the coreaction accelerator copper nanospheres,the dissolved oxygen in the detection solution was activated into a great number of reactive oxygen species（ROS）,and the ECL emission of the system was significantly promoted.The linear range of the proposed biosensor for miRNA-21 detection was 100 amol/L～1 nmol/L,and the limit of detection was 58.9amol/L.