Study On The Construction Of Electrochemiluminescence Biosensor Based On Nucleic Acid Amplification And Co-reactive Amplification Strategy

The electrochemiluminescence(ECL)biosensor is a new type of biosensor,which combines biomolecules such as DNA,aptamers,antibodys as molecular recognition elements and the ECL analytical technology as detection tool.Owing to their high sensitivity,good selectivity,fast response,simple operation and low cost,ECL biosensors play an important role in the disease-related nucleic acid molecules,protein detection.Generally,the ECL intensity could be significantly enhaced when co-reactants are introduced into ECL system,resulted the detection sensitivity of ECL biosensors improving.However,some co-reactants are unstable and difficult to be labeled,increasing the difficulty of loading,and there are some shortcomings such as long distance for electron transfer,high energy loss and poor stability when interacting with the luminophor.Accordingly,in this thesis,combined with nucleic acid amplification technology and a variety of nano-composite materials,multiple ECL biosensors are contructed for the sensitive detection of a variety of disease-related biological molecules such as micro RNAs,proteins by synthesizing novel self-enhanced ECL luminophor and introducing new co-reaction accelerator to improve the efficiency of interaction between ECL luminophor and co-reactants.In our paper,research work is carried out from the aspects as follows:Part 1 A sensitive ECL biosensor based on target-catalyzed hairpin assembly and intramolecular/intermolecular co-reaction for signal amplificationUp to now,the conbine of ECL and nucleic acid amplification strategy have been widely developed to achieve the sensitive detection of mi RNAs.However,most of the nucleic acid amplification techniques introduce enzymes that are affected by a number of some negative factors,including high cost,multiple assay steps and exogenous reagents dependence.While the target-catalyzed hairpin assembly(T-CHA)with enzyme-free not only exhibits the high sensitivity,short analysis time and simplified operation,but also recycles the target to improve the sensitivity.In addition,it is particularly important to introduce suitable co-reactants in the construction of ECL biosensors,while effectively increasing the efficiency of the co-reactants and luminophore.The self-enhanced luminophore(PEI-Ru(II))was synthesized by covalently crosslinking the ECL-active luminophore and coreactant into one molecule,which shortening the electronic transmission distance of PEI and Ru(II),reducing the energy loss between Ru(II)and polyethyleneimine(PEI),and enhancing the efficiency of the luminophore and co-reactants.Therefore,the stronger and more stable ECL signal was obtained.The new coreactant,thiosemicarbazone(TSC)was first introduced into the PEI-Ru(II)system to form dual amplification strategies with intramolecular/intermolecular co-reaction,and the T-CHA was incorporated to construct the enzyme-free biosensor to detect mi R-21 with a linear response(1.0×10-16 mol/L to 1.0×10-11 mol/L)with a relatively low detection limit of 30 amol/L.Part 2 A sensitive ECL biosensor based on perylene derivatives as a novel co-reaction accelerator for signal amplificationWith controllable size and emission wavelength,high photoluminescence yield,and good chemical stability,quantum dots(QDs)have been widely used as a kind of ECL emitters for biomarkers analysis.It was worth noting that the immobilization of QDs was the most important step in solid-state QDs based-ECL biosensors fabrication.Due to the remarkable conductivity and large specific surface area,graphene can be as the desirable matrix for QDs anchoring to form the graphene-Cd Te(G-Cd Te)nanocomposites.At the same time,as the most commonly used coreactant of QDs,persulfate(S2O82-),was difficult to immobilize on the biosensor due to its special property,and was often placed in the solution by increasing its concentration to enhance the ECL signal of QDs,which limited its application.The introduction of co-reaction accelerator in the S2O82-/QDs system can resolve this problem.The L-lysine covalently binded 3,4,9,10-perylenetetracarboxylic acid(PTC-Lys),as a kind of perylene derivatives,could interact with the co-reactant of S2O82-to promote the more oxidant mediators of SO4·-,which could further react with QDs to produce excited state species QDs* for emitting light.Based on PTC-Lys as a new co-reaction accelerator,an ECL aptasensor was designed and exhibited ultra-high sensitivity to quantify the concentration of thrombin(TB)from 1.0×10-16 mol/L to 1.0×10-8 mol/L with a detection limit of 34 amol/L.Part 3 A sensitive ECL biosensor based on the composite material of metal organic skeleton material and quantum dots as multifunctional signal probesMetal-organic frameworks(MOFs)with 3D network structure are a class of crystalline microporous materials formed from transition metal ions or clusters and polytopic organic linkers.Due to their large surface area,MOFs was widely used as an ideal carrier material.In this work,in order to increase the load capacity of Cd Te QDs,the methods of “intra-filling”,“inner surface modification” and “outer surface modification”were employed to modify Cd Te QDs in or on the inner and outer surfaces of isoreticular metal organic frameworks-3(IRMOF-3),which prepared the Cd Te@IRMOF-3/Cd Te QDs composite materials.It was worth noting that IRMOF-3 could not only act as a immobilized matrix for Cd Te QDs,but also act as a new co-reaction accelerator to promote S2O82-to produce more strong oxidant intermediates SO4·-,which enhanced the ECL signal of Cd Te QDs.As a result,a highly sensitive immunosensor was constructed to detect cardiac troponin I(c Tn I)with a wide response range(1.1 fg/m L ~ 11 ng/m L)and a very low detection limit(0.46 fg/m L)based on the Cd Te@IRMOF-3/Cd Te QDs composite as a multi-function signal probe.