Design And Application Of Ratiometric Electrochemiluminescence Biosensor

Electrochemiluminescence（ECL）biosensing method is a detection method based on ECL signal response caused by biomolecular recognition.In this method,bioactive molecules are used as recognition elements,ECL reagents/materials are used as signal indicators,and the concentration of target molecules is measured by converting biochemical interactions into quantifiable ECL signals.It combines the advantages of electrochemical method and chemiluminescence method.Compared with other optical analysis methods,it has many unique advantages,such as high sensitivity,high selectivity,low background signal width,dynamic detection range and convenient operation.Among ECL biosensors,ratiometric ECL biosensors are a relatively new ECL construction strategy.Ratiometric ECL biosensors not only have the inherent advantages of conventional ECL detection,but also can effectively reduce instrumental or environmental impact,especially in low concentration targets or complex biological systems can provide more accurate analysis results,so it has attracted more and more research interest.Ratiometric ECL biosensing methods are mainly divided into wavelength-resolved ECL ratiometry and potential-resolved ECL ratiometry.So far,it has been widely used in the detection of ionic,microRNA,protein,Cells.However,the common ECL ratiometry method usually requires a good energy transfer efficiency between donor and recipient,or a wide and easily discernable luminescent potential,and common reactants shared by anode and cathode luminescent materials.These conditions are the main factors limiting their development.Therefore,the development of a new ratiometric biosensor is of great significance to expand the application of ECL sensor in the field of biological analysis.In this paper,two kinds of ratiometric microRNA ECL sensors are designed by using internal standard method and ratio method of electrochemiluminescence signal/electrochemical signal,combined with nucleic acid amplification strategy,providing a new idea for the development of ratiometric biosensors.1.The ratiometric microRNA sensor based on internal standard method was constructed and successfully used for quantitative analysis of microRNA-21.The sensor used the cathode ECL signal of Au-g-C₃N₄ nanosheet as the internal reference signal,the anode ECL signal of luminol molecule as the monitoring signal,and hydrogen peroxide as the co-reaction agent.When the detection solution contained microRNA-21,the probe DNA chain modified on the electrode surface catalyzed hairpin assembly reaction,and the Probe DNA chain labeled with Pt nanoclusters was brought to the electrode surface.With the increase of microRNA-21 concentration,Pt nanoclusters assembled on the electrode increased,and the anodic ECL signal intensity of luminol catalyzed by the Pt nanoclusters also increased,while the cathodic ECL signal of Au-g-C₃N₄ nanosheets remained unchanged.Sensitive detection of target microRNA-21 concentration can be achieved by monitoring the ratio response of anode signal intensity to internal reference signal intensity.2.A ratiometric microRNA sensor based on the ratio of electrochemiluminescence to electrochemical signal was designed for sensitive detection of microRNA-21.In this method,luminol molecules were selected as ECL signal transmitter,and ferrocene was used as EC signal transmitter.Under the initial condition,ferrocene as a quencher can quench the ECL signal value.When microRNA-21 was present,it triggered proximity ligation reaction with both capture chains and further formed MNAzyme with the substrate chain.With the help of Mg²⁺,MNAzyme can catalyze the cleavage of the substrate DNA chain,part of the DNA fragment labeled ferrocene is released from the electrode surface,and the remaining G-quadruplex DNA chain on the electrode surface and hemin form a hemin/G-quadruplex structure.With the increase of microRNA-21concentration,ferrocene content on the electrode decreased,and EC signal also decreased.The hemin/G-quadruplex structure increased with the increase of microRNA-21 concentration,and the catalytic effect of H₂O₂ also increased,which effectively enhanced the ECL signal of luminol.By monitoring the ratio of the two signals,high-precision detection of microRNA-21 was realized.