Preparation And Application Of Electrochemiluminescence Aptamer Sensor Based On Metal Nanoclusters And Luminol

Electrochemiluminescence（ECL）technology is widely applied in the field of bioas-say because of its high sensitivity,wide dynamic range,simple operation and good stability.In recent years,researchers have combined stripping voltammetry and chemiluminescence to propose a novel electrochemiluminescence technique——electrochemical stripping luminescence.It has better sensitivity and selectivity compared to ECL.At the same time,researchers have developed ratiometric ECL sensors.It can improve the reliability of its detection by avoiding the interference and error of environmental factors on single signal in the traditional electrochemiluminescent sensors.However,ratiometric ECL sensors mostly use two eligible luminophors that conform to specific conditions,limiting the ap-plication and development of the sensors.Therefore,it is necessary to further expand the application of electrochemical stripping chemiluminescent in bioassays and to explore a ratiometric ECL sensor based on a single luminophore.In this thesis,a highly sensitive and selective electrochemical stripping luminescence sensor and a dual-potential ratiometric electrochemiluminescence sensor were successfully constructed based on luminol.It used metal nanoclusters as signal enhancement elements to achieve sensitive and reliable detection of the tumor marker CEA.1.Electrochemical stripping chemiluminescent sensor based on copper nanoclusters for detection of carcinoembryonic antigenAs a new type of nanocatalytic material,copper nanoclusters（Cu NCs）have received more and more attention in various fields such as biosensors and catalysis.In this work,an electrochemical stripping chemiluminescent（ESCL）aptamer sensor based on Cu NCs was constructed for the detection of carcinoembryonic antigen（CEA）.The Cu NCs were gen-erated using DNA duplex（the CEA aptamer and its complementary strand）as template.During the ESCL detection process,Cu NCs catalyzed the reduction of H₂O₂ and were gradually electrochemically oxidized to Cu²⁺,which both promoted the decomposition of H₂O₂ and thus greatly improved the ECL of luminol.CEA could specifically bind with its aptamer,so the DNA duplex was damaged,causing a decrease of Cu NCs and lower ECL signals.Under the optimized conditions,the sensor achieved a high sensitivity detection of CEA with a linear range of 0.2 fg/m L to 1 ng/m L and a detection limit of 66.67 ag/m L（S/N=3）.In addition,the sensor exhibited good specificity,stability and reproducibility,and the detection of actual samples obtained satisfactory results,indicating that the provided strat-egy has potential application prospects in clinical detection.2.Electrochemical stripping chemiluminescent coupled with recycling amplification strategy for sensitive detection of carcinoembryonic antigenAs a new generation of electrochemiluminescence（ECL）,the electrochemical stripping chemiluminescent（ESCL）technology synthesized the advantages of chemiluminescence and stripping voltammetry,which has attracted more and more attention in biosensor field.Here,Ag nanoclusters（NCs）were prepared using DNA double strands as template,and firstly explored to construct ESCL aptamer sensor for the detection of carcinoembryonic antigen（CEA）.During the detection process,we found that the Ag clusters and the stripped Ag⁺could respectively catalyze the electrochemical reduction and oxidation of H₂O₂,accelerating the decomposition of H₂O₂ to generate reactive oxygen species（ROSs）and thus effectively enhancing the ECL intensity of luminol.Coupled with the exonuclease-I assisted recycling amplification technology,the developed ESCL aptamer sensor achieved the sensitive detection of CEA,exhibiting a broad linear response from 100 ag/m L to 10 ng/m L with a low detection limit of 38.73 ag/m L（S/N=3）.In addition,the sensor had a good performance in the determination of CEA in the human serum with the relative standard deviation（RSD）lower than 5%.This work provides an effective method for constructing simple,fast and sensitive biosensors.3.Construction of ratiometric electrochemiluminescence sensor for sensitive detec-tion of carcinoembryonic antigen using luminol as single luminophorCompared with conventional ECL sensors,ratiometric electrochemiluminescence sensors can effectively eliminate environmental interference to achieve accurate detection.Nonetheless,most of these sensors use two eligible luminophors that meet specific condi-tions,increasing the complexity of the system and limiting their practical clinical applica-tion.In this work,we prepared palladium nanoclusters using the CEA aptamer and its complementary duplex DNA as the template,and the palladium nanoclusters can both promote luminol to produce cathodic ECL and quench its anodic ECL,based on which a dual-potential ratiometric ECL sensor was constructed using luminol as single lumino-phore.During the detection process,CEA can disrupt the double-stranded structure,lead-ing to decrease in the content of palladium nanoclusters,which triggers a remarkable de-crease in the ratio of cathodic ECL to anodic ECL(ECL_cathodic/ECL_anodic),thus achieving a sensitive detection of CEA.In addition,the ECL_cathode/ECL_anode is independent of the H₂O₂concentration,which significantly improves the reliability of the detection.The developed ratiometric ECL aptamer sensor exhibited a ultra-sensitive detection toward CEA with a wide linear range of 100 ag/m L to 10 ng/m L and the minimum detection limit was 87.1ag/m L（S/N=3）.In conclusion,this work provided new ideas and theoretical support for the construction of ratiometric electrochemiluminescent sensors and provided methods and technical support for the early diagnosis of cancer.