Construction And Research Of Novel Magnetic Electrochemical Immunosensors Based On Optical Multi-Metal Nanocomposites For Oral Cancer Biomarkers

Objective Based on the problems of low sensitivity,single sensing configuration,poor immobilization effect and complex detection faced by electrochemical immunosensor for protein tumor markers（PTM）related to oral squamous cell carcinoma（OSCC）,this paper started from the active interface for immobilization of biological components,signal labels and immunoassay mode to establish a series of electrochemical immunosensing system for simple and rapid detection of PTM with high sensitivity,stability and specificity.It provides an efficient and precise methodological basis for the identification and quantitative detection of trace substances in biological sample,and new ideas for the detection of low concentration substances.Methods In this paper,the optical multi-metal nanocomposites（OMMN）combining ECL active materials with multi-metal nanocomposites were prepared by mechanical alloying（MA）as signal labels.After field emission scanning electron microscope（FESEM）,inverted fluorescence microscope（IFM）,X-ray diffraction（XRD）for micro-morphology characterization and elemental analysis,ECL and electrochemical performance studies,we selected a series of OMMN—optical binary metal Ru-doped（Bi-Ru）,trimetallic Ru-doped（Tri-Ru）and binary metal Lu-doped（Bi-Lu）nanocomposites with excellent photoelectric behavior as signal label.At the same time,we built a series of functional magnetic interfaces—magnetic graphene oxide（MGO）,gold nanoparticles（Au NPs）/magnetic redox graphene（Mr GO）（Au Mr GO）and graphene oxide/poly-L-cysteine/Au@Fe₃O₄（GO/PL-Cys/MAu/MGCE）used as sensing interfaces to immobilize immune molecule.Through the specific binding of antigen and antibody,the single PTM P16 protein(P_p16),c-Myc protein(AG_c-Myc),positive feedback dual PTM AG_c-Mycand cancerous inhibitor of protein phosphatase 2A（CIP2A）were identified and detected.The modified electrodes were characterized by FESEM,transmission electron microscopy（TEM）and fourier transform infrared（FTIR）.Cyclic voltammetry（CV）and electrochemical impedance spectroscopy（EIS）were used to monitor the process of sensor construction.ECL and differential pulse voltammetry（DPV）were used to detect targets sensitively.The analytical performances of the three sensors are systematically studied through methodological research and feasibility validation.Results Quantitative detection ranges of the three electrochemical immunosensors were 0.1 ng/L～0.1μg/L,10 pg/L～0.1μg/L,5 pg/L～5 ng/L and 50 pg/L～50 ng/L,with detection limits of 0.5 ng/L,5 pg/L,2.5 pg/L and 25 pg/L respectively.Systematic methodological validation and feasibility analysis confirmed that the three electrochemical immunosensors had good stability,reproducibility and selectivity.They were applied to the detection of standard-added blood and saliva samples,respectively.The recoveries ranged from 96.0%to 110.0%with RSD less than 5.90%.Conclusions In this paper,the OMMN prepared by MA had excellent ECL and electrochemical performance,and can be widely used as signal labels in sensor detection systems.A series of functional magnetic electrodes as active interfaces for immobilizing immune molecules,integrated separation,enrichment and detection.It realized multiple amplification of detection signals on a more convenient basis,achieving the purpose of highly sensitive detection of OSCC related PTM.Single and multiple markers at low concentrations can be detected specifically and conveniently while ensuring sensitivity,which provides a new idea for detecting TM.They have satisfactory recovery rate in PTM detection of various standard biological samples,which proves that the constructed series of electrochemical immunosensors had good application potential in practical detection.