Preparation And Application Of Immunosensor Based On The Cathodic Electrochemiluminescence Of Luminol

Electrochemiluminescence（ECL）is chemiluminescence induced by electrochemical reaction,which has been widely used in the field of bioassay analysis and clinical medicine because of its high sensitivity,high selectivity,low background signal and rapid response.The ECL immunosensor is a product developed by combining electrochemiluminescence technology and specific recognition of immune response,which can monitor and analyze cancer markers with high sensitivity.In addition,nanocomposites with high performance provide new ideas for the research and development of immunoassay,and play a vital role in the development of highly sensitive immunosensors.In recent years,luminol has been widely used due to its non-toxic,low excitation potential,chemical and thermal stability,and high luminous efficiency.It is worth noting that luminol can produce the anodic electrochemiluminescence and can also achieve the cathodic electrochemiluminescence at low potentials,avoiding damage to modified electrodes and biomolecules at high potentials.Based on this,we prepared several nanocatalytic materials,which can enhance luminol’s electrochemiluminescence,such as graphene-platinum nanoparticle composites,titanium carbide-gold nanoparticle composites,etc.,and constructed luminol electrochemiluminescence immunosensors based on these nanomaterials to detect carcinoembryonic antigen（CEA）.The specific work is as follows:1.Construction of carcinoembryonic antigen immunosensor based on the cathodic electrochemiluminescence of luminolAn ionic liquid functionalized graphene-loaded porous platinum（GR-IL/p Pt） nanostructure was used as a platform to construct a label-free ECL sensor for detecting carcinoembryonic antigen（CEA）.The composite has a highly porous structure and good biocompatibility,and exhibits good electrocatalytic activity for the reduction of H₂O₂,which help to increase the load density of CEA antibodies and amplify the cathodic ECL signal of luminol.After incubation of CEA with CEA antibodies,luminol’s cathodic ECL signal decreased due to the low conductivity of the immune complex.Under the optimal experimental conditions,the ECL immunosensor has achieved high sensitivity detection of CEA with a linear range from 0.001 fg m L^-1to 1 ng m L^-1and a detection limit of 0.0003 fg m L^-1（S/N=3）.In addition,the sensor has good specificity,stability and repeatability,and the test results of actual samples are also satisfactory,indicating that the proposed strategy has potential application prospects in clinical testing.2.Construction of immunosensor based on Mo₂C@C nanospheres for enhancing cathodic electrochemiluminescence of luminolMo₂C@C nanospheres were synthesized by hydrothermal synthesis,and CEA’s secondary antibody（Ab₂）was labeled on the amino-functionalized nanospheres.In the presence of CEA,the Mo₂C@C nanosphere loaded on the electrode surface can accelerate the electron transfer speed between the solution and the electrode surface due to its excellent conductivity,good stability and high specific surface area.More importantly,it can efficiently catalyze the reduction of H₂O₂,thus greatly improving the intensity of luminol cathodic ECL and achieving sensitive detection of CEA.In the range of 1 fg m L^-1～1 ng m L^-1,the ECL intensity of the immunosensor has a linear relationship with the logarithm of CEA concentration.The linear regression equation is ECL=6167.8+953.96lg c _CEA/pg m L^-1,the correlation coefficient is 0.99751,and the detection limit reached 0.33fg m L^-1.The sensor has high selectivity,good stability and good reproducibility,and the detection results of clinical actual samples are within acceptable ranges.This work provides a new way to improve the efficiency of luminol cathodic ECL,and has great application potential in biological analysis and early diagnosis of cancer.3.A bipotential proportional electrochemiluminescence sensor for detecting of carcinoembryonic antigen based on luminol single luminophorIn order to reduce the signal fluctuation caused by environmental changes in single mode and improve the accuracy and sensitivity of the analysis method,this paper used the proportion of luminol cathodic-anodic luminous signal as the detection signal,a dual-potential proportional ECL sensor for sensitive detection of CEA was constructed,which overcame the limitation of the general proportional sensor that requires two kinds of light emitters and expanded the application of proportional luminous sensors.Luminol exhibited anodic and cathodic luminescence on Ti₃C₂MXenes-Au NPs hybrids and graphene-ionic liquid-platinum（GR-IL-Pt）composites,respectively.Using GR-IL-Pt composite material as a platform to fix primary antibody（Ab₁）,luminol can produce a strong cathodic electrochemiluminescence(ECL_cathodic)on this material.At the presence of CEA,Ab₂labeled Ti₃C₂MXenes-Au NPs nanomaterials can significantly enhance luminol’s anodic electrochemiluminescence(ECL_anodic).At the same time,the ECL_cathodicreduced due to its own steric effect,which makes the ECL_anodic/ECL_cathodicproportion significantly increased.More importantly,ECL_anodic/ECL_cathodicwas independented of the concentration of the co-reactant H₂O₂,which greatly improved the reliability of the test.The proposed ECL sensor had high sensitivity for CEA detection,ranging from 0.1 pg m L^-1to 10 ng m L^-1with a detection limit as low as 34.58 fg m L^-1（S/N=3）.In addition,this strategy showed good practicability in detecting CEA in patient serum,providing new ideas for ECL bioanalysis.