Construction Of A Highly Sensitive Electrochemiluminescence Sensor Chip And Its Application In Biomarker Detection

Biomarkers are a class of substances discovered by people that can objectively represent the health of living organisms,and their accurate detection is of great significance for disease diagnosis and prevention.Electrochemiluminescence(ECL)biosensor is a highly sensitive sensing and analysis method that uses biomarkers as targets,uses biosensitive materials as identification elements,and is based on the ECL response principle of materials.Compared with the related analysis methods based on large-scale instruments in the field of clinical diagnosis,the biomarker analysis method of ECL biosensor has significant advantages such as convenient operation,low cost,less sample consumption and fast analysis speed,so it has obvious advantages in the field of biomarker detection.In this paper,we use three common biological recognition elements(peptide,antigen-antibody,nucleic acid aptamer)and the most widely used metal complex ECL luminophore Ru(bpy)2(mcpbpy)Cl2,which is convenient to use,low-cost,disposable screen-printed electrodes(SPE),constructed three ECL biosensor chips based on different recognition mechanisms,and studied their effects on biomarkers(caspase proteases-3(caspase-3),carcinoembryonic antigen(CEA)and thrombin(TB)detection.The specific research contents are as follows:1.Caspase proteases(caspases)are a class of caspase-specific proteases closely related to apoptosis.Monitoring the activity of caspase-3 is of great significance for understanding its role in the process of apoptosis,as well as the diagnosis and treatment of related diseases.We developed a novel ternary self-enhancing ECL material(RuPEI@PCN-333(Al)),and constructed a single-use ECL sensor chip for detecting caspase-3 activity based on the peptide cleavage-mediated principle.The luminescent material adopts MOF(PCN-333(Al))to immobilize the luminescent reagent Ru(bpy)2(mcpbpy)Cl2,and uses covalent bonds to connect the co-reactant polyethyleneimine(PEI)with the luminescent reagent,which promotes the material the self-enhanced ECL luminous efficiency.A ferrocene(Fc)-modified polypeptide with caspase-3-specific cleavage site Asp-Glu-Val-Asp(DEVD)and capable of quenching Ru2+ ECL signal was assembled on the surface of SPE electrode using Au-S bond to construct disposable ECL sensor chip.In the presence of caspase-3,due to the specific cleavage of the peptide,the Fc-containing peptide is released from the electrode surface,and the "Turn-ON" response of the ECL signal is realized.Its detection limit is as low as 0.017 pg/m L,with excellent selectivity,reproducibility and stability.It was successfully used to detect the apoptosis of MOLM-13 cells induced by two anticancer drugs,daunorubicin and doxorubicin.2.Carcinoembryonic antigen(CEA)is a glycoprotein produced by human tumor cells and is a disease marker commonly used in clinical diagnosis and treatment of cancer.Usually in the serum of cancer patients,CEA levels are significantly elevated.Monitoring the concentration of CEA in serum is of great significance for early diagnosis of disease and understanding of cancer process.We developed a novel quaternary self-enhancing ECL material(Au NPs-Ru-Arg@NH2-Ti3C2-MXene),and constructed a disposable ECL sensor chip for detecting CEA concentration based on the principle of antigen-antibody immune recognition.The material uses an aminomodified transition metal carbide two-dimensional nanolayered material(NH2-Ti3C2-MXene)as a carrier,and uses a covalent bond to combine the luminescent reagent Ru(bpy)2(mcpbpy)Cl2 with the co-reactant L-arginine(L-Arg)connection,and then using electrostatic interaction to introduce Au NPs to prepare quaternary self-enhanced ECL materials.The CEA antibody(anti-CEA)was dropped onto the surface of the SPE electrode to construct a disposable ECL sensor chip.In the presence of CEA,a "TurnOFF" response of ECL signaling is achieved due to the insulating effect of protein molecules and steric hindrance caused by antigen-antibody bioconjugation.With a detection limit as low as 1.5 pg/m L,excellent stability,reproducibility,and selectivity,it can be successfully used to detect the concentration of CEA in serum samples from healthy and cancer patients.3.Thrombin(TB)is a multifunctional serine protease that plays an important role in regulating various physiological processes such as hemostasis.Abnormal concentrations are often involved in various diseases such as cardiovascular disease,inflammatory response,and thromboembolism.Therefore,researching an efficient TB detection method has important practical value for the early diagnosis and prevention of the disease.In this work,an electrochemiluminescence(ECL)sensor chip for sensitive detection of thrombin(TB)was prepared using a screen-printed electrode(SPE)as a working electrode and an aptamer as a specific recognition moiety.To produce an ECL sensor chip,a layer of p L-Cys was immobilized on the surface of SPE by the cyclic voltammetry scanning method,a layer of Au NPs was assembled through an Au-S bond and hairpin DNA was further immobilized on the electrode surface.Ru(bpy)2(mcpbpy)2+,as a luminescent reagent,was covalently bound to ss DNA to prepare a luminescent probe ss DNA-Ru.The probe hybridized with TB aptamer to form a capture probe.In the presence of TB,the TB aptamer in the capture probe bound to TB,causing the release of ss DNA-Ru that could bind to hairpin DNA on the electrode surface.After the solution was added dropwise to the sensor chip,the hairpin DNA was base-paired with free ss DNA-Ru to form a double strand.The luminescent reagent was assembled on the electrode surface,and p L-Cys acted as a co-reactant to realize the "Turn-ON" response of the ECL signal.It has a detection limit as low as 0.2 f M,excellent stability,reproducibility,and selectivity,and was successfully used to detect TB concentrations in human serum samples.