Construction Of Electrochemiluminescence Biosensor Based On Functional Nanomaterials And Their Applications

Electrochemiluminescence（ECL）also called electrogenerated chemiluminescence.It refers to the substance generated on the electrode surface during the luminescence process that undergoes an electron transfer reaction and forms an excited state to emit light.ECL is a perfect combination of electrochemistry and spectroscopy.Therefore,ECL not only holds the sensitivity and wide dynamic range inherited from conventional chemiluminescence（CL）but also exhibits several advantages of electrochemical methods including simple instrument and equipment,simple operation,fast analysis,becoming a valuable and powerful analysis technology in many fields.Cancer is a life-threatening disease that kills millions of people every year due to its high metastatic spread and lethality,seriously affecting human health and public safety.Early and accurate tumor diagnosis is of great significance to improve the the therapeutic effect and survival rate of tumor patients.Nanomaterials provide a new approach for the research and application of electrochemiluminescence sensors due to their special nanostructure,strong adsorption capacity,and large specific surface area.Applying it to the construction of electrochemiluminescence immunosensors can greatly increase the immobilization capacity of biomolecules（such as antigens,antibodies,etc.）,accelerate electron transfer,and enhance ECL.The paper mainly from the following five aspects to improve the sensitivity or accuracy of the method:Firstly,to improve the ECL efficiency,thereby improving the sensitivity of biosensors,the nanoluminophore with excellent performance is synthesized.Secondly,to improve the detection sensitivity of biosensors,the electrochemiluminescence resonance energy transfer technology is used to detect biomarkers.Thirdly,the construction of a ratio-based immunosensor,which effectively eliminates the interference caused by the internal environment and improves the accuracy of the detection method.Fourthly,using spatially-resolved technology to simultaneously detect multiple biomarkers to improve detection throughput and shorten analysis time.Fifth,the co-reactant K₂S₂O₈ and dissolved oxygen are fully utilized in one detection system,which avoids the defects of the existing multiplex immunoassay and improves the detection sensitivity and accuracy.The main research contents and results of this paper are as follows:1.Gold nanoclusters（Au NCs）are wrapped with 6-aza-2-thiothymine（ATT）.The aggregation of Au NCs protected by ATT induces electrochemiluminescence（ECL）to enhance the ECL of Au NCs.A new dual-quenching ECL immunosensor based on solid-state ATT-Au NCs（SS-ATT-Au NCs）and aggregation-induced emission（AIE）was designed for sensitive determination of prostate-specific antigen（PSA）.Moreover,the synthesized CeO₂-PEI@Ag composite can be used not only as the carrier to immobilize secondary antibody（Ab2）,but also to improve the efficiency of the ECL sensing platform based on the resonance energy transfer between SS-ATT-Au NCs and CeO₂-PEI@Ag.With the optimal conditions,the prepared dual-quenching aggregation-induced electrochemiluminescence resonance energy transfer（AIECL-RET）immunosensor shows the linear range from 1×10^-5 ng/m L to 200 ng/m L,and the detection limit is 2.2 fg/m L（S/N=3）.The result shows that the prepared AIECL-RET sensor can be successfully applied to detect PSA in human serum with the advantages of high stability and good selectivity.The study not only enriches the ECL research strategies,but also provides feasible methods for basic clinical testing,and shows the potential applications of ECL sensors in clinical bioassays.2.Electrochemiluminescence resonance energy transfer（ECL-RET）is a promising signal mechanism and has broad application prospects in the field of biological detection.However,the limitation of the energy transfer efficiency of ECL-RET is still a great challenge.This study proposed an efficient ECL-RET mode,which combined the luminescent materials of tris（4,4’-dicarboxylicacid-2,2’-bipyridyl）ruthenium（II）(Ru（dcbpy）₃²⁺)（energy donors）and tin dioxide and tin disulfide quantum dots（SnO₂/Sn S₂QDs）（energy acceptors）into the isoreticular metal organic framework-3（IRMOF-3）material to form a composite.In this mode,the distance between the energy donor and the acceptor is greatly shortened,reducing the energy loss,and thereby effectively improving RET efficiency and further significantly improving the ECL signal.The obtained composite（SnO₂/Sn S₂QDs-Ru@IRMOF-3）is combined with sandwich immunoreaction to construct an ECL immunosensor for the sensitive detection of procalcitonin（PCT）.Under the optimized experimental conditions,the proposed PCT biosensor exhibited a linear concentration range of 10^-4–200 ng/m L,with a detection limit of 0.029 pg/m L（S/N=3）.In conclusion,the biosensor had broad application prospects in biological analysis and clinical diagnosis due to its high sensitivity,good specificity,and good stability.3.Alzheimer’s disease（AD）threatens the health of elderly people.Amyloid-βprotein(Aβ₄₂)is considered to be a critical protein in the development of AD and a predictive biomarker for the diagnosis of AD.In this study,a dual-wavelength ratiometric electrochemiluminescence（DWR-ECL）sensor was developed for sensitive detection of Aβ₄₂.A Ru@TiO₂@Au nanomaterial was used as the energy receptor,and gold nanoparticles（Au NPs）-modified graphitic carbon nitride nanosheets（g-C₃N₄NSs）were employed as the energy donor.Au-g-C₃N₄NSs exhibits a highly stable ECL signal with an emission center at 460 nm,which overlaps with the Ru@TiO₂@Au UV-vis absorption spectrum also well stimulates the emission of Ru（bpy）₃²⁺at 620 nm,manufacturing ECL resonance energy transfer（RET）with a good performance.Therefore,an ECL-RET system was established.Au NPs functionalized the surface of g-C₃N₄NSs,not only providing a good substrate to fix primary antibodies（Ab1）（Au-N）but also increasing the electron transport rate of g-C₃N₄NSs on the surface of the electrode.Furthermore,titanium dioxide nanoparticles（TiO₂NPs）due to high specific surface area were applied to load numerous Ru（bpy）₃²⁺and Au NPs,and the obtained Ru@TiO₂@Au nanomaterials were introduced to combine secondary antibody（Ab2）,thus remarkably amplifying the ECL signal.Under optimal experiments,on the basis of the ratio of log(I_{460 nm}/I_{620 nm})（I is the ECL intensity）,the linearity range of Aβ₄₂ is from 1×10^-5 to 200 ng/m L with a limit of detection（LOD）of 2.6 fg/m L（S/N=3）.The results indicate that the designed DW-RECL immunosensor has satisfactory stability,high sensitivity,good reproducibility,and excellent specificity,and it can be applied in clinical diagnosis.4.Neuron-specific enolase（NSE）is the first choice marker for monitoring small cell lung cancer and neuroblastoma.A sensing strategy of potential resolved electrochemiluminescence（PRECL）is designed for sensitive detection of NSE.In this work,a novel ECL nanoluminescence group,namely,N-（4-aminobutyl）-N-ethylisoluminol functionalized sulfur quantum dots（ABEI-SQDs）is synthesized,using K₂S₂O₈ as a co-reactant,and three completely potential-resolved bicolor ECL luminescence are observed,namely,three electrochemiluminescence at-1.75 V（ECL-1）,-0.64 V（ECL-2）,and+0.70 V（ECL-3）,the center of ECL-1 peak is at 675 nm,and the centers of ECL-2 peak and ECL-3 peak is at 450 nm.Based on the above characteristics,a free labeled potential ratio ECL immunosensor is developed to detect NSE by assembling of ABEI-SQDs and antibodies of NSE layer by layer on chitosan-modified glassy carbon electrode.In the presence of NSE,the intensity（I₁）of ECL-1 decreased,while the intensity（I₂）of ECL-2 and the intensity（I₃）of ECL-3 changed slightly.In the absence of K₂S₂O₈,the ratio of the luminescence intensity of ECL-2 to ECL-3 remained unchanged.In the presence of K₂S₂O₈,it is found that the intensity of ECL-2 changed,but the intensity of ECL-3 remained unchanged,which the contribution of dissolved oxygen to ECL-2 could be calculated.The systematic errors and background interference is eliminated that based on the ratio of the intensity of ECL-1 to the intensity of ECL-2.However,it is found that deducting the effect of dissolved oxygen on ECL-2 from ECL-2 can better reflect the rationality of the method and obtain better detection results.Therefore,a more accurate self-calibration analysis method is established.Under the optimized experimental conditions,NSE shows a good linear relationship in the range of 1×10^-5-200ng/m L with a detection limit of 2.7 fg/m L（S/N=3）.The proposed potential ratio ECL immunosensor can effectively eliminate the system error or background signal caused by environmental changes by self-calibrating the two signals,and improve the detection reliability.The method has good reproducibility and stability,and provides a successful paradigm for other biomolecule detection and analysis.5.In this paper,a new potential-resolved ECL strategy based on Ti-ETTC-MOL and ABEI@Pt is constructed for simultaneous determination of human chorionic gonadotropin（HCG）and alpha-fetoprotein（AFP）.Ti-ETTC-MOL with aggregation-induced enhanced electrochemiluminescence is used as the cathode signal probe,and ABEI@Pt is used as the anode signal probe.Electrodeposited gold nanoparticles are deposited on the surface of a spatially resolved double-disk glassy carbon electrode to capture HCG-Ab1 and AFP-Ab1（Au-N or Au-S）respectively,and then HCG and AFP antigens,secondary antibody complexes（Ti-ETTC-MOL-HCG-Ab2 and ABEI@Pt-AFP-Ab2）are gradually connected to the electrode.Under the optimized experimental conditions,the linear range of HCG is 1×10^-4-200 m IU/m L,and that of AFP is 1×10^-4-200 ng/m L.With the increasing concentration of HCG and AFP antigen,the ECL signals of-1.98 V from Ti-ETTC-MOL and the ECL signals of+0.70 V from ABEI@Pt increase simultaneously,thus realizing synchronous immunoassay of HCG and AFP.The detection limits of HCG and AFP are 31 n IU/m L and 25 fg/m L（S/N=3）,respectively.The integration of Ti-ETTC-MOL/S₂O₈^2-and ABEI@Pt/O₂ system avoids the defects of the existing multiplex immunoassay,thus providing an attractive ECL platform for simultaneous determination of multiple biomarkers.The thesis consists of six chapters.Chapter 1 is the introduction,Chapter 2 for an aggregation-induced electrochemiluminescence resonance energy transfer with dual quenchers for sensitive detection of prostate specific antigen,Chapter 3 for a high efficiency resonant energy transfer electrochemiluminescence sensor based on SnO₂/Sn S₂QDs-Ru@IRMOF-3composite material was constructed for sensitive detection of procalcitonin,Chapter 4 for design of a dual-wavelength ratiometric electrochemiluminescence immunosensor for sensitive detection of amyloid?βprotein in human serum,Chapter 5 for the construction of ratio potential-resolved electrochemiluminescence immunosensor and its application to detect neuron-specific enolase and Chapter 6 for the simultaneous detection of human chorionic gonadotropin（HCG）and alpha-fetoprotein（AFP）by potential-resolved aggregation-induced electrochemiluminescence.The Chapter from 2 to 6 is my research work.