Near Infrared Electrochemiluminescence Of Functional Gold Nanoclusters And Biosensing Application

As a highly sensitive analytical technology,electrochemiluminescence（ECL）detection has attracted more and more interest.ECL refers to the phenomenon that some active intermediates are produced by electrochemical methods,and then react with each other to produce light.ECL is the combination of chemical method and electrochemical method,and thus possesses a series of advantages,such as high sensitivity,wide linear range,good reproducibility and simple instrument operation.At present,ECL has been widely used in immunoassay,nucleic acid analysis,small molecule and cell analysis.Gold nanoclusters（AuNCs）,as a new class of ECL emitters,have attracted a lot of attention in the fields of cell analysis,bioimaging and biosensors owing to their excellent biocompatibility,low environmental harm and good photostability.The functionalization of AuNCs can improve its ECL performance,expand its ECL wavelength for realizing near infrared electrochemiluminescence（NIR-ECL）.In this thesis,a series of functional AuNCs were constructed to study their NIR-ECL performance,and several highly sensitive ECL immunoassay methods were developed for the detection of disease markers.The related works are as follows:（1）Using N-acetyl-L-cysteine（NAC）and cysteamine（Cys）as model capping agents,a dual-stabilizer-capped strategy was proposed to achieve red-shifted ECL in the NIR via surface engineering water soluble and biocompatible AuNCs.The dual-stabilizer-capped NAC/Cys-AuNCs were environment-friendly and exhibited efficient ECL emission at around860 nm when utilizing triethanolamine as a coreactant,thus providing an NIR-ECL tag for bioanalysis in aqueous medium.Benefiting from the low background of NIR emission and the good biocompatibility of the dual-stabilizer-capped NAC/Cys-AuNCs,a“signal on”-type NIR-ECL immunosensing method was proposed.Using carcinoembryonic antigen（CEA）as a model analyte and NAC/Cys-AuNCs as a tag to label the signal antibody,this method exhibited a linear range from 1 fg/m L to 0.5 ng/m L with a limit of detection of 0.33 fg/m L（S/N=3）.（2）A promising modification strategy for improving the ECL performance of AuNCs as a water-soluble luminophore was proposed.Upon the introduction of L-cysteine（L-Cys）onto the surface of glutathione（GSH）-stabilized AuNCs（GSH-AuNCs）,the dual-thiol bond between L-Cys and GSH was formed to limit the intramolecular motion and non-radiative relaxation of excited state from the capping agents,which resulted in the enhancement of monochromatic ECL emission of GSH-AuNCs with a redshifted wavelength.By utilizing the coreatant of triethylamine（TEA）,the ECL of L-Cys/GSH-AuNCs was about 1.5-fold stronger than that of GSH-AuNCs,and the emission wavelength redshifted from 660 nm to 780 nm at relatively low potential,which could decrease the interference in bioassay and the photochemical damage in nondestructive detection.As a proof of application,a sandwich-type immunosensing method for CYFRA 21-1 was proposed with L-Cys/GSH-AuNCs as the signal tag,which displayed a wide linear range from 0.2 fg/m L to 2 ng/m L and a limit of detection down to 0.067 fg/m L at 3S/N.（3）A strategy to regulate the ECL emisssion of AuNCs in NIR was proposed by doping the NAC and Cys stabilized NAC/Cys-AuNCs with cobalt ions(Co²⁺).The Co²⁺doping NAC/Cys-AuNCs(Co²⁺@NAC/Cys-AuNCs)could not only generate tunable hole-injection channels for the ECL emission,but also reduce the superficial defects to promote electron transfer through the synergetic effect of Au and Co inside Co²⁺@NAC/Cys-AuNCs.Thus,the Co²⁺@NAC/Cys-AuNCs displayed enhanced NIR-ECL intensity with the triggering potential around+0.95 V,which was 0.2 V more negative than+1.15 V of NAC/Cys-AuNCs,and increased the ECL efficiency to 33.8%from 15.6%of NAC/Cys-AuNCs(vs Ru（bpy）₃²⁺/TPr A).In addition,the doping strategy changed the surface state of NAC/Cys-AuNCs,which resulted in the ECL emission wavelength of Co²⁺@NAC/Cys-AuNCs blue-shifted for 50 nm to NIR 820 nm.Benefitting from the enhanced NIR-ECL emission and the excellent biocompatibility of Co²⁺@NAC/Cys-AuNCs,as well as the low background interference and photochemical injuring in nondestructive analysis of NIR ECL emission,a“signal-on”NIR-ECL immunoassay strategy was proposed.Using neuron specific enolase（NSE）as a model analyte,the NIR-ECL immunosensor constructed with Co²⁺@NAC/Cys-AuNCs as the tags exhibited a wide linear range from 0.5 fg/m L to 1 ng/m L with a limit of detection（LOD）of 0.16 fg/m L（S/N=3）.（4）A NIR-ECL immunosensor for the detection of NSE was proposed by utilizing NAC/Cys-AuNCs as NIR-ECL emitters and ZIF-67/Pd NCs hybrid as the coreaction accelerator.The NIR-ECL emission could reduce the photochemical damage to the samples with high strong susceptibility and suitability.Compared to NAC/Cys-AuNCs,the presence of ZIF-67/Pd NCs displayed 2.7-folds enhanced ECL emission when utilizing TEA as the coreactant due to the unique catalytic performance of ZIF-67/Pd NC,which acclerated the oxidation of coreactant TEA to form more reducing intermediate radical of TEA^·+.By introducing HWRGWVC（HWR）heptapeptide to nanoparticle surfaces to immoblize capture antibody and labelling the secondary antibody respectively,a“signal on”NIR-ECL immunosensor for NSE detection was proposed,which showed a linear range from 0.1 fg/m L to 5 ng/m L and a low limit of detection（0.033 fg/m L）（S/N=3）.（5）Hollow double-shell CuCo₂O₄@Cu₂O（HDS-CuCo₂O₄@Cu₂O）heterostructures were systhesized by in situ forming unique Cu₂O nanoparticles（NPs）on layered-hollow CuCo₂O₄nanospheres（NSs）due to the perfect-matched lattice spacing.The heterostructures possessed short charge transfer distance,high interfacial charge transfer efficiency and effective“electron-hole”separation performance.Thus it could be used as an attractive coreaction accelerator to promote the formation of sufficient coreactant intermediate radicals,and thus improved the NIR-ECL performance of L-methionine stabilized AuNCs（L-Met-AuNCs）,which led to 3-folds stronger NIR-ECL response.The NIR-ECL emission around 830 nm of L-met-AuNCs could decrease the photochemical damage to even realize a nondestructive detection with improved susceptibility and circumambient adaptability.Utilizing HWRGWVC（HWR）heptapeptide as antibody immobilizer to functionalize HDS-CuCo₂O₄@Cu₂O and L-Met-AuNCs for immobilization of capture antibody and labelling of the secondary antibody,a NIR-ECL immunosensing method was proposed for the ultrasensitive detection of CYFRA 21-1,which achieved a linear range from 2 fg/m L to 50ng/m L and a LOD of 0.67 fg/m L（S/N=3）.