Construction Of Electrochemiluminescence Biosensor Based On Gold Nanoclusters And The Application In Biomolecular Detection

Electrochemiluminescence（ECL）is a new analytical technique that combines electrochemical technology with chemiluminescence.It possesses the high controllability in electrochemistry and high sensitivity in chemiluminescence,demonstrating enormous potential in analytical detection.Combining the high sensitivity of ECL technology with the high specificity of biometrics,ECL biosensor can achieve the quantitative detection by identifying different biological targets and converting ECL output signal.Therefore,ECL biosensor have the advantages of simple construction,fast response speed,high sensitivity and good selectivity,which are widely used in clinical diagnosis,food safety,environmental monitoring,and other biochemical analysis fields.The development of materials science and nanotechnology provides technical support and material foundation for the development of new ECL luminescent materials.Gold nanoclusters（Au NCs）with good optical properties and low biological toxicity are an ideal new type of ECL emitter.However,obtaining Au NCs with efficient ECL performance remains a challenging issue.Hence,this work aims to improve the ECL efficiency of Au NCs and construct ECL biosensors based on high-performance Au NCs and compound nanomaterials for specific and sensitive detection of various disease biomarkers.The main researchs are included as follows:（1）Electrochemiluminescence biosensor based on thiol-gold nanoclusters for simultaneous detection of glutathione and copper ionsThe inefficient electron transfer of Au NCs in electrochemical excitation limited the ECL efficiency,hindering the further application in biological detection.In view of the synthesis of Au NCs depended on the stabilization of the outer ligands,a series of thiol or amino ligands were applied to prepare monodisperse Au NCs for exploring the influence of the outer ligands on the ECL performance of Au NCs.Impressively,Au NCs with high Au-S bonding ratio presented unexceptionable ECL performance due to fast electron transfer from massive Au-S electronic pathways in electrochemical excitation,thus proposing a novel thiol ligand ECL enhancement strategy.A“on-off-on”type ECL biosensing platform was constructed based on thiol Capt-Au NCs as high-efficient ECL emitter for simultaneous detection of glutathione（GSH）and copper ions(Cu²⁺).GSH-triggered competing chain displacement reaction and Cu²⁺-specific-DNAzyme walker were used as nucleic acid signal amplification strategy.As a result,the ECL biosensor achieved simultaneous trace detection of GSH with a detection limit of 148 pmol/L and Cu²⁺with a detection limit of 91.5 fmol/L,broadening the application in multicomponent clinical diagnosis.（2）Non-enzymatic electrochemiluminescence biosensor based on gold nanoclusters signal probes for sensitive detection of ochratoxin AThe traditional sensor construction methods used ECL emitter as the electrode substrate for subsequent electrode assembly and nucleic acid amplification reactions.However,the stability of the substrate materials Au NCs with good water solubility was affected by repeated assembly reactions,thereby reducing the detection ability of the sensor.Herein,the signal probes formed by cross-linking ECL emitter Au NCs with DNA were introduced into the electrode through nucleic acid specific hybridization reaction,increasing the local concentration of the ECL emitter on the electrode surface while avoiding the influence of homogeneous molecules on the substrate.Impressively,the silver nanoflower（Ag NFs）synthesized by electrodeposition in situ could not only act as stable substrate materials for immobilizing DNA tracks,but also as co-reaction accelerator to enhance the ECL efficiency of Au NCs/S₂O₈^2-system.An ECL biosensor was constructed for ultrasensitive detection of carcinogenic marker ochratoxin A（OTA）based on non-enzymatic DNA walker as the effective amplification strategy.The target related bipedal DNA walker moved along stable 2D DNA tracks,improving the walking efficiency.Thus,biosensor achieved sensitive detection of OTA in the range of 10 fg/m L to 100 ng/m L,with a detection limit as low as 3.19 fg/m L.Significantly,this assay had been successfully applied to the practical analysis of OTA in edible vegetable oil samples,exhibiting great application ability in trace food analysis.（3）Electrochemiluminescence biosensor based on gold nanoclusters-hydroxyl double salt compound nanomaterial for detection of soluble urokinase type plasminogen activator receptorECL consists of two processes:electrochemical process and luminescence process.Thus,the development of an effective and simple strategy to promote two processes is of great significance for the overall improvement of ECL efficiency of Au NCs.In this work,Au NCs were immobilized on the surface of zinc-containing hydroxyl double salt hydroxy double salt（Zn-HDS）nanomaterials to prepare gold nanocluster-hydroxyl double salt compound nanomaterial（Zn-HDS@Au NCs）.Impressively,Zn-HDS could not only accelerate the electrochemical reduction of Au NCs in the electrochemical process,but also reduce the non radiative transition and improve optical radiative transition in the luminescence process,resulting in the ECL strength and efficiency of Zn-HDS@Au NCs had been increased by 3.9 times and 9.9 times compared with pure Au NCs.Thus,ECL biosensor was constructed for detection of soluble urokinase type plasminogen activator receptor（su PAR）based on Zn-HDS@Au NCs.The conversion and amplification of the target substance were achieved through specific immune reactions and catalytic hairpin self-assembly reactions.The stable DNA cubic track and positively charged SH-PEG-NH₃⁺sufficiently ensured the efficient walking of the DNA walker.Consequently,the biosensor method was used for the first time to achieve sensitive detection of su PAR ranging from 700 fg/m L to 80 ng/m L with a detection limit of 652 fg/m L,showing good application prospects in clinical monitoring and disease diagnosis.