Copper-based Nanozyme Synthesis And Application In Bioelectrochemical Analysis

Human beings are very concerned about their health,and disease is the main threat to health.For many common and serious diseases,the earlier testing is available the better for subsequent treatment.On this basis,the corresponding biomarkers were proposed and validated.Sensitive detection of biomarkers is great significance for clinical disease diagnosis.There are many methods of biomarkers detection,including immunoassay,proteomics,mass spectrometry and molecular biology.In order to achieve the rapid analysis and tracking of the target,biosensing technology came into being,it combines analytical chemistry,life science,informatics,physics and other disciplines and technologies.For the DNA sensor,immunosensor,cell sensor or microbial sensor,the beacon molecule plays an important role.Using nanozyme as a beacon molecule will provide excellent conditions for sensitive detection of biomarkers.Nanozymes have attracted the attention of researchers because of their excellent catalytic performance and structural controllability.Among them,copper-based nanozymes show a good enzyme-like activity due to their efficient conversion between valence states of copper ions.In this paper,these copper-based nanozymes were synthesized by copper ion modified metal organic framework structure,cuprous oxide modified reduced graphene oxide,cysteine-histidine-Cumodifiedcuprousoxidenanoparticlesand cysteine-histidine-copper nanoparticle aggreates.These nanomaterials are used for the sensitive detection of bacterial endotoxin,glycated albumin,mucin-1 protein and Alzheimer's disease amyloid?₄₂oligomer,respectively.Finally,it provides theoretical support for the application of nanozyme in clinical detection.This dissertation is divided into six chapters.The Chapter-One is introduction and the Chapter-Six provides the summary and prospect.The other four sections are as follows:The second chapter:This work reports an electrochemical sensor based on Cu²⁺-modified metal-organic framework nanopaticles(Cu²⁺-NMOFs)of dual function for detection of bacterial lipopolysaccharide(LPS).The sensor was constructed with C18 alkane thiol chains tethered to gold nanoparticles/reduced graphene oxide(Au NPs/er GO)as LPS probe.The LPS can be immobilized over C18 alkane thiol chains via hydrophobic interactions firstly,and the Cu²⁺-NMOFs played a vital role of recognition unit were captured by anionic groups of the carbohydrate portions from LPS molecules based on the specific interactions.More importantly,the Cu²⁺-NMOFs also act as a catalytic unit for electrical signal.In the detection buffer solution with H₂O₂and dopamine,the Cu²⁺-NMOFs can catalyze dopamine oxidation to generate aminochrome,resulting in a highly electrochemical oxidation signal.The electrochemical sensor based on Cu²⁺-NMOFs of dual function was investigated by differential pulse voltammetry(DPV)and the stripping peak currents of dopamine oxidizted to aminochrome was used to monitor the level of LPS.The developed assay for LPS detection demonstrated a wide linear range of 1.5 pg·m L^-1to 750 ng·m L^-1with limit of detection down to 0.61 pg·m L^-1.It is worth mentioning that the electrochemical sensor has fewer modification steps and uses only Cu²⁺-NMOFs to realize dual function,the target LPS recognition and electrocatalysis for detection system.In addition,the fabricated sensor can be also applied to detect LPS in mouse blood serum.Thus,the proposed method provides a new method for LPS determination based on the catalytic peroxidase-mimicking NMOFs with the advantages of simplicity,label-free,and excellent specificity.The third chapter:In this work,an electrochemical ratiometric assay for glycated albumin(GA)was designed by combining the nanozyme electrocatalytic activity of Cu₂O-modified reduced graphene oxide nanocomposites(Cu₂O-r GO NCs)and the adsorption-desorption characteristics of DNA tripods on electrode surfaces.In this study,Cu₂O-r GO NCs were fabricated by simultaneous reduction of copper chloride and graphene oxide.Specifically,the prepared Cu₂O-r GO NCs display outstanding electrocatalytic activity to glucose oxidation and peroxidase-like catalytic activity toward 3,3',5,5'-tetramethylbenzidine in the presence of H₂O₂.Meanwhile,they have excellent electrical conductivity and adsorption-desorption properties of DNA.The methylene blue-labelled DNA tripod(T-DNA)could be captured on a glassy carbon electrode modified with Cu₂O-r GO NCs(Cu₂O-r GO/GCE).The T-DNA/Cu₂O-r GO NCs/GCE included a DNA probe-attached redox reporter(methylene blue)and glucose oxidize reporter(catalysed by Cu₂O-r GO NC nanozymes)to generate two signals,achieving good dual-signalling ratiometric GA assay.GA exhibited strong competitive capacity towards the T-DNA/Cu₂O-r GO NCs/GCE to combine T-DNA,causing sensitive changes in the ratio of two measured electrochemical currents.In particular,the ratio of the peak currents of glucose oxidation and methylene blue reduction(I_Glu/I_MB)was used to monitor GA's level and ultimately improve the accuracy of the method.The electrochemical sensor showed high sensitivity with a low detection limit of 0.07?g·m L^-1and a wide linear range of 0.2 to 1500?g·m L^-1.The proposed sensor was also used to measure the GA expression level in the blood serum of a diabetic mouse model.The fourth chapter:In this study,an electrochemical sensor for mucin-1(MUC1)was constructed based on the gold-modified reduced graphene oxide nanocomposite(Au@r GO)and the cysteine-histidine-Cu@cuprous oxide jujube-like nanozyme(CH-Cu@J-Cu₂O).At first,gold-modified reduced graphene oxide nanocomposites were immobilized on a glassy carbon electrode as substrate electrode.Then,the DNA aptamer was assembled on the surface of Au@r GO/GCE via Au-S bond.Successively,MUC1 was modified on electrode surface through the specific interaction between protein and aptamer.Finally,the antibody-modified CH-Cu@J-Cu₂O(Ab/CH-Cu@J-Cu₂O)was captured through the immune reaction.More importantly,the Ab/CH-Cu@J-Cu₂O contain a cysteine-histidine-Cu unit and a jujube-like Cu₂O unit,which can catalyze dopamine oxidation to generate aminochrome,resulting in a strong electrochemical oxidation signal.The electrochemical sensor based on CH-Cu@J-Cu₂O that enhanced catalytic was investigated by differential pulse voltammetry,and the stripping peak currents of dopamine oxidized to aminochrome were used to monitor the level of MUC1.The electrochemical sensor detects MUC1in the dynamic range spanning 4 orders of magnitude with high specificity,and the limit of detection is 0.085 pg·m L^-1,owing to the specific identification and enhance catalytic performance of Ab/CH-Cu@J-Cu₂O.The expression of MUC1 protein on the surface of MCF-7 cells,were further analyzed,indicating the proposed strategy offered a good model for the detection of protein markers.The fifth chapter:Amyloid-?(A?)oligomers have high toxicity during A?aggregation and are a potent therapeutic targets and diagnostic markers for the treatment of Alzheimer's disease(AD).This work is based on the laccase mimicking nano-aggregates as beacons to achieve simple and highly specific detection of A?oligomers.First,a peptide chain of amino acids that can specifically recognize A?oligomer is fixed on the surface of the electrode to achieve the target capture.Then,the DNA aptamer modified nano-aggregate beacon is bound to the target site,which can oxidize hydroquinone and generate electrochemical signal.Sensitive detection of A?oligomer was realized by the intensity of electrochemical signal.A wide linear range of 0.01 to 10 n M with a low detection limit of 0.0036 n M was obtained.The biosensor displayed excellent selectivity toward A?₄₂oligomer in contrast to other possible interfering analogs(A?₄₂monomer,A?₄₂fibril)at same concentrations.The recoveries for A?₄₂oligomer spiked the serum sample was 93.0?105.3%.This strategy has good specificity,repeatability and stability,and which is expected to be a new method to achieve sensitive detection of A?₄₂oligomers in clinical examinationIn summary,the preparation of copper-based nanozymes with high catalytic activity,as well as the composition,structure and morphology of these nanozymes have been systematically studied in this work,and these nanozymes were modified into multi-catalytic site structures,which can be used as beacons to amplify electrochemical signals.These studies provide theoretical and experimental basis for the design and synthesis of nanomaterials with low cost,high stability and high electrocatalytic activity.It is helpful to speed up the application of copper-based nanozyme in electrocatalysis.