Construction Of Novel Nanozyme Array Sensor And Its Application In Disease Diagnosi

Sensor array can generate differential response signals by the cross-reactions between multiple sensing units and analytes.Combined with data processing methods such as machine learning algorithm,simultaneous determination and differentiation of multiple analytes can be realized with simple operation,rapid response and high detection throughput.Graphene quantum dots（GQDs）is an emerging carbon-based nanomaterial with outstanding optical and electrical properties,as well as unique mimic enzymatic catalytic activity,and has been widely used in biosensing,disease diagnosis and treatment.In this paper,two different optical sensor arrays are designed based on the peroxidase activity of GQDs for the identification and detection of biomarkers（thiols and exosomes）to realize early diagnosis of diseases.In this thesis,two works was carried out as follows:1.Construction of metal ion-regulated GQD nanozyme-based sensor array for thiol differentiation and disease identificationIn this work,a three-channel nanozyme sensor array has been constructed using terephthalic acid functionalized graphene quantum dots（TPA@GQDs）as a basic material.By regulating the catalytic activity of TPA@GQDs with three metal ions(Fe²⁺,Cu²⁺and Zn²⁺),six types of thiols can be accurately differentiation.As a nanozyme,TPA@GQDs possess the capability to catalyzes the oxidation of 3,3’,5,5’-tetramethylbenzidine（TMB）by hydrogen peroxide（H₂O₂）,producing characteristic absorption peaks at 650 nm.Three transition metal ions(Fe²⁺,Cu²⁺and Zn²⁺)with different Fenton activities can coordinate with abundant hydroxyl groups on the surface of TPA@GQDs to induce the formation of metal ion-regulated catalytic active centers,resulting in the increase of the catalytic activity of metal ion-TPA@GQDs ensembles to various degrees.However,in the presence of thiols,the strong affinities between thiols and metal ions promote the release of hydroxyl groups,which inhibits the catalytic activity of the sensing unit to divese degrees.In a TMB-H₂O₂system,each sensing unit can generate differential colorimetric signals（i.e.,fingerprints）for various thiol analytes.Six thiols（cysteine,glutathione,homocysteine,dithiothreitol,6-mercaptohexanol and thioglycollic acid）are accurately indentified through machine learning algorithm with a detection limit of 50 n M.In addition,discrimination of the same thiol with different concentrations and thiol mixtures have also been achieved using the nanozyme-based sensor array with good anti-interference performance.Further,to evaluate the feasibility of the nanozyme sensor array in practical applications,accurate identification of a variety of diseases has been realized according to the difference of thiol content in actual samples.The nanozyme sensor array is simple to operate and low cost,which shows great application potential in early diagnosis of diseases.2.DNA flower structure encapsulatd Fe²⁺/TPA@GQDs-based colorimetric/photothermal dual-mode biosensor for exosome detection and cancer diagnosisIn this work,based on the dual aptamers recognition strategy,a"magnetic bead capture probe-exosomes-Fe²⁺/TPA@GQDs@DFs recognition probe"sandwich structure is constructed for colorimetric/photothermal dual-mode exosome detection.In the sandwich structure,Ep CAM-modified magnetic beads are used as exosome capture probes,and Fe²⁺/TPA@GQDs@DFs serves as exosome recognition probes.In addition,by simply changing the the aptamers in capture probes,a sensor array is constructed for cancer diagnosis.Specifically,the complementary sequence of CD63 exosome aptamer is firstly designed into the circular template to obtain DNA flowers（DFs）structure based on rolling circle amplification（RCA）and DNA self-assembly.In the process of RCA,Fe²⁺modified TPA@GQDs(Fe²⁺/TPA@GQDs)nanozyme is encapsulated into DFs(Fe²⁺/TPA@GQDs@DFs),which not only serves as exosome recognition probe,but also improves the stability of Fe²⁺/TPA@GQDs simultaneously.The aptamer Ep CAM is anchored on the magnetic beads to capture exosomes（separated from He La cells）,in which a sandwich structure of"magnetic bead capture probe-exosomes-Fe²⁺/TPA@GQDs@DFs recognition probe"is thus constructed based on dual aptamer recognition strategy.In the acidic TMB-H₂O₂ system,DFs are disintegrated to release Fe²⁺/TPA@GQDs with intense peroxidase-like activity,for the catalyzed oxidation of TMB along with the characteristic absorption peak of at 650 nm,which further exhibit photothermal effect under near infrared laser irradiation.In this way,a colorimetric/photothermal dual-mode biosensor has been designed for the detection of He La derived exosomes.Based on the catalytical activity promotion by photothermal performance,the detection sensitivity in colorimetric mode is improved significantly with a limit of detection of 1300 particles m L^-1,which can be applied to the accurate determination of exosomes in real human serum successfully.Furthermore,considering the differences in the types and expressions of exosomal proteins derived from different serums,three aptamers（HER2,MUC1 and GPC3）anchored magnetic beads are selected as capture probes to construct a three-channel sensor array based on the sandwich structure of"magnetic bead capture probe-exosomes-Fe²⁺/TPA@GQDs@DFs recognition probe",in which the discrimination of different types of cancer and the indenitfication of liver cancer from hepatitis are achieved,providing new avenues for early diagnosis of cancer.