Investigation Of Protein Biosensing Based On One-dimensional Peroxidase Mimics

Enzyme-linked immunosorbent assay?ELISA?is one of the most commonly used methods for qualitative and quantitative analysis of target proteins molecules by specific binding of antigens and antibodies and special markers.It requires an antigen or antibody to be attached to an enzyme?e.g.,horseradish peroxidase,HRP?as a reporter group for signal amplification.However,the natural enzymes have a lot of intrinsic drawbacks,such as high cost,laborious preparation,difficult storage,and easy denaturation by the external environment.Nanozyme,emerged as“artificial enzyme”with catalytic activity,has been widely used in various fields,such as biology,medicine,and environment,due to the advantages of high catalytic efficiency,convenient preparation,and stable properties.Herein,we proposed an ultrasensitive protein colorimetric assays based on one-dimensional inorganic nanozymes.The contents of this thesis are as follows:One dimensional?1D?core-shell Fe₃O₄@C nanowires were synthesized by a solvent-thermal method.Then,the morphology and the structure of the Fe₃O₄@C nanowires were characterized by scanning electron microscopy?SEM?and transmission electron microscopy?TEM?,respectively.More importantly,the prepared nanocomposites showed excellent peroxidase-like activity through TMB reaction system,and the signal-to-noise ratio reached 8 times.In addition,the influence factors,such as pH,temperature,and the concentration,were studied,and the optimal conditions of the catalytic activity were obtained.By comparison,the Michaelis-Menten constant of the prepared Fe₃O₄@C nanowires was 670 and 17times lower than that of HRP and Fe₃O₄ NPs with the H₂O₂ as substrate,respectively,suggesting a higher affinity and catalytic activity.Moreover,it was also found that the catalytic mechanism follows a ping-pong mechanism.Based on the peroxidase-like activity of the Fe₃O₄@C nanocatalyst and nucleic acid aptamers,we developed a nanozyme-linked aptamer sorbent assay?NLASA?for detection of platelet-derived growth factor-BB?PDGF-BB?.Under the optimized experimental conditions,the proposed biosensor showed an ultrasensitive detection of PDGF-BB with a dynamic range spanning 7 orders of magnitude,with detection limit as low as 10 fM.Moreover,the color change of the TMB reaction system corresponding to different concentration of the target could be easily observed by naked eyes.To further improve the detection limit,we immobilized a DNAzyme by incorporating of hemin into a G-quadruplex?G4?sequence onto the surface of Fe₃O₄@C nanowires to form hybrid artificial enzymes.As a result,the detection limit has been further lowered to 50 aM due to the synergetic catalytic effects.Furthermore,NLASA assay could effectively detect PDGF-BB as low as 100 fM in 50%human serum,where both the colorimetric signal and the background were almost unchanged compared to that in buffer systems,offering new opportunities for protein detection in clinical diagnosis.We developed a protein sensor array based upon the peroxidase-like catalytic activities of three 1D inorganic nanozymes.Three kinds of 1D core-shell nanocomposites,including PPy@MoS₂@Pd,PPy@MoS₂@Au,and PPy@MoS₂@Ag,were prepared and characterized by SEM.In addition,the peroxidase-like activity,the influencing factors and the catalytic mechanism of the nanozymes were also investigated.Based on the interactions between eight kinds of representative proteins and three kinds of the prepared 1D nanozymes,the effect of proteins on the catalytic activity of different nanomaterials were studied,and the protein sensor array was initially successfully constructed.