HRP Catalysis Based Construction Of Single Particle Probe And Single Molecule Study

The conventional measurements are calculated to obtain the average results of countless molecules, and the results often represent overall similarities. Different from ensemble assays, single particle or molecule measurements could unearth the individual characteristics of single particle or molecule which may be hidden deep in the overall averaging. In this study, we use reaction catalyzed by Horseradish Peroxidase(HRP) to construct single-particle plasmonic probe and single molecule study.(1) Construction of single particle plasmonic probe for detection of hydrogen peroxide(H2O2)Alpha-synuclein(α-Syn) is in close proximity to synaptic vesicles, and is considered to play a significant role in a series of nerve injury and neurodegenerative diseases, leading to neurotoxicity. A previous research proposed hydroxyl radical is produced during the process of α-Syn accumulation through electron spin resonance assay, and indicated reactive oxygen species damage caused by this process could result in the death of substantia nigral neurons. However, the mechanism of how H2O2 influence the accumulation of α-Syn is still controversial.In order to resolve this problem, we take advantage of gold nanoparticle(Au NPs) to design an ultrasensitive single nanoparticle plasmonic probe to detect H2O2. One of the most important optical properties of Au NPs is localized surface plasmon resonance(LSPR). This unique optical properties bring about many useful features of Au NPs, for example, high scattering light with excellent stability, negligible bleaching, and above all it is very sensitive to the change of dielectric environments. We modify HRP on the surface of Au NPs, HRP then catalyze the polymerization of aniline to generate an ordered polyaniline(PANi) shell on the surface of Au NPs in the presence of H2O2,which causes the change of LSPR response of Au NPs. We detect this process through dark field microscope(DFM) and spectrometer detection, the detection limit is 8 n M. Then, we use this probe to detect H2O2 to clarify its role in the aggregation of α-Syn, and the results show that exogenous H2O2 plays a dominant role for the accumulation of α-Syn in vitro.(2) Single molecule study of HRPSince the pioneer work of Xie group researching on cholesterol oxidase(COx), single molecule enzymology has become an independent research field and research hotspot due to the capacity of revealling the dynamic interactions between single enzyme and the substrate, which is hidden in ensemble averaging. A previous literature suggested the activity of HRP was noncompetitive inhibited by its catalytic productresorufin. In order to study the kinetic dynamics of this reaction, we construct a single enzyme assay of HRP using DNA origami due to its easy addressability and modification. In order to co-localize enzyme site and the product generation site. The triangular DNA origami is fluorescent labelled, and the single HRP is fixed onto it simultaneously. We extract the fluorescence intensity time trajectory of single HRP catalyzing single turnover and analyze it mathematically, then propose a kinetic model of the catalytic process of single HRP.(3) Single molecule study of cascade reactionLife has evolved to organize ordered cascade reactions for enzymes to function effectively. Many researches propose various strategies for fabricating spatial confined multi-enzyme systems to research on the distance-dependence for activity of cascade enzymes, which neglect removing the unbind enzyme, leading to inaccurate information of cascade reactions. In order to obtain the exact catalytic kinetics of cascade reactions, we construct a single molecule system for this assay. In this study, triangular DNA origami is used to precise control the interspace of cascade pairGlucose Oxidase(GOx) and Horseradish Peroxidase(HRP). The constructed structures are purified through glycerol rate-zonal centrifugation to remove unbind enzymes.Then we confine the enzyme-origami complex onto the surface of glass coverslips and observe the real-time generation of products. The fluorescent products are generated only when the cascade pair is intact and the intensity trajectory of products directly reflect the catalytic dynamics of the cascade enzymes.