Studies And Andapplications Of Enzyme/Cell Nano-Assembling Based On Aluminana Nochannel Array

Enzymatic catalysis reaction is an important path for the exogenous and endogenous metabolism in organisms.Using nanomaterials to simulate biological enzyme assmebling,investigate the biotransformation and metabolic mechanisms of synthetic chemicals and environmental toxicants possess fundamental studies and practical applications for the development of life sciences and new drugs,toxicology research and evaluation.Meanwhile,the research of interaction mechanism between enzyme and nanomaterials and the nature of the enzyme metabolic reaction also provide technical support for the assessment of environmental chemicals toxicity,the increasment of gas invovled catalytic reactions efficiency,and the study of pathogenic mechanisms of gaseous pollutants.In this dissertation,based on enzymes/cells nano-assembling,three-phase gas-solid-liquid interface was constructed on alumina nanochannel arrays which can be used for glucose oxidase and lung epithelial cells immolization.Various analytical techniques such as electrochemistry,spectroscopy,mass spectrometry can be used to study the kinetic mechanism of the enzyme reaction in which oxygen directly participates in glucose oxidation from the gas phase and the cell cytoxicity mechanism in which respiratory toxicants contact with cell directly.The details are as follows:1.The construction and application of gas-solid-liquid three-phase interface:the low solubility of gases in aqueous solution is the major kinetic limitation of gas involved reactions.To improve the kinetic efficiency of the enzymatic reaction with gas involved,a porous anodic alumina?PAA?nanochannel membrane with different wettability was constructed for glucose oxidase?GOx?immobilization,which contacted with glucose aqueous solution on one side,while the other side get in touch with the gas phase directly.Interestingly,it was observed that the O₂ could participate in the enzymatic reaction directly from gas phase through the fabricated nanochannels,and the hydrophobic interface was more favorable for the enzymatic reaction due to the rearrangement of GOx structure as well as the high gas adhesion.As a result,the catalytic efficiency of GOx in the proposed interface was increased up to 80-fold compared with that of the free state in traditional aqueous air-saturated electrolyte.This controlled wettability gas-solid-liquid three-phase interface was constructed for enzyme immobilization,and the gaseous molecules from gas phase could participate in the catalytic reaction directly,which could improve the kinetic efficiency of the reaction with gas involved and solve lower solubility of the gas in solution.Therefore,this triphase interface with controlled wettability provides a novel strategy for gas involved biocatalysts,industry catalysis and synthesis.2.The gas pollution exposed cell model based on gas-to-liquid-liquid three-phase interface:The development of a novel gas pollution exposed cell model for the detection and evaluation of biological toxicity mechanisms of gaseous pollutants plays an important role in the study of toxic mechanisms,signal transmission and development mechanism of environmental substance.Thus,Human pulmonary alveolar epithelial cell?HPAEC?were cultured at nanobiochip with a gas-solid-liquid three-phase interface,at which HPAEC could be contact with gas pollution directly,and the expression of proteins and inflammatory factors were studied after exposure to gas pollution to elucidate the cytotoxic mechanism of pollutants exposure.Compared with conventional gas-liquid interface,the viability of cell at the prepared three-phase interface was almost enhanced up to 3 folds.Additionally,it was observed that the inflammatory responses of HPAEC induced by gas pollution from gas phase directly via passing through the prepared nanochannels showed obvious variation,which was assessed by gene expression profiles,phase I/II activity,secretion of inflammatory factor.After exposure to gas pollution from gas phase,the increment of inflammatory factors IL-2,IL-6 and TNF-?were almost two times larger than that induced by gas pollution from solution,which illustrated that the prepared nanobiochip could be utilized for inflammation response analysis of HPAEC more sensitively.Furthermore,the inhibitor of enzyme was introduced to evaluate the potential application of the nanobiochip.This disposable nanobiochip may enable development of integrated cell culture and toxicity models and provide low-cost alternatives to clinical researches for drug screening and toxicology application.3.Cytochrome 1A1 assembly on electrode surface with electrocatalytic behavior:The behavior of direct electrochemical and electrochemical catalytic kinetics reaction of enzyme was researched by the functionalized nanomaterial.Thus,a butyric acid?PBA?functionalized nitrogen-doped graphene nanocomposite?PB/NG?was constructed to modified the electrode for cytochrome CYP1A1 immobilization,and the rapid electron transfer between CYP 1A1 and the electrode was achieved.A pair of pronounced stable redox peak at-0.456 and-0.512 V?vs SCE?were displayed in electrochemical cyclic voltammogram,with an apparent electron transfer constant of 0.53 s^-1.The increment of the peak current with the addition of BaP to the air-saturated buffer further confirmed the electrochemical catalytic behavior of CYP1A1 for BaP,the catalytic reaction constant(K_m^app)and the heterogeneous reaction rate constant are 25.6?M and 1.9 s^-1,respectively.At the same time,the production of metabolites hydroxylated benzopyrenes was also domenstrated by high performance liquid chromatography?HPLC?and gas chromatography-mass spectrometry?GC/MS?techniques.Finally,using this electrochemically driven enzyme catalysis technique,the inhibitory action of the inhibitor?-napthoflavone on the catalytic activity of CYP1A1 was further studied.This work provides new methods and means for the development of drugs and the detection of chemical toxicants in foods and the environment.4.The detection of nanobody based on photoelectrochemical immunosensor:Herein,A simple and sensitive photoelectrochemical immunosensor based on TiO₂ nanotube arrays?TNAs?was proposed for the sensitive detection of Serum cysteine protease inhibitor C?CysC?.The TNAs which deposited by electrochemical anodization displayed a high and stable photocurrent response under irradiation.After coupling CysC-specific nanobody to TNA?Nb/TNA?with glutaraldehyde,the proposed immunosensor for CysC can be utilized for tracking the photocurrent change of Nb/TNA caused by immunoreactions between CysC and the immobilized CysC-specific Nb.This allowed for the determination of CysC with a calibration range from 0.72 pM to 7.19 nM.The variation of the photocurrent was in a linear relationship with the logarithm of the CysC concentration in the range of 0.72 pM-3.6 nM.The immunosensor had a correlation coefficient of 0.97 and a detection limit of0.14 pM at a signal-to-noise ratio of 3.The proposed immunosensor showed satisfactory intra-and inter-assay accuracy,high selectivity and good stability.As a result,this proposed strategy would offer a novel and simple approach for the detection of immunoreactions.