Preparation And Application Of Single-cell Enzyme Sensor

This thesis utilizes the single cell enzyme sensor we used to quantitatively measure the matter single eosinophil cell released after it was stimulated with drugs.This thesis is divided into two part:In Chapter I :Single cell enzyme sensors were made, characterized and used simply. In Chapter II :Single eosinophil cell was activated with PMA and released H₂O₂ and we determined H₂O₂ quantitatively using single enzyme sensor with a dual microelectrode.In Chapter I: A array of microcells with the volume nanoliter were fabricated by the way of lithography and etching. Single neutrophil was converted into microcell by pushing slide and immobilized with denatured collagen on the bottom of the microcell. Scondly,Dig in PBS buffer can dissolve cholosterol on the cell membrane and microholes were formed on it. Finally a dual electrode was integrated with it. So the single cell enzyme sensor was made completely.A dual electrode consists of two electrode, one is Au disk working electrode with its radius 10μm ,the other is Ag/AgCl microreference electrode with its radius 50 μm. The radius of the tip of dual electrode is less than 100μm. So that this dual electrode can insert into the microcell we made. The pripincle of single cell enzyme sensor is that H₂O₂ reacts with H₂Q with the enzyme-MPO in single neutrophil cell and the rate of this reaction is fastened greatly.The product of this reaction-BQ was deoxidized on dual electrode.In this way,we detect H₂O₂ quantitivately.The reproduction of the single cell is very good. There are two difficulities during this expcrient.One is that the preparation of dual electrode and the other is the preparation of microcell array with the way of lithography and etching and single cell is immobilized on the bottom of the microcell with the denatured collagen.In Chapter II: A new way of detection of the substrate of single eosinophil release with single cell enzyme sensor was developped. Firstly,single eosinophil was converted into single cell enzyme sensor and activated with reaction mixture containing chemical substance-PMA.Secondly,it released matter-H₂O₂ detected with single cell enzyme sensor.The nanoliter injector was home-bulit.The activating time...