Improving The Selectivity In Single Cell Analysis And Sample Loading Performance On Chip-based CE

Chip-based capillary electrophoresis(chip-based CE)has emerged as a main area of development in the field of micrototal analysis systems(μTAS) and known for it's high speed analysis,minute sample and reagent consumption and easy to integrate.However,chip-based CE limits the column capacity due to the short separation channel.It is difficult to separate and identify all the intracellular species interested.Sample injection is crucial because it determines the quantity and the shape of a sample plug,both of which are closely related to separation. Recently,there is a growing interest in delivering variable-volume sample plugs into separation channel.However,four or more electrodes with complicated electric power supplies and implementation are needed to vary the volume of the sample plugs with multiple electrokinetic pinching techniques.The present work reports an ultrasensitive method for the determination of intracellular superoxide(O₂^?)in individual HepG2 cells by chip-based CE after labeling with a specific probe dihydroethidium(DHE)and proposes a variable-volume sampling scheme for chip-based free-zone electrophoresis using negative pressure combined with electrokinetic force for the first time.In Chapter 1,Intracellular labeling method and sample loading method used for chip based CE are reviewed.In Chapter 2,Dihydroethidium(DHE)was used as the specific fluorescent probe to react with intracellular superoxide(O₂^?)to form the fluorescent 2-hydroxyethidium.The electrophoretic behavior of 2-hydroxyethidium and ethidium cation(E⁺),which was recently reported to be generate from photochemical oxidation of DHE and interfere the determination of O₂^?with fluorescence microscopic technique,was studied. The excellent resolution between 2-hydroxyethidium and ethidium cation(E⁺) can be achieved within 20 s.An extremely low detection limit of 2.0 amol was achieved which is more than three orders of magnitude lower than that obtained by using HPLC-EC and five orders of magnitude lower than that obtained by employing HPLC fluorescence analysis.Different from the HPLC analysis results,only 2-hydroxyethidium but not E⁺ was detected,indicating that photooxidation of DHE to E⁺ was avoided by using the suggested single-cell analysis method.In Chapter 3,A variable-volume sample loading scheme for chip-based free zone CE was developed by negative pressure combined with electrokinetic force.This was achieved by using a low-cost microvacuum pump and a single potential supply at a constant voltage.By adjusting the subambient pressure applied to the headspace of sample waste reservoir (SW)and the electric field between B and BW,the charged sample in the sample flow can be driven into the separation channel.The injected sample plug length is in proportion with the loading time and the loading voltage. Once the vacuum in SW reservoir was released to activate electrophoretic separation,flows from S and B to SW were immediately terminated by the back flow induced by the difference of the liquid levels in the reservoirs to prevent sample leakage during the separation stage.It is easy to freely choose the sample plug volume in this method by simply changing the loading time.The system has been proved to possess an exciting potential for improving sensitivity of chip-based CE.