Measure Of H₂O₂ Released From Single Cells And Constructe A Cell-based Biosensor To Detect L-lactate

This thesis is consisted of two parts: In chapter I, H₂O₂ was quantitatively measured, which was released from single cells after stimulation with drugs. In chapter II, a new cell-based sensor was constructed, the sensor was used to measure L-lactate.In chapter I: H₂O₂ was measured by voltammetry using a microcell with a positionable dual electrode. Dual microelectrodes with tip diameters of <200μm were made with theta glass capillaries. A platinum microelectrode and a Ag/AgCl reference electrode were inserted into the two channels of theta glass capillary, the microelectrodes could be easily placed into an electrochemical microcell to analyze compounds in the solution. The microcell array was constructed by photolithographic technique. The extraction of human neutrophils released H₂O₂ after incubation with PMA, and the released H₂O₂ could be detected with dual microelectrodes by voltammetry. The whole procedures of single cells analysis, such as transference of single cells and PMA, incubaton and voltammetry-detection, were integrated on a single chip. The average released amount of H₂O₂ was determined by using the standard calibration method. The single cell released amount of H₂O₂ determined in our experiments is in the range of 21 - 70 fmol/cell.In chapter II: A new cell-based biosensor was constructed to quantitatively measure of L-lactate. We integrated the working, counter and reference electrodes on a single ITO plate. A PDMS layer with micro-hole was fabricated on the same glass chip to form a micro electrochemical cell. In the experiments, cells was immobilized on the bottom of an electrochemical cell. The cells were perforated with digitonin to form enough micropores on the cell membrane. When these immobilized cells were immersed in the buffer containing reaction substrates, the substrates could enter the cells via these micropores by diffusion. Lactate dehydrogenase inside the cells could convert L-lactate and K₃Fe(CN)₆ to pyruvate and K₄Fe(CN)₆ in proportion, Then L-lactate and K₄Fe(CN)₆ were diffused back into the solution surrounding the cell's surface. The amount of L-lactate could therefore be determined by measuring the amount of the K₄Fe(CN)₆ indirectly, involving the amperometric determination of K₄Fe(CN)₆ with +550 mV (vs. Ag/AgCl).