New Method For Single Cell Analysis And Adrenaline Cell Sensor

In chapter one of this thesis, the techniques of single-cell analysis were reviewed briefly. These techniques were capillary electrophoresis (CE) over the past two years, microfluidic chip including the cell-culture, cell manipulation and detection of trance material in single cells and image analysis such as fluorescence microscopy, laser scanning confocal microscopy (LSCM) and total internal reflection fluorescence microscopy (TIRFM).In chapter two, an electrochemical method with a microfluidic device was developed for analysis of single cells. In this method, cell injection, loading and cell lysis, and electrokinetic transportation and detection of intercellular species were integrated in a microfluidic chip with a double-T injector coupled with an end-channel amperometric detector. A single cell was loaded at the double-T injector on the microfluidic chip by using electrical field. Then, the docked cell was lysed by a direct current electric field of 220 V/cm. The analyte of interest inside the cell was electrokinetically transported to the detection end of separation channel and was electrochemically detected. External standardization was used to quantify the analyte of interest in individual cells. Ascorbic acid (AA) in single wheat callus cells was chosen as the model compound. AA could be directly detected at a carbon fiber disk bundle electrode. The selectivity of electrochemical detection made the electropherogram simple. The technique described here could, in principle, be applied to a variety of electroactive species within single cells.In chapter three, determination of mRNA was carried out by fluorescence resonance energy transfer (FRET). In this method, two fluorescence nucleic acid probes were hybridized with target mRNA and the FRET between the two probes was detected. The concentration of the target mRNA could be determined through measuring the fluorescence intensity of FRET. The method investigated here was applied to determine mRNA in the extracts of MGC 803 cells.In chapter four, high-throughput single-cell analysis of mRNA was developed based on FRET. In this method, cells were first perforated with digitonin to improvepermeability. In this case, the probes could diffuse into cells easily. After the probes were hybridizui with target mRXT \, the FRET image was taken. The fluorescence intensity of FRET of individual cells was obtained using software MetaMorph. Based on the fluorescence intensity, the mRNA amount in single cells could be acquired. Since the FRET images of thirty cells could be taken simultaneously, the analysis throughput was high. This method was applied to determine CEA mRNA in single MGC 803 cells.In chapter five, a method for determination of glucose in individual cells was developed based on double enzymes reaction. In this method, a single cell was loaded in a capillary and then a solution containing 10 mmol/L (SDS), glucose oxidase (GOD), horseradish peroxidase (HRP) and 10-Acetyl-3,7-dihydroxy- phenoxazine (ADHP) was introduced into the capillary. After the cell was lysed by SDS, oxygen was reduced to H2O2 by GOD in the presence of glucose released from the lysed cell. At the same time, ADHP was converted to fluorescent resorufin by HRP. The amount of glucose in the cell could be obtained through detecting the fluorescence intensity of resorufin. This method was used to determine glucose amount in single MGC 803 cells.In chapter six, we investigated the cell sensor of epinephrine based on the cell signaling transduction. The principle is: After epinephrine combined with adrenergic receptor on the cell surface, the stimulated G protein within cells causes the signaling transduction to generate glucose. The generated glucose is released from the cells into the solution. The glucose in the solution is then detected using the method described in chapter five. The amount of epinephrine can be measured through determining glucose concentration. Since the glucose concentration is higher than the epinephrine concentration, implying amplification of the signal. It was found that the signal was amplified 38 times. This cell sensor was used to determine epinephrine.