Single-Cell Analysis Based On Capillary Electrophoresis

In the chapter one, first of all, the principle of capillary electrophoresis (CE) was introduced briefly. Then the analysis of single cells by CE including the single cell sampling, detection methods, derivatization methods and methods of lysing cell were reviewed in detail. Finally, the work for capillary electrophoresis with electrochemiluminescence detection was summarized.In the chapter two, dopamine (DA), epinephrine (E) and ascorbic acid (AA) were separated and detected by capillary electrophoresis with electrochemical detection (ED) at a carbon fiber microdisk bundle electrode. The effects of the running buffer pH, running buffer concentration and separation voltage on CE-ED were studied. The optimum conditions of separation and detection were 17 mmol·L-1 Na2B4O7 (pH 9.04) for the buffer solution, 10 kV for the separation voltage, 5 kV and 10 s for the injection voltage and the injection time, and 0.45 V (vs. SCE) for the detection potential. The limits of detection (signal to noise, S/N =3) of the analytes were 1.2×10-7 g·mL-1 for dopamine, 1.7×10-7 g·mL-1 for epinephrine and 1.4×10-7 g·mL-1 for ascorbic acid. The method was used to detected dopamine, epinephrine and ascorbic acid in the mixture of their injections, The recoveries were DA 97%, EP 96%和AA 105%.In the chapter three, capillary electrophoresis was employed for the analysis of ascorbic acid in individual rat peritoneal mast cells using an end-column amperometric detector with a carbon fiber microdisk bundle electrode. The optimum conditions of separation and detection were 1.83×10-2 mol·L-1 NaH2PO4-1.70×10-3 mol·L-1 Na2HPO4 (pH 7.8) for the buffer solution, 20 kV for the separation voltage, and 0.8 V (vs. SCE) for the detection potential. The limit of detection is 1.7×10-6 mol·L-1 (S/N = 3) and the linear range is 5.0×10-6 -5.0×10-4 mol·L-1 with a correlation coefficient of 0.9962 for the injection voltage of 5 kV and the injection time of 10 s. The response for a series of seven injections of 5.0×10-5 mol·L-1 AA resulted in a relative standard deviation of 0.85% for the migration time, and 1.8% for the electrophoretic peak current, respectively.Without any pretreatment of the capillary, individual rat peritoneal mast cells could be drawn into the capillary easily. Then the single cell was lysed by 0.1% SDS. The amount of AA in individual rat peritoneal mast cells was detected to be 2.4-7.1 fmol. This is the first report of AA in individual rat peritoneal mast cells.In the chapter four, AA in individual rat hepatocytes was determined by capillary electrophoresis with electrochemical end-column amperometric detection at a new kind of homemade carbon fiber microdisk bundle electrode (CFMBE).The new CFMBE was more beneficial to environmental protection because of free hydrargyrum in the creating process. Moreover it was created more simply and used expediently, so it was very suited to CE/ED. The recovery of AA was between 91% and 97%, and the amount of AA in single rat hepatocytes ranged from 28 to 63 fmol. This is the first report of AA in individual rat hepatocytes.In the chapter five, a new method of the detection of AA in individual rat hepatocytes was developed by combining capillary electrophoresis with electrochemiluminescence (ECL) based on tris(2,2′-bipyridine) ruthenium(Ⅱ) (Ru(bpy)32+). The optimum conditions of separation and detection are 7.32×10-2 mol·L-1 NaH2PO4-1.16×10-2 mol·L-1 Na2HPO4 (pH 8.0) for the buffer solution, 15 kV for the separation voltage, 12 kV and 10 s for the injection voltage and the injection time, and 1.2 V(vs. Ag/AgCl)for the detection potential. The limit of detection is 1.0×10-8 mol·L-1 (S/N = 3) and the linear range is 1.0×10-8-1.0×10-4 mol·L-1 for the injection voltage of 12 kV and the injection time of 10 s. The response for a series of seven injections of 1.0×10-5 mol·L-1 AA resulted in a relative standard deviation of 0.38% for the migration time, and 2.6% for the ECL intensity, respectively. The amount of AA in individual rat hepatocytes ranged from 16 to 62 fmol. It's very closed to the amount of AA in the extract of rat hepatocytes 37 fmol. It's also closed to the amount of AA in individual rat hepatocytes based on capillary electrophoresis with electrochemical end-column amperometric detection in the chapter four 28 to 63 fmol.