毛细管电泳单细胞分析
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摘要
第一章,首先对毛细管电泳的基本原理进行了简单的介绍。然后对毛细管电泳应用于单细胞分析(包括单细胞采样,单细胞分析检测方法,单细胞衍生技术及单细胞溶膜等)进行了详细的综述,并介绍了电化学发光检测技术在毛细管电泳的应用。
第二章,我们采用了毛细管电泳柱端安培检测的方法,以碳纤维簇微盘电极为工作电极分离并检测了多巴胺(dopamine, DA)、肾上腺素(epinephrine, E)和抗坏血酸(ascorbic acid, AA)。考察了缓冲液的pH值、缓冲液浓度、分离电压等实验参数对分离、检测的影响。最佳实验条件是,缓冲液为pH=9.04的17 mmol·L-1 Na2B4O7,分离电压为10 kV,检测电势为0.45 V(vs. SCE)。5 kV电渗进样10 s的条件下,各组分的检测限(S/N=3)分别为:多巴胺1.2×10-7 g·mL-1,肾上腺素1.7×10-7g·mL-1和抗坏血酸1.4×10-7 g·mL-1,并以此方法检测了盐酸多巴胺注射液、盐酸肾上腺素注射液和维生素C注射液的混合液中多巴胺、肾上腺素和抗坏血酸的含量,样品的加标回收率分别为DA 97%,E 96%和AA 105%。
第三章,采用毛细管电泳柱端安培检测的方法,以碳纤维簇微盘电极为工作电极测定了单个大鼠腹腔肥大细胞中抗坏血酸的含量。检测抗坏血酸的最佳实验条件是,缓冲液pH=7.8的1.83×10-2 mol·L-1 Na2HPO4-1.70×10-3 mol·L-1 NaH2PO4,分离电压为20 kV,检测电势为0.8 V(vs. SCE),在此条件下,5.0 kV进样10 s,得到了抗坏血酸的线性范围(5.0×10-6 -5.0×10-4 mol·L-1),线性回归系数为0.9962,并以电泳峰电流与噪声比值(S/N)为3时得到浓度检测限为1.7×10-6 mol·L-1。将5.0×10-5 mol·L-1抗坏血酸标准溶液连续进样7次,得到抗坏血酸的迁移时间tm和峰电流ip的相对标准偏差(RSD)分别为0.85%和1.8%。在这一方法中,单个细胞通过电迁移引入分离毛细管的进样端,在2.0 kv的电压下引入0.1%的SDS作为细胞溶膜液,溶膜后细胞中的组分在毛细管中得到分离并检测。用上述方法测得单个大鼠腹腔肥大细胞中抗坏血酸的含量为2.4-7.1 fmol,这是对CE/柱端安培检测单个大鼠腹腔肥大细胞中抗坏血酸的首次报道。
第四章,采用了一种自制的碳纤维簇微盘电极,以CE/柱端安培检测测定了单个大鼠肝细胞中的抗坏血酸。这种新型的碳纤维簇微盘电极灵敏度高,体积更小,适用于在多种仪器的狭窄空间内使用(例如MPI-A型毛细管电泳电化学发光检测仪和MPI-M型微流控芯片化学发光分析系统)。并且克服了传统电极中需要用汞做导体的缺点,因而对环境无污染。我们以此电极为工作电极检测了单个大鼠肝细胞中的抗坏血酸。该方法测得的抗坏血酸的回收率是91-97%,单个大鼠肝细胞中抗坏血酸的含量为28-63 fmol,这是对CE/柱端安培检测单个大鼠肝细胞中抗坏血酸的首次报道。
第五章,我们采用毛细管电泳-电化学发光来检测单个肝细胞中抗坏血酸的含量,我们所使用的发光试剂是Ru(bpy)32+。确定了检测抗坏血酸的最佳实验条件是:缓冲液为pH=8.0的7.32×10-2 mol·L-1 NaH2PO4-1.16×10-2 mol·L-1 Na2HPO4,分离电压为15 kV,检测电势为1.2 V(vs. Ag/AgCl)。使用内径25μm,长60 cm的毛细管,在12 kV电渗进样10 s的条件下,检测抗坏血酸的线性范围为1.0×10-8-1.0×10-4 mol·L-1,检测限可达1.0×10-8 mol·L-1 (S/N=3)。7次平行测定的迁移时间与峰电流的相对标准偏差分别为0.38%和2.6%。这是将CE-ECL用于单细胞分析的首次报道。检测单个大鼠肝细胞中抗坏血酸的含量为16-62 fmol,与测得的肝细胞提取液中平均每个细胞中抗坏血酸的含量37 fmol·cell-1很接近,且与第四章中用CE/柱端安培检测测得的单个大鼠肝细胞中抗坏血酸的含量28-63 fmol相一致。
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.
第二章,我们采用了毛细管电泳柱端安培检测的方法,以碳纤维簇微盘电极为工作电极分离并检测了多巴胺(dopamine, DA)、肾上腺素(epinephrine, E)和抗坏血酸(ascorbic acid, AA)。考察了缓冲液的pH值、缓冲液浓度、分离电压等实验参数对分离、检测的影响。最佳实验条件是,缓冲液为pH=9.04的17 mmol·L-1 Na2B4O7,分离电压为10 kV,检测电势为0.45 V(vs. SCE)。5 kV电渗进样10 s的条件下,各组分的检测限(S/N=3)分别为:多巴胺1.2×10-7 g·mL-1,肾上腺素1.7×10-7g·mL-1和抗坏血酸1.4×10-7 g·mL-1,并以此方法检测了盐酸多巴胺注射液、盐酸肾上腺素注射液和维生素C注射液的混合液中多巴胺、肾上腺素和抗坏血酸的含量,样品的加标回收率分别为DA 97%,E 96%和AA 105%。
第三章,采用毛细管电泳柱端安培检测的方法,以碳纤维簇微盘电极为工作电极测定了单个大鼠腹腔肥大细胞中抗坏血酸的含量。检测抗坏血酸的最佳实验条件是,缓冲液pH=7.8的1.83×10-2 mol·L-1 Na2HPO4-1.70×10-3 mol·L-1 NaH2PO4,分离电压为20 kV,检测电势为0.8 V(vs. SCE),在此条件下,5.0 kV进样10 s,得到了抗坏血酸的线性范围(5.0×10-6 -5.0×10-4 mol·L-1),线性回归系数为0.9962,并以电泳峰电流与噪声比值(S/N)为3时得到浓度检测限为1.7×10-6 mol·L-1。将5.0×10-5 mol·L-1抗坏血酸标准溶液连续进样7次,得到抗坏血酸的迁移时间tm和峰电流ip的相对标准偏差(RSD)分别为0.85%和1.8%。在这一方法中,单个细胞通过电迁移引入分离毛细管的进样端,在2.0 kv的电压下引入0.1%的SDS作为细胞溶膜液,溶膜后细胞中的组分在毛细管中得到分离并检测。用上述方法测得单个大鼠腹腔肥大细胞中抗坏血酸的含量为2.4-7.1 fmol,这是对CE/柱端安培检测单个大鼠腹腔肥大细胞中抗坏血酸的首次报道。
第四章,采用了一种自制的碳纤维簇微盘电极,以CE/柱端安培检测测定了单个大鼠肝细胞中的抗坏血酸。这种新型的碳纤维簇微盘电极灵敏度高,体积更小,适用于在多种仪器的狭窄空间内使用(例如MPI-A型毛细管电泳电化学发光检测仪和MPI-M型微流控芯片化学发光分析系统)。并且克服了传统电极中需要用汞做导体的缺点,因而对环境无污染。我们以此电极为工作电极检测了单个大鼠肝细胞中的抗坏血酸。该方法测得的抗坏血酸的回收率是91-97%,单个大鼠肝细胞中抗坏血酸的含量为28-63 fmol,这是对CE/柱端安培检测单个大鼠肝细胞中抗坏血酸的首次报道。
第五章,我们采用毛细管电泳-电化学发光来检测单个肝细胞中抗坏血酸的含量,我们所使用的发光试剂是Ru(bpy)32+。确定了检测抗坏血酸的最佳实验条件是:缓冲液为pH=8.0的7.32×10-2 mol·L-1 NaH2PO4-1.16×10-2 mol·L-1 Na2HPO4,分离电压为15 kV,检测电势为1.2 V(vs. Ag/AgCl)。使用内径25μm,长60 cm的毛细管,在12 kV电渗进样10 s的条件下,检测抗坏血酸的线性范围为1.0×10-8-1.0×10-4 mol·L-1,检测限可达1.0×10-8 mol·L-1 (S/N=3)。7次平行测定的迁移时间与峰电流的相对标准偏差分别为0.38%和2.6%。这是将CE-ECL用于单细胞分析的首次报道。检测单个大鼠肝细胞中抗坏血酸的含量为16-62 fmol,与测得的肝细胞提取液中平均每个细胞中抗坏血酸的含量37 fmol·cell-1很接近,且与第四章中用CE/柱端安培检测测得的单个大鼠肝细胞中抗坏血酸的含量28-63 fmol相一致。
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.
引文
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