董 谦
论文题目：毛细管电泳/安培检测生物物质及单细胞分析
作者简介：董 谦，女，1973年08月出生，1998年09月师从于山东大学金文睿教授，于2001年06月获博士学位。
摘 要
本文第一章首先对毛细管电泳（CE）的基本原理及与其联用的常见检测器（紫外、激光诱导荧光和电化学检测器）进行了简单的介绍。其次对CE电化学检测中应用较少的电导和电势检测器的原理和典型的装置进行了简述。然后详细的综述了应用最多的安培检测及其应用。这一部分主要涉及到理论，技术，电极，方法，应用，微型化CE及单细胞分析。本章共引用文献267篇。
第二章涉及到乳酸钠、丙酮酸钠及转铁蛋白的CZE/柱端安培检测。迄今为止尚未见有关报道。我们发现乳酸钠及丙酮酸钠在碳纤维电极上的阳极线形扫描伏安图上没有明显的氧化峰信号，因此乳酸钠和丙酮酸钠的直接伏安测定无法进行。但是，如果CZE与柱端安培检测联用，可以实现对乳酸钠及丙酮酸钠的直接检测。我们采用自行设计的CZE/柱端安培电化学检测系统，用碳纤维作为工作电极，确定了检测乳酸钠和丙酮酸钠的最佳实验条件是：缓冲液为pH＝7.2的3.6×10-3 mol/L
Na2HPO4-1.4×10-3 mol/L NaH2PO4，分离电压为18.0 kV，检测电势为1.60 V（vs.SCE）。在内径30 µm，长63 cm的毛细管，6.0
kV电渗进样5 s的条件下，检测乳酸钠的线性范围为1.7×10-6-8.2×10-4 mol/L，检测限可达7.6×10-7 mol/L (1.7 fmol，S/N＝3)；检测丙酮酸钠的线性范围为1.0×10-5 -5.0×10-3 mol/L，检测限可达8.0×10-6 mol/L (24 fmol，S/N＝3)。对8.44´10-5 mol/L乳酸钠平行6次测定的迁移时间（tm）与峰电流（ip）的相对标准偏差分别为1.8％，3.3％。对1.00´10-4 mol/L丙酮酸钠平行6次测定的tm与ip的相对标准偏差分别为2.0％，5.7％。并将此方法应用于测定人唾液中的乳酸钠和人汗液中的丙酮酸钠。此外还用这套检测系统，在缓冲液为7.5×10-4 mol/L Tris-3.4×10-4 mol/L HCl，分离电压为20 kV，检测电势为1.90 V的条件下测定了人血清中的转铁蛋白的含量。人血清中5-羟色胺不会干扰人血清中转铁蛋白的测定。测定结果与文献值一致。
第三章研究的是CZE/柱端安培检测19种常见的氨基酸，采用萘-2，3-二缩醛（NDA）及NaCN柱前衍生的方法。此法所得的氨基酸衍生产物的稳定性好，在碳纤维电极上电化学响应较灵敏。用这种方法文献中仅检测了丙氨酸、谷氨酸和天冬氨酸等3种氨基酸。检测19种氨基酸衍生产物的最佳实验条件是：缓冲液为1.25´10-2 mol/L硼砂- 3.13´10-3 mol/L NaOH，分离电压为18 kV，检测电势为1.15 V。在内径20 µm，长80 cm的毛细管，6.0
kV电渗进样5 s的条件下，可很好的分离10种氨基酸（精氨酸、赖氨酸、鸟氨酸、色氨酸、丝氨酸、丙氨酸、甘氨酸、半胱氨酸、谷氨酸和天冬氨酸）。分别检测单个氨基酸所得的氨基酸的检测限在1.7´10-7 mol/L-1.8´10-6 mol/L之间。我们发现了前人在氨基酸的检测中从未谈及的一种现象，即在测定氨基酸混合物时某些氨基酸的电化学响应与测定单个氨基酸时不同。当检测不同种类及不同数量的氨基酸的混合物时，某些氨基酸衍生产物的电化学响应有时会升高而有时会降低。它们相应的检测限及其线性范围也将随之发生变化。这种现象是由于当混合物中的氨基酸与NDA及NaCN进行衍生反应时，各种氨基酸之间的相互竞争。因此在检测实际样品时，不能用单个氨基酸测定的工作曲线定量样品中的氨基酸，而应该用标准加入法进行样品中氨基酸的定量。在最佳分离及检测条件下，测得啤酒样品中精氨酸、色氨酸、苯丙氨酸、酪氨酸、缬氨酸、丙氨酸及甘氨酸7种氨基酸。测定这7种氨基酸的回收率在91%~109 %之间。
第四章研究了用CZE/柱端安培检测法，在pH 7.0的Na2HPO4-NaH2PO4缓冲液中用Au/Hg电极检测单个巨噬细胞中的谷胱甘肽（GSH）。在这一方法中，毛细管的进样端口不需作任何腐蚀或削尖的处理，可用电迁移的方法方便的将单个全细胞导入分离毛细管中。检测巨噬细胞中的GSH不需要任何衍生或预处理步骤，单细胞的定量测定不会受衍生反应的效率的影响，也不会受部分进样造成的进样效率以及样品在样品池中保存状况的影响。我们发现，文献中常用于破膜的低渗缓冲液无法使巨噬细胞破膜，而用0.01 mol/L NaOH可使巨噬细胞破膜。我们还发现，如用文献中报道的用于单细胞分析中的工作曲线法定量，所得单细胞中GSH的量小于文献值，因此不能用工作曲线法定量单个巨噬细胞中GSH的含量。我们提出了一种新的文献中从未报道的定量单细胞中物质的方法，即在每次单细胞测定后再测定标准溶液作为外标来定量单细胞中的物质。用这种方法所得的单个巨噬细胞中GSH的平均含量为5.8 fmol，与文献值一致。实验结果指出，巨噬细胞中GSH含量的分布不符合高斯分布，它反映了细胞之间的差别，这种现象是只有通过单细胞的检测才能发现的。
第五章中采用细胞电穿孔这种将物质导入细胞中的技术，实现了将双氯灭痛这种药物导入人血红细胞，并对单个血红细胞中导入的双氯灭痛的含量用CZE/柱端安培检测法进行测定。细胞电穿孔技术是给细胞施加脉冲电压，使细胞膜穿孔，提高水分子及某些大分子对细胞膜的通透性。同时细胞膜仍然完整存在，细胞不破膜溶解，细胞膜在电穿孔后在几秒或几分钟内还可修复。细胞经过这样的电穿孔过程后仍然是存活的细胞。为此我们设计了一种简单的RC电脉冲发生器及电穿孔室。RC电脉冲发生器主要由充电和放电部分组成。电穿孔室主要由两根互相平行的铂丝组成，两铂丝间的间距为375 mm。欲穿孔的血红细胞悬浮液滴于电穿孔室的两铂丝电极之间，由RC电脉冲发生器给血红细胞施加电脉冲。我们还提出了一种判断细胞是否发生了电穿孔的方法，即在细胞悬浮液中加入台盼兰（一种染料），如果细胞变兰即可认为细胞已电穿孔并已导入所加药物。在研究了血红细胞发生可逆性电穿孔的最佳条件时发现，血红细胞在含有双氯灭痛和不含有双氯灭痛的两种溶液中发生可逆性电穿孔的条件是不同的。当溶液中含有双氯灭痛时，血红细胞发生可逆性电穿孔的脉冲电压低于溶液中不含有双氯灭痛时的情况。我们还发现用碳纤维微盘电极代替单根碳纤维电极检测双氯灭痛时检测限较低。从单细胞分析的结果得出，电穿孔法导入血红细胞中双氯灭痛的平均含量为4.21 mmol/L，相对标准偏差为10％，这说明我们所设计的电穿孔装置，电穿孔的方法及单细胞分析的方法是确实可行。
第六章中我们用CZE/柱端安培检测法检测了单个人血红细胞（人细胞中直径最小且体积也最小的细胞）中的氨基酸。文献中已报道了测定单个嗜铬细胞及单个巨噬细胞中的氨基酸，含量都在fmol水平。由于血红细胞中氨基酸的平均含量很低（小于50 amol），若采用柱前衍生的方法将不能使单个细胞中的物质全部进样到毛细管中，从而无法测定单个细胞中的氨基酸。若采用柱上衍生的方法，虽然能使单细胞全部进样到毛细管中，但柱上衍生的稀释倍数为100倍，会使血红细胞中氨基酸的浓度被稀释至CZE/柱端安培检测的检测限以下，也不适用于检测血红细胞中的氨基酸。为此我们提出了一种新的细胞内衍生的方法。这种方法采用电穿孔方法将衍生试剂强制导入血红细胞中，使氨基酸的衍生反应在细胞内进行。血红细胞电穿孔后的直径由穿孔前的约8 mm变为穿孔后的约10 mm，细胞内样品的稀释小于2倍，从而实现了血红细胞中氨基酸的检测。
论文主题词：毛细管电泳，安培检测，生物物质，单细胞分析。
Abstract
In the chapter one of this thesis, first of all, the principle of capillary electrophoresis (CE) and the detection (Ultraviolet, Laser-induced fluorescence and electrochemical detection) which are often used with CE are introduced simply. The conductivity, potentiometric and amperomatric detection are three models of the CE electrochemical detection. The conductivity and potentiometric detection were seldom used, so the principles and typical equipment of are introduced simply. The mean part in this chapter is CE ampermetric detection and its applications. The equipment, the principles, the methods of detection and analysis, the applications of different electrode, microchip CE, continuous separation with micro-fabricated electrophoresis and single cell analysis of CE amperometric detection are introduced in detail. 267 references are referred.
In the chapter two, lactate, pyruvate and transferrin are determinated using CZE/end-column amperometric detection. Up to now, there were no articles about this aspect being published. It is difficult to determine lactate and pyruvate using direct electrochemical technique, because obvious and useful electrochemical signals such as peaks can not be obtained on its voltamograms. However, we found that lactate and pyruvate can be oxidized at carbon fiber electrodes. The behavior can be used for direct determination of lactate and pyruvate, if capillary zone electrophoresis (CZE) is used. In this work, CZE was employed for the determination of lactate, pyruvate and transferrin using the end-column amperometric detection at a carbon fiber array microdisk electrode. The optimum conditions of separation and detection for lactate and pyruvate are 3.6×10-3 mol/L Na2HPO4-1.4×10-3 mol/L NaH2PO4 (pH 7.2) for the buffer solution, 18 kV for the separation voltage and 1.60 V vs. saturated calomel electrode (SCE) for the detection potential. The limit of detection of lactate is 7.6×10-7 mol/L(1.7 fmol, S/N=3) and the linear range is 1.7×10-6-8.2×10-4 mol/L for the injection voltage of 6 kV and the injection time of 5 s. The limit of detection of pyruvate is 8.0×10-6 mol/L (24 fmol, S/N=3) and the linear range is 1.0×10-5 -5.0×10-3 mol/L for the injection voltage of 6 kV and the injection time of 5 s. The response for a series of six injections of 8.44×10-5 mol/L lactate resulted in a relative standard deviation of 1.8% for the migration time, tm, and 3.3% for the electrophoretic peak current, ip, respectively. The response for a series of six injections of 1.00×10-4 mol/L pyruvate resulted in a relative standard deviation of 2.0% for tm and 5.7% for ip, respectively. Concentration of lactate in human saliva, pyruvate in human sweat and transferrin in human serum can be readily and directly determined by using this CZE-electrochemical detection system at a carbon fiber array microdisk electrode without any pre-treatment. Serotonin in human serum can not interfere with the detection of transferrin. The results are agreed with the literature value.
In the chapter three, capillary zone electrophoresis
with electrochemical detection was employed for the analysis of 19 amino acids.
Amino acids were derivated with naphthalene-2, 3-dicarboxaldehyde (NDA) before
detection. NDA-amino acids can be oxidized on the carbon fiber microdisk
electrode. Only 3 amino acids (Alanine, glutamic acid and aspartic acid)
derivated with NDA and NaCN have ever been investigated by CE/amperometric
detection using the carbon firber microelectrode. In this work, 19 amino acids
are analysis by using CZE/amperometric detection. NDA-Amino acids were detected
in 1.25´10-2
mol/L borate- 3.13´10-3
mol/L NaOH for buffer solution, 18 kV for the separation voltage for the
capillary of 80 cm length, and 1.15 V for the detection potential. The limit of
detection (LOD) for individual amino acids is between 1.7´10-7 mol/L and
1.8´10-6
mol/L (according to the ratio of signal to noise of 3). 10 amino acids (Arg,
Lys Orn, Try, Ser, Ala, Gly, Cys, Glu, Asp) can be separated well in the
presence of other amino acids. The response of the amino acids in the mixtures
of different amino acids is different from that of individual amino acids.
Therefore, different mixtures of amino acids can result in different
electrochemical response of amino acids. LOD of amino acids as well as the
linear relationship exists between the peak current detected and the
concentration are also different. This might due to competitive between amino
acids in the derivatization reaction with NDA, when they are mixed together.
Therefore, the calibration curve can not be used for quantifying the
concentrations of the amino acids in the beer, while the standard addition
method can solve this problem. Amino acids (Arg, Try, Phe, Tyr, Val, Ala and
Gly) in the beer sample were determined qualitatively and quantitatively using
standard addition method. The recoveries of the method for the seven amino
acids are between 91% and 109 %.
In the chapter four, glutathione (GSH) in individual mouse peritoneal macrophages was determined by capillary zone electrophoresis with electrochemical end-column amperometric detection at a gold/mercury amalgam microelectrode. Without any pretreatment of the capillary, individual macrophages could be drawn into the capillary simply. The whole cell injection and no need of derivatization reaction lead more accurate and precise results. It was found that macrophages could not be lysed in the acid and neutral solutions. But it can be lysed in 0.01 mol/l NaOH. It was also found that the usual calibration curve of standard GSH could not be used for the quantification of GSH in individual macrophages. A new method for the quantified detection of substances in single cell was put forward in this work, that is the content of substance in individual cells can be quantified by comparison of the peak current against those of standard substance injected after each cell run. The average amount of GSH in an individual mouse peritoneal macrophage is 5.8 fmol, which is consistent with the literature value. A histogram of the data reflects true differences between cells. This phenomenon can be found only in single cell analysis.
In the chapter five, a method for introduced the
diclofenac sodium (a drug) into individual human erythrocytes by
electroporation was developed. The
diclofenac sodium in single erythrocyte was detected using CZE with
electrochemical detection at a carbon fiber microdisk microelectrode. Cell electroporation happened when the cells were put in
the electric field, and then they were opeareted by the electric pulse.
Excessive pulse voltage can lead to the breakdown of the cell membrane. Hence
pore formation and much enhanced permeability to macromolecules. After
electroporation, cells are still alive. In this work, a simple RC
electric-pulse generator and electroporation
cell were made for the cell electroporation. The cells that generated
reversible electroporation can be discriminated using trypan blue, The optimum conditions of separation and detection
for the diclofenac sodium is 1.25×10-2 mol/L Na2B4O7-3.13×10-3
mol/L NaOH for the buffer, 20 kV for the separation voltage and 1.0 V for the
detection potential. The mean concentration of
diclofenac sodium introduced into cell was 4.21 mmol/L.
In the chapter six, amino acids in individual human erythrocyte was detected by using intracellular derivatization and CZE/amperometric detection. Human erythrocyte is the smallest cell (diameter 8 mm, volume 87 fL) in the human cells. The contents of amino acids in single cell detected in the references were fmol. The mass contents of amino acids in erythrocyte are less than 50 amol. If the amino acids in erythrocyte are detected using precolumn derivatization with NDA and NaCN before separation and detection, not all of the substances in the single cell were injected into CZE/amperometric detection. Therefore, precolumn derivatization is not fitted for the analysis of amino acids in single erythrocyte. Using the on-column derivatization method, in which the concentration of sample will be diluted 100, the concentration of amino acids in erythrocyte will decrease under the LOD of CZE/end-column amperometric detection. Therefore, on-column derivatization is also not fitted for the detection of amino acids in single erythrocyte. A new intracellular derivatization method for single cell analysis was put forward in this work. The derivatization reagents were introduced into erythrocytes by force when the reversible electroporation of erythrocyte happened. Then the derivatization of amino acids and NDA in the present of NaCN happened within the erythorcyte. The derivatization of amino acids with NDA and NaCN can finish in the environment of physiological buffer saline fast enough. After electroporation, the diameter of erythrocyte changed from 8 mm to 10 mm. The dilution of sample in erythrocyte was less than 2, far less than other derivatization for single cell analysis before.
