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Determination of Glutathione in a Single Human Hepatocarcinoma Cell Using a Microfluidic Device Coupled with Electrochemical Detection
CHINESE JOURNAL OF CHROMATOGRAPHY Volume 25, Issue 6, November 2007 Online English edition of the Chinese language journal Cite this article as: Chin J Chromatogr, 2007, 25(6): 799–803.
RESEARCH PAPER
Determination of Glutathione in a Single Human Hepatocarcinoma Cell Using a Microfluidic Device Coupled with Electrochemical Detection WANG Wenlei1,2, JIN Wenrui1,* 1 2
School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China Shandong Province Environmental Monitoring Centre, Jinan 250013, China
Abstract: A method for the determination of glutathione (GSH) in a single human hepatocarcinoma cell, using a microfluidic chip coupled with electrochemical detection, was developed. In this method, the cell injection, loading, and cytolysis, as well as the transportation and detection of intracellular GSH were integrated in a microfluidic chip with a double-T injector and an end-channel amperometric detector. A single cell was loaded in the double-T injector on the microfluidic chip, using liquid pressure. The docked cell was perforated in the electrophoretic buffer containing digitonin and was lysed under a direct current electric field. The GSH from the single cell was electrokinetically transported to the separation channel and was electrochemically detected at an Au/Hg electrode. The GSH in the single cell was quantitatively determined using a calibration curve. Key Words: microfluidic chip; electrochemical detection; glutathione; single cell; hepatocarcinoma cell
The microfluidic chip is a powerful tool for the analysis of a single cell [1–4]. Biochemical components in single cell can be determined by using a microfluidic device. Glutathione (GSH) is a major intracellular thiol in mammals [5,6]. It is known that GSH is a tripeptide involved in many biological processes [7,8]. A microfluidic chip with laser-induced fluorescence (LIF) detection was developed by Fang’s group [9] to determine glutathione in a single cell. They also applied a microfluidic chip with LIF detection to simultaneously determine GSH and reactive oxygen species (ROS) inside the single cell [10–12]. Moreover, it was also reported that GSH in a single cell was determined by using capillary electrophoresis (CE) with LIF detection [13,14]. In all methods using LIF detection, GSH had to be reacted with fluorescent reagents before analysis. The incompleteness and nonuniformity of the derivatization reaction could lead to deviations. It was previously reported that GSH in a single cell was determined using CE with electrochemical detection (ECD), in this group [15–18]. In this study, a microfluidic chip with electrochemical detection was
developed to directly determine GSH in a single human hepatocarcinoma (HH) cell. No derivatization was needed in this method.
1 Materials and methods 1.1 Chemicals The GSH (content > 98%) was purchased from Sigma (St. Louis, MO, USA). Other chemicals (analytical grade) were purchased from standard reagent suppliers. The stock solution of GSH (1.00 × 102 mol/L) was prepared by dissolving an appropriate amount of GSH in a solution of Na2EDTA (2.00 × 102 mol/L). The running buffer was 0.020 mol/L Na2HPO4-NaH2PO4, containing 2.0 × 103 mol/L Na2EDTA and 1.00 × 104 mol/L digitonin (pH 7.4). The running buffer was deaerated with N2 for 15 min before use. 1.2 Apparatus A high-voltage power supply (Model 9323-HVPS, Beijing Institute of New Technology, Beijing, China) coupled with a laboratory-built relay box (Fig. 1) was used to provide high
Received June 11, 2007; revised August 20, 2007 *Corresponding author. Tel: +86-531-88361318, E-mail: [email protected] This work was supported by the National Natural Science Foundation of China (No. 20235010). Copyright © 2007, Chinese Chemical Society and Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Published by Elsevier BV. All rights reserved.
WANG Wenlei et al. / Chinese Journal of Chromatography, 2007, 25(6): 799–803
potential. ECD at a constant potential was performed on an electrochemical analyzer (Model CHI802a, CH Instruments, Austin, TX, USA). ECD was carried out with a three-electrode system, which consisted of an Au/Hg electrode as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a Pt wire as the auxiliary electrode. The Pt wire also served as the ground for the high voltage across the separation channel. The Au/Hg electrode was similar to that used in the authors’ previous work [15]. The microfluidic chip-ECD system, including three electrodes, was housed in a Faraday cage, to minimize the noise interference from external sources.
Fig. 1
Circuit diagram of voltage divider
1. to buffer reservoir; 2. to sample reservoir; 3. to waste reservoir; 4. to detection reservoir; 5. high-voltage power supply.
The microfluidic chip with an ECD system was similar to the system reported in the previous work [19]. Briefly, a microfluidic chip with a “double-T” injector (Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, China) (Fig. 2) was used in this work. Three micropipette tips (4 mm i.d. and 6 mm tall) were stuck on the chip surrounding the access holes with epoxy, serving as reservoirs. The depth and width of the channel were 20 µm and 60 µm, respectively. The length of the double-T injector was 200 µm. The lengths of the separation channel, sample channel, and sample waste channel were 50, 5, and 5 mm, respectively. The distance between the buffer reservoir and double-T injector
Fig. 2 Microfluidic chip-electrochemical detection system 1. buffer reservoir; 2. sample reservoir; 3. waste reservoir; 4. microfluidic chip; 5. glass slide; 6. working electrode; 7. reference electrode port; 8. auxiliary electrode; 9. cathode of separation voltage; 10. epoxy resin; 11. electrochemical cell and waste reservoir.
was 5 mm. The chip (4) was fixed on a microscope slide (5) with epoxy glue. Three Pt wires were inserted into the buffer reservoir, (1) as the anode of high voltage for separation, the sample reservoir (2) as the anode of loading voltage, and the waste sample reservoir (3) as the cathode of loading voltage. 1.3 Cell preparation The HH cells (BEL-7402) were provided by the School of Medicine, Shandong University (Jinan, China). The HH cells were suspended in ~3 mL culture medium. After it was centrifuged for 10 min at 1000 r/min, the supernatant was discarded. Following this, 2 mL running buffer was added, and the cell mixture was disrupted gently by pipetting. After it was centrifuged for 10 min at 1000 r/min, the supernatant was discarded again. This step was repeated twice. Then, the cells were suspended in 1 mL running buffer. The cell suspension was used for single-cell analysis. The number of cells in the cell suspension was 9.5 × 105, which was counted using a hemocytometer (Shanghai Medical Optical Instrument Plant, Shanghai, China). To prepare the cell extract, after 100 ȝL of 1.00 × 103 mol/L digitonin was added to 1 mL of cell suspension, the mixture was sonicated for 10 min. Thus, the cell extract with a cell concentration of 8.6 × 105/mL was obtained. 1.4 Determination of standard GSH using microfluidic chip-ECD The channels of the microchip were flushed with 0.1 mol/L NaOH, for 60 min, water for 10 min, and running buffer for 20 min, before use. A voltage of 1000 V was applied across the separation channel, and a detection potential of 0.2 V was applied at the working electrode. After the electro-osmotic flow reached a constant value, the standard solution was electrokinetically injected for 20 s. At that time, a voltage of 500 V was applied between the sample reservoir and the waste sample reservoir. The waste sample reservoir was grounded, and both the waste reservoir and the buffer reservoir were floated. Then, a separation voltage of 1000 V was applied to the buffer reservoir. The waste reservoir remained on the ground, and the sample reservoir and the waste sample reservoir were floated. The signal on the working electrode at the end of the separation channel was recorded. All potentials of the working electrode were against SCE. 1.5 Determination of GSH in a single HH cell using microfluidic chip-ECD The channels of the microchip were flushed with 0.1 mol/L NaOH for 5 min, water for 5 min, and running buffer for 5 min. Then 10 µL of cell suspension was diluted to 1 mL with the running buffer and injected into the sample reservoir using a syringe. After 3 to 4 min, an HH cell was propelled into the double-T injector. When the profile of the cell became fuzzy (~20 min), a voltage of 1000 V was applied across the separation channel. Subsequently, the cell was lysed immediately, and the recording of the electropherogram started.
WANG Wenlei et al. / Chinese Journal of Chromatography, 2007, 25(6): 799–803
2 Results and discussion 2.1 Detection of GSH in the extract of HH cell In the authors’ previous works [15–18,20], in which GSH was determined using CE-ECD, it was found that the phosphate buffer (pH 7.4) was the best choice for the determination of GSH in a single cell. Thus, the phosphate buffer of 0.020 mol/L Na2HPO4-NaH2PO4, containing 2.0 × 103 mol/L Na2EDTA and 1.00 × 104 mol/L digitonin (pH 7.4) was selected as the running buffer in this study. It was found that GSH was well detected when 1000 V for the separation voltage and 0.20 V for the detection potential were applied. Figure 3-a shows the electropherogram of 1.00 × 104 mol/L GSH. The standard calibration curve between peak area and concentration was obtained with the linear range of 1.00 × 105–5.00 × 104 mol/L and correlation coefficient of 0.9998. The limit of detection (LOD) calculated from the peak area at the low end of its linear range was 4.8 × 106 mol/L when the signal-to-noise ratio was 3. The relative standard deviations (n = 6) for the migration time and the peak area were 1.6% and 4.4%, respectively.
Fig. 3
Electropherograms of (a) 1.00 × 104 mol/L GSH and (b) HH cell extract
Conditions: 2.0 × 103 mol/L Na2EDTA-1.00 × 104 mol/L digitonin-0.020 mol/L Na2HPO4-NaH2PO4 (pH 7.4); separation voltage, 1.00 kV; injection voltage, 500 V; injection time, 10 s; detection potential, 0.20 V (vs. SCE).
Figure 3-b shows the electropherogram for the extract of HH cells. Only one peak was found in the electropherogram. It has been demonstrated [18] that when a potential of 0.20 V was applied at the Au/Hg electrode, compounds such as, dopa, dopamine, serotonin, epinephrine, and norepinephrine, which could be directly oxidized at the electrode, could not be detected at the Au/Hg electrode, whereas, GSH and cysteine could be still detected. Moreover, the concentration of cysteine in the cell extract was lower than its LOD [17]. Thus, the peak for cysteine was not found in the electropherogram in
this study. When comparing Fig. 3-b with Fig. 3-a, it was found that the migration times of the two peaks were the same. Thus, the peak in Fig. 3-b was identified as the peak for GSH in a single HH cell. The average concentration of GSH in the cell extract was 2.13 × 105 mol/L, which was calculated using the standard calibration curve. The recovery was between 97% and 103%, when a certain amount of standard GSH was added to the cell extract. According to the determined cell concentration in the cell extract, the average content of GSH in one HH cell was calculated to be 2.5 × 1014 mol. This result was in agreement with that in the previous work [17]. 2.2 Single cell analysis In a microfluidic chip, cells were often driven from the sample reservoir to the channel by using an electric field. Sometimes, cells were driven using hydrodynamic force [21]. For cells with a diameter much smaller than the size of the channel, it was easy to load cells using electro-osmosis [22]. In this study, it was difficult to load a cell using electro-osmosis because the diameter of a cell (~24 µm) was larger than the channel depth (~20 µm). Thus, the authors used a syringe to transport a single cell into the double-T injector with the help of pressure. For this work, the cells were suspended in the running buffer without Cl– (0.020 mol/L phosphate buffer, pH = 7.4) instead of the normal saline because Cl– ions in the normal saline would produce an electrophoretic peak for electrochemical detection. Thus, only one peak of GSH was found in the electropherogram for a single HH cell, which made the single-cell analysis simple. To accelerate the lysis of HH cells in the channel, digitonin that was bound to cholesterol on the cell membrane to form micropores [23,24], was added to the cell suspension, before loading. When the profile of the cell docked in the double-T injector became fuzzy (~20 min), a voltage of 1000 V was applied across the separation channel. At that time, the cell was lysed immediately and the GSH was released from the cell, driven into the end of the separation channel, and detected at the Au/Hg electrode. Figure 4 shows the electropherograms for two single HH cells. Only one electrophoretic peak appeared on each electropherogram. The migration time was the same as that of the standard solution. After each run, the separation channel was flushed for 5 min, with 0.1 mol/L NaOH, and for another 5 min with the running buffer. Using this flushing method, the reproducibility of the electropherogram in a single cell analysis was improved, and the accumulation of the cellular debris and the substances in cells adsorbed on the channel were eliminated. The width of the peak half-height in single cell analysis was 3.3–4.1 s, which was narrower than that of the standard solution, because the plug length of the standard solution, injected in the double-T injector, was longer than the cell diameter. The concentration calibration curve could also be used to quantify GSH. To determine the mass linear range, the actual loaded volume of the standard solution had to be measured. Moreover, when the double-T injector was used,
WANG Wenlei et al. / Chinese Journal of Chromatography, 2007, 25(6): 799–803
the dispersion effect and leakage effect had to be considered [25]. For the microchip in this study, the actual injected volume of standard solution was 339 pL, which was obtained using epi-fluorescence microscopy [19]. On the basis of the injected volume and the concentration calibration curve, the mass linear range was 3.39 × 1015–1.70 × 1013 mol, with a correlation coefficient of 0.9998. The mass LOD was 1.7 × 10–15 mol. The contents of GSH in six single cells were 1.27, 1.89, 1.59, 2.24, 3.06, and 2.18 × 1014 mol, respectively. The corresponding average value in the cell extract (2.5 × 1014 mol) was within the results obtained in single cells, which meant that the method for single-cell analysis was reliable.
[3] Erickson D, Li D Q. Anal Chim Acta, 2004, 507: 11 [4] Vilkner T, Janasek D, Manz A. Anal Chem, 2004, 76: 3373 [5] Meister A, Tate S S. Annu Rev Biochem, 1976, 45: 559 [6] Meister A, Anderson M E. Annu Rev Biochem, 1983, 52: 711 [7] Black M. Annu Rev Med, 1984, 35: 577 [8] Curello S, Ceconi C, Carqnoni A, Cornacchiari A, Ferrari R, Albertini A. Clin Chem, 1987, 33: 1448 [9] Gao J, Yin X F, Fang Z L. Lab Chip, 2004, 4: 47 [10] Ling Y Y, Yin X F, Fang Z L. Electrophoresis, 2005, 26: 4759 [11] Sun Y, Yin X F, Ling Y Y, Fang Z L. Anal Bioanal Chem, 2005, 382: 1472 [12] Sun Y, Yin X F. J Chromatogr A, 2006, 1117: 228 [13] Hogan B L, Yeung E S. Anal Chem, 1992, 64: 2841 [14] Orwar O, Fishman H A, Ziv N E, Scheller R H, Zare R N. Anal Chem, 1995, 67: 4261 [15] Jin W R, Li W, Xu Q. Electrophoresis, 2000, 21: 774 [16] Jin W, Dong Q, Ye X, Yu D. Anal Biochem, 2000, 285: 255 [17] Wang W, Xin H, Shao H L, Jin W R. J Chromatogr B, 2003, 789: 425 [18] Jin W, Li X, Gao N. Anal Chem, 2003, 75: 3 859 [19] Xia F Q, Jin W R, Yin X F, Fang Z L. J Chromatogr A, 2005, 1063: 227 [20] Jin W, Wang Y. Anal Chim Acta, 1997, 343: 231 [21] Burns M A, Johnson B N, Brahmasandra S N, Handique K, Webster J R, Krishnan M, Sammarco T S, Man P M, Jones D, Heldsinger D, Mastrangelo C H, Buke D T. Science, 1998, 282:
Fig. 4 Electropherograms of two single HH cells Conditions as in Fig. 3.
References [1] Andersson H, van den Berg A. Sens Actuators B Chem, 2003, 92: 315 [2] Ozkan M, Wang M, Ozkan C, Flynn R, Birkbeck A, Esener S. Biomed Microdevices, 2003, 5: 61
484 [22] Li P C H, Harrison D J. Anal Chem, 1997, 69: 1564 [23] Roehrig S, Tabbert A, Ferrando-May E. Anal Biochem, 2003, 318: 244 [24] Gao N, Wang W L, Zhang X L, Jin W R, Yin X F, Fang Z L. Anal Chem, 2006, 78: 3213 [25] Shultz-Lockyear L L, Colyer C L, Roy K I, Harrison D J. Electrophoresis, 1999, 20: 529
RESEARCH PAPER
Determination of Glutathione in a Single Human Hepatocarcinoma Cell Using a Microfluidic Device Coupled with Electrochemical Detection WANG Wenlei1,2, JIN Wenrui1,* 1 2
School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China Shandong Province Environmental Monitoring Centre, Jinan 250013, China
Abstract: A method for the determination of glutathione (GSH) in a single human hepatocarcinoma cell, using a microfluidic chip coupled with electrochemical detection, was developed. In this method, the cell injection, loading, and cytolysis, as well as the transportation and detection of intracellular GSH were integrated in a microfluidic chip with a double-T injector and an end-channel amperometric detector. A single cell was loaded in the double-T injector on the microfluidic chip, using liquid pressure. The docked cell was perforated in the electrophoretic buffer containing digitonin and was lysed under a direct current electric field. The GSH from the single cell was electrokinetically transported to the separation channel and was electrochemically detected at an Au/Hg electrode. The GSH in the single cell was quantitatively determined using a calibration curve. Key Words: microfluidic chip; electrochemical detection; glutathione; single cell; hepatocarcinoma cell
The microfluidic chip is a powerful tool for the analysis of a single cell [1–4]. Biochemical components in single cell can be determined by using a microfluidic device. Glutathione (GSH) is a major intracellular thiol in mammals [5,6]. It is known that GSH is a tripeptide involved in many biological processes [7,8]. A microfluidic chip with laser-induced fluorescence (LIF) detection was developed by Fang’s group [9] to determine glutathione in a single cell. They also applied a microfluidic chip with LIF detection to simultaneously determine GSH and reactive oxygen species (ROS) inside the single cell [10–12]. Moreover, it was also reported that GSH in a single cell was determined by using capillary electrophoresis (CE) with LIF detection [13,14]. In all methods using LIF detection, GSH had to be reacted with fluorescent reagents before analysis. The incompleteness and nonuniformity of the derivatization reaction could lead to deviations. It was previously reported that GSH in a single cell was determined using CE with electrochemical detection (ECD), in this group [15–18]. In this study, a microfluidic chip with electrochemical detection was
developed to directly determine GSH in a single human hepatocarcinoma (HH) cell. No derivatization was needed in this method.
1 Materials and methods 1.1 Chemicals The GSH (content > 98%) was purchased from Sigma (St. Louis, MO, USA). Other chemicals (analytical grade) were purchased from standard reagent suppliers. The stock solution of GSH (1.00 × 102 mol/L) was prepared by dissolving an appropriate amount of GSH in a solution of Na2EDTA (2.00 × 102 mol/L). The running buffer was 0.020 mol/L Na2HPO4-NaH2PO4, containing 2.0 × 103 mol/L Na2EDTA and 1.00 × 104 mol/L digitonin (pH 7.4). The running buffer was deaerated with N2 for 15 min before use. 1.2 Apparatus A high-voltage power supply (Model 9323-HVPS, Beijing Institute of New Technology, Beijing, China) coupled with a laboratory-built relay box (Fig. 1) was used to provide high
Received June 11, 2007; revised August 20, 2007 *Corresponding author. Tel: +86-531-88361318, E-mail: [email protected] This work was supported by the National Natural Science Foundation of China (No. 20235010). Copyright © 2007, Chinese Chemical Society and Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Published by Elsevier BV. All rights reserved.
WANG Wenlei et al. / Chinese Journal of Chromatography, 2007, 25(6): 799–803
potential. ECD at a constant potential was performed on an electrochemical analyzer (Model CHI802a, CH Instruments, Austin, TX, USA). ECD was carried out with a three-electrode system, which consisted of an Au/Hg electrode as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a Pt wire as the auxiliary electrode. The Pt wire also served as the ground for the high voltage across the separation channel. The Au/Hg electrode was similar to that used in the authors’ previous work [15]. The microfluidic chip-ECD system, including three electrodes, was housed in a Faraday cage, to minimize the noise interference from external sources.
Fig. 1
Circuit diagram of voltage divider
1. to buffer reservoir; 2. to sample reservoir; 3. to waste reservoir; 4. to detection reservoir; 5. high-voltage power supply.
The microfluidic chip with an ECD system was similar to the system reported in the previous work [19]. Briefly, a microfluidic chip with a “double-T” injector (Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, China) (Fig. 2) was used in this work. Three micropipette tips (4 mm i.d. and 6 mm tall) were stuck on the chip surrounding the access holes with epoxy, serving as reservoirs. The depth and width of the channel were 20 µm and 60 µm, respectively. The length of the double-T injector was 200 µm. The lengths of the separation channel, sample channel, and sample waste channel were 50, 5, and 5 mm, respectively. The distance between the buffer reservoir and double-T injector
Fig. 2 Microfluidic chip-electrochemical detection system 1. buffer reservoir; 2. sample reservoir; 3. waste reservoir; 4. microfluidic chip; 5. glass slide; 6. working electrode; 7. reference electrode port; 8. auxiliary electrode; 9. cathode of separation voltage; 10. epoxy resin; 11. electrochemical cell and waste reservoir.
was 5 mm. The chip (4) was fixed on a microscope slide (5) with epoxy glue. Three Pt wires were inserted into the buffer reservoir, (1) as the anode of high voltage for separation, the sample reservoir (2) as the anode of loading voltage, and the waste sample reservoir (3) as the cathode of loading voltage. 1.3 Cell preparation The HH cells (BEL-7402) were provided by the School of Medicine, Shandong University (Jinan, China). The HH cells were suspended in ~3 mL culture medium. After it was centrifuged for 10 min at 1000 r/min, the supernatant was discarded. Following this, 2 mL running buffer was added, and the cell mixture was disrupted gently by pipetting. After it was centrifuged for 10 min at 1000 r/min, the supernatant was discarded again. This step was repeated twice. Then, the cells were suspended in 1 mL running buffer. The cell suspension was used for single-cell analysis. The number of cells in the cell suspension was 9.5 × 105, which was counted using a hemocytometer (Shanghai Medical Optical Instrument Plant, Shanghai, China). To prepare the cell extract, after 100 ȝL of 1.00 × 103 mol/L digitonin was added to 1 mL of cell suspension, the mixture was sonicated for 10 min. Thus, the cell extract with a cell concentration of 8.6 × 105/mL was obtained. 1.4 Determination of standard GSH using microfluidic chip-ECD The channels of the microchip were flushed with 0.1 mol/L NaOH, for 60 min, water for 10 min, and running buffer for 20 min, before use. A voltage of 1000 V was applied across the separation channel, and a detection potential of 0.2 V was applied at the working electrode. After the electro-osmotic flow reached a constant value, the standard solution was electrokinetically injected for 20 s. At that time, a voltage of 500 V was applied between the sample reservoir and the waste sample reservoir. The waste sample reservoir was grounded, and both the waste reservoir and the buffer reservoir were floated. Then, a separation voltage of 1000 V was applied to the buffer reservoir. The waste reservoir remained on the ground, and the sample reservoir and the waste sample reservoir were floated. The signal on the working electrode at the end of the separation channel was recorded. All potentials of the working electrode were against SCE. 1.5 Determination of GSH in a single HH cell using microfluidic chip-ECD The channels of the microchip were flushed with 0.1 mol/L NaOH for 5 min, water for 5 min, and running buffer for 5 min. Then 10 µL of cell suspension was diluted to 1 mL with the running buffer and injected into the sample reservoir using a syringe. After 3 to 4 min, an HH cell was propelled into the double-T injector. When the profile of the cell became fuzzy (~20 min), a voltage of 1000 V was applied across the separation channel. Subsequently, the cell was lysed immediately, and the recording of the electropherogram started.
WANG Wenlei et al. / Chinese Journal of Chromatography, 2007, 25(6): 799–803
2 Results and discussion 2.1 Detection of GSH in the extract of HH cell In the authors’ previous works [15–18,20], in which GSH was determined using CE-ECD, it was found that the phosphate buffer (pH 7.4) was the best choice for the determination of GSH in a single cell. Thus, the phosphate buffer of 0.020 mol/L Na2HPO4-NaH2PO4, containing 2.0 × 103 mol/L Na2EDTA and 1.00 × 104 mol/L digitonin (pH 7.4) was selected as the running buffer in this study. It was found that GSH was well detected when 1000 V for the separation voltage and 0.20 V for the detection potential were applied. Figure 3-a shows the electropherogram of 1.00 × 104 mol/L GSH. The standard calibration curve between peak area and concentration was obtained with the linear range of 1.00 × 105–5.00 × 104 mol/L and correlation coefficient of 0.9998. The limit of detection (LOD) calculated from the peak area at the low end of its linear range was 4.8 × 106 mol/L when the signal-to-noise ratio was 3. The relative standard deviations (n = 6) for the migration time and the peak area were 1.6% and 4.4%, respectively.
Fig. 3
Electropherograms of (a) 1.00 × 104 mol/L GSH and (b) HH cell extract
Conditions: 2.0 × 103 mol/L Na2EDTA-1.00 × 104 mol/L digitonin-0.020 mol/L Na2HPO4-NaH2PO4 (pH 7.4); separation voltage, 1.00 kV; injection voltage, 500 V; injection time, 10 s; detection potential, 0.20 V (vs. SCE).
Figure 3-b shows the electropherogram for the extract of HH cells. Only one peak was found in the electropherogram. It has been demonstrated [18] that when a potential of 0.20 V was applied at the Au/Hg electrode, compounds such as, dopa, dopamine, serotonin, epinephrine, and norepinephrine, which could be directly oxidized at the electrode, could not be detected at the Au/Hg electrode, whereas, GSH and cysteine could be still detected. Moreover, the concentration of cysteine in the cell extract was lower than its LOD [17]. Thus, the peak for cysteine was not found in the electropherogram in
this study. When comparing Fig. 3-b with Fig. 3-a, it was found that the migration times of the two peaks were the same. Thus, the peak in Fig. 3-b was identified as the peak for GSH in a single HH cell. The average concentration of GSH in the cell extract was 2.13 × 105 mol/L, which was calculated using the standard calibration curve. The recovery was between 97% and 103%, when a certain amount of standard GSH was added to the cell extract. According to the determined cell concentration in the cell extract, the average content of GSH in one HH cell was calculated to be 2.5 × 1014 mol. This result was in agreement with that in the previous work [17]. 2.2 Single cell analysis In a microfluidic chip, cells were often driven from the sample reservoir to the channel by using an electric field. Sometimes, cells were driven using hydrodynamic force [21]. For cells with a diameter much smaller than the size of the channel, it was easy to load cells using electro-osmosis [22]. In this study, it was difficult to load a cell using electro-osmosis because the diameter of a cell (~24 µm) was larger than the channel depth (~20 µm). Thus, the authors used a syringe to transport a single cell into the double-T injector with the help of pressure. For this work, the cells were suspended in the running buffer without Cl– (0.020 mol/L phosphate buffer, pH = 7.4) instead of the normal saline because Cl– ions in the normal saline would produce an electrophoretic peak for electrochemical detection. Thus, only one peak of GSH was found in the electropherogram for a single HH cell, which made the single-cell analysis simple. To accelerate the lysis of HH cells in the channel, digitonin that was bound to cholesterol on the cell membrane to form micropores [23,24], was added to the cell suspension, before loading. When the profile of the cell docked in the double-T injector became fuzzy (~20 min), a voltage of 1000 V was applied across the separation channel. At that time, the cell was lysed immediately and the GSH was released from the cell, driven into the end of the separation channel, and detected at the Au/Hg electrode. Figure 4 shows the electropherograms for two single HH cells. Only one electrophoretic peak appeared on each electropherogram. The migration time was the same as that of the standard solution. After each run, the separation channel was flushed for 5 min, with 0.1 mol/L NaOH, and for another 5 min with the running buffer. Using this flushing method, the reproducibility of the electropherogram in a single cell analysis was improved, and the accumulation of the cellular debris and the substances in cells adsorbed on the channel were eliminated. The width of the peak half-height in single cell analysis was 3.3–4.1 s, which was narrower than that of the standard solution, because the plug length of the standard solution, injected in the double-T injector, was longer than the cell diameter. The concentration calibration curve could also be used to quantify GSH. To determine the mass linear range, the actual loaded volume of the standard solution had to be measured. Moreover, when the double-T injector was used,
WANG Wenlei et al. / Chinese Journal of Chromatography, 2007, 25(6): 799–803
the dispersion effect and leakage effect had to be considered [25]. For the microchip in this study, the actual injected volume of standard solution was 339 pL, which was obtained using epi-fluorescence microscopy [19]. On the basis of the injected volume and the concentration calibration curve, the mass linear range was 3.39 × 1015–1.70 × 1013 mol, with a correlation coefficient of 0.9998. The mass LOD was 1.7 × 10–15 mol. The contents of GSH in six single cells were 1.27, 1.89, 1.59, 2.24, 3.06, and 2.18 × 1014 mol, respectively. The corresponding average value in the cell extract (2.5 × 1014 mol) was within the results obtained in single cells, which meant that the method for single-cell analysis was reliable.
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Fig. 4 Electropherograms of two single HH cells Conditions as in Fig. 3.
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