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Compact polytetrafluoroethylene assembly-type capillary electrophoresis with chemiluminescence detection
Journal of Chromatography A, 1125 (2006) 144–146
Short communication
Compact polytetrafluoroethylene assembly-type capillary electrophoresis with chemiluminescence detection Kazuhiko Tsukagoshi ∗ , Shingo Ishida, Yuichi Oda, Keiichi Noda, Riichiro Nakajima Department of Chemical Engineering and Materials Science, Faculty of Engineering, Doshisha University, Kyotanabe 610-0321, Japan Received 9 May 2006; received in revised form 14 June 2006; accepted 28 June 2006
Abstract We have developed a compact polytetrafluoroethylene (PTFE) assembly-type capillary electrophoresis with chemiluminescence (CL) detection system. Luminol–microperoxidase–hydrogen peroxide chemiluminescence reaction was adopted. The device is rectangular in shape (60 mm × 40 mm × 30 mm) and includes three reservoirs (sample, migration buffer, and detection reservoirs) with electrodes. The detection reservoir includes an optical fiber to transport light at the capillary tip to a photomultiplier tube. Isoluminol isothiocyanate (ILITC) was analyzed as a model using this device with fused-silica or polytetrafluoroethylene capillary tubes 10 cm in length. We also used the sample reservoir as a reactor for an immune reaction between anti-human serum albumin immobilized on glass beads and isoluminol isothiocyanate-labeled human serum albumin. The present polytetrafluoroethylene assembly with the capillary tube was useful as a palm-sized analysis device for separation and detection, as well as a reactor. © 2006 Elsevier B.V. All rights reserved. Keywords: Capillary electrophoresis; Chemiluminescence detection; Miniaturization; Polytetrafluoroethylene assembly
1. Introduction Capillary electrophoresis (CE) is one of the most powerful and conceptually simple separation techniques for the analysis of complex mixtures, due to its high resolution, relatively short analysis time, and low operational costs as compared with HPLC [1,2]. The ability to analyze nanoliter-scale samples makes CE ideal for extremely volume-limited biological microenvironments. In addition, chemiluminescence (CL) reactions have been characterized in a variety of species for over a century. CL detection has many advantages in various applications, as demonstrated in FIA [3] and HPLC [4]. These include: (1) high detection sensitivity; (2) a wide linear range of response signal, which is beneficial for quantitative analyte determination; (3) inexpensive reagent and apparatus as well as easy and rapid measurement; and (4) no light source or spectroscopes are needed, allowing the instrument configuration to be very simple.
∗
Corresponding author. Tel.: +81 774 65 6595; fax: +81 744 65 6803. E-mail address: [email protected] (K. Tsukagoshi).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.06.101
Over the past decade, the applicability of CL detection in CE has been demonstrated [5]. Several CL reagents, such as luminol, acridinium, peroxyoxalate, and the Ru(II) complex, have been utilized. However, compared with other detection methods that are widely incorporated in CE, CL detection is an evolving technique. Therefore, it is necessary to focus on the development of new CE–CL detection systems with simpler and more efficient instrumentation than existing systems. Several groups have used optical fibers to effectively catch CL at the tip of the capillary and transport the light to the face of the photomultiplier tube [6,7]. A combination of microchip-capillary electrophoresis with CL detection, where quarts and plastic microchips were used, was also reported [8–10]. Polytetrafluoroethylene (PTFE) is one of the most useful and known materials in chemical experiments. It is generally characterized by inertness to chemicals, insulator, easiness to process, and economic surplus. The feature was thought to suit a material for compact CE–CL detection devise. In the present study, we developed a compact polytetrafluoroethylene assembly-type CE with CL detection. The palm-sized device includes three reservoirs with electrodes and represents miniaturization of a CE–CL detection system using a fused-silica or PTFE capillary tube, and not micro-channels fabricated in a microchip.
K. Tsukagoshi et al. / J. Chromatogr. A 1125 (2006) 144–146
145
Fig. 1. Schematic diagram of a compact PTFE assembly-type CE with CL detection device.
2. Experimental All reagents used were commercially available and of analytical grade. Deionized water was distilled for use. Luminol– microperoxidase–hydrogen peroxide CL reaction was adopted. Microperoxidase and hydrogen peroxide were purchased from Nacalai Tesque. Isoluminol isothiocyanate (ILITC) were purchased from Tokyo Chemical Industry Co. Human serum albumin (HSA; MW, 66 000) and rabbit anti-human serum IgG (anti-HSA; MW, 150 000) were purchased from Sigma Chemical Co. and Wako Pure Chemical Industries Ltd., respectively. Labeling of HSA using ILITC was carried out as described previously [2]. The ILITC-labeled HSA solution was subjected to column separation for purification to remove excess ILITC. Anti-HSA was also immobilized on glass beads (1.0 mm in diameter) according to the procedure reported in our previous paper [10]. A schematic diagram of the present CE with CL detection device is shown in Fig. 1. The device constructed of PTFE is rectangular in shape (60 mm × 40 mm × 30 mm) and includes three reservoirs (each inner volume, ca. 1 ml) with platinum electrodes: i.e., sample, migration buffer, and detection reservoirs. The detection reservoir also includes an optical fiber to transport light at the capillary tip to the photomultiplier tube. The distance between the optical fiber face and the capillary outlet
was ca. 0.5 mm. A fused-silica capillary or PTFE capillary tube 10 cm in length and 100 m I.D. was used. The sample reservoir contained ILITC in 10 mM carbonate buffer (pH 10.8). The buffer reservoir contained 10 mM carbonate buffer (pH 10.8) containing 4 M microperoxidase. The detection reservoir contained 10 mM carbonate buffer (pH 10.8) containing 400 mM hydrogen peroxide. As shown in Fig. 1, sample load was carried out by applying a voltage of 1.7 kV to the sample reservoir for 10 s, with the detection reservoir at ground. Then, the inlet part of the capillary tube was moved and positioned from the sample reservoir to the buffer reservoir. Subsequently, analysis was performed by application of a voltage of 1.7 kV to the buffer reservoir, with the detection reservoir at ground. ILITC as a model sample migrated in the capillary tube and mixed with microperoxidase and hydrogen peroxide at the tip of the capillary tube to produce CL. The CL was captured by the optical fiber and transported to the photomultiplier tube. 3. Results and discussion The electropherograms obtained with fused-silica and PTFE capillary tubes are shown in Fig. 2. The components, hydrolyzed ILITC and natural ILITC, were separated on both electropherograms. We confirmed that the electropherograms were reproducibly observed at least up to the repeated fifth-measurement without changing any buffers. The first peak may have been
Fig. 2. Electropherograms obtained using fused-silica and PTFE capillaries. Conditions: capillary, 10 cm in length and 100 m I.D.; applied voltage, 1.7 kV; migration buffer, 10 mM carbonate buffer (pH 10.8) containing 4 M microperoxidase; hydrogen peroxide solution, 10 mM carbonate buffer (pH 10.8) containing 400 mM hydrogen peroxide; and sample, 5 × 10−7 M ILITC.
146
K. Tsukagoshi et al. / J. Chromatogr. A 1125 (2006) 144–146
due to hydrolyzed ILITC and the second peak due to ILITC. The isothiocyanate group must be partly decomposed through hydrolysis to an amino group, which may possess a positive charge. This positive charge caused the migration time to be shorter. Fused-silica capillary tubes feature higher electroosmotic flow than PTFE capillary tubes [11]. Even in this experiment, migration times of hydrolyzed ILITC and ILITC in fused-silica capillary tubes were about two-fold shorter than those in PTFE capillary tubes. In addition, the electropherogram obtained using PTFE capillary tubes gave better resolution than those obtained using fused-silica capillary tubes. We constructed ILITC calibration curves using fused-silica and PTFE capillary tubes. The concentration of ILITC was estimated from the sample weight ignoring the existence of hydrolyzed ILITC. In both capillary tubes, ILITC was linearly determined over the range of 5 × 10−8 –1 × 10−5 M. The correlation coefficients were 0.999 and the relative standard deviations calculated for 5 × 10−7 and 5 × 10−6 M ILITC (n = 8) were less than 5%. Next, we tentatively used the sample reservoir in the present device as a reactor. Anti-HSA immobilized or untreated glass beads (0.600 g; ca. 600 pieces) were added to the sample reservoir. Subsequently, 1.3 × 10−7 M ILITC-labeled HSA solution (0.60 ml) was added to bring about an immune reaction between the immobilized anti-HSA and the labeled HSA for 6 h. After the reaction, an aliquot of the supernatant (reactant) was injected into the capillary as a sample, and analyzed in a similar way to obtain a CL peak of ILITC-labeled HSA on the electropherogram. There was no difference in CL intensity of ILITC-labeled HSA between reaction in the presence of untreated glass beads and in the absence of glass beads. Thus, there was no nonspecific adsorption of HSA onto the untreated glass surface. On the other hand, the CL intensity of ILITC-labeled HSA decreased in the presence of anti-HSA immobilized glass beads, not changing migration times. Using the obtained CL intensity and the calibration curve of ILITC-labeled HSA (determinable range, 5 × 10−8 –5 × 10−6 M; relative standard deviations for 5 × 10−7 and 5 × 10−6 M ILITC-labeled HSA (n = 8) <7%), we estimated the amount of ILITC-labeled HSA that reacted with anti-HSA immobilized on the glass beads through immune reaction. Our observations indicated that ca. 6.6 × 10−11 mol HSA
reacted with anti-HSA on the glass beads under the present conditions, i.e., the adsorption capacity was estimated to be ca. 1.1 × 10−13 mol HSA per glass bead. We have developed a compact PTFE assembly-type CE with CL detection. The device is palm-sized and includes three reservoirs with electrodes. The device represents miniaturization of the CE–CL detection system using a fused-silica or PTFE capillary tube without using a micro-channel fabricated on a microchip. The micro-channels are often subject to problems, such as blockage. In the device described here, fused-silica and PTFE capillary tubes can be exchanged easily and rapidly. Furthermore, the inner wall of fused-silica capillary tube can easily be modified with reference to other previous reports, and the modified capillary tubes can easily be applied to the present device. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. This study was also supported by the Academic Frontier Research Project on “New Frontier of Biomedical Engineering Research” of the Ministry of Education, Culture, Sports, Science, and Technology, Japan. References [1] S.D. Mendonsa, M.T. Bowser, J. Am. Chem. Soc. 127 (2005) 9382. [2] K. Tsukagoshi, T. Nakamura, R. Nakajima, Anal. Chem. 74 (2002) 4109. [3] A. Roda, M. Guardigli, E. Michelini, M. Mirasoli, P. Pasini, Anal. Chem. 75 (2003) 462A. [4] T. Fukushima, N. Usui, T. Santa, K. Imai, J. Pharm. Biomed. Anal. 30 (2003) 1655. [5] K. Tsukagoshi, K. Nakahama, R. Nakajima, Anal. Chem. 76 (2004) 4410. [6] X.-J. Huang, Q.-S. Pu, Z.-L. Fang, Analyst 126 (2001) 281. [7] M. Hashimoto, K. Tsukagoshi, R. Nakajima, K. Riichiro, Kondo, J. Chromatogr. A 832 (1999) 191. [8] R. Su, J.-M. Lin, K. Uchiyama, M. Yamada, Talanta 64 (2004) 1024. [9] B.-F. Liu, M. Ozaki, Y. Utsumi, T. Hattori, S. Terabe, Anal. Chem. 75 (2003) 36. [10] K. Tsukagoshi, N. Jinno, R. Nakajima, Anal. Chem. 77 (2005) 1684. [11] M. Macka, W.-C. Yang, P. Zakaria, A. Shitangkoon, E.F. Hilder, P. Andersson, P. Nesterenko, P.R. Haddad, J. Chromatogr. A 1039 (2004) 193.
Short communication
Compact polytetrafluoroethylene assembly-type capillary electrophoresis with chemiluminescence detection Kazuhiko Tsukagoshi ∗ , Shingo Ishida, Yuichi Oda, Keiichi Noda, Riichiro Nakajima Department of Chemical Engineering and Materials Science, Faculty of Engineering, Doshisha University, Kyotanabe 610-0321, Japan Received 9 May 2006; received in revised form 14 June 2006; accepted 28 June 2006
Abstract We have developed a compact polytetrafluoroethylene (PTFE) assembly-type capillary electrophoresis with chemiluminescence (CL) detection system. Luminol–microperoxidase–hydrogen peroxide chemiluminescence reaction was adopted. The device is rectangular in shape (60 mm × 40 mm × 30 mm) and includes three reservoirs (sample, migration buffer, and detection reservoirs) with electrodes. The detection reservoir includes an optical fiber to transport light at the capillary tip to a photomultiplier tube. Isoluminol isothiocyanate (ILITC) was analyzed as a model using this device with fused-silica or polytetrafluoroethylene capillary tubes 10 cm in length. We also used the sample reservoir as a reactor for an immune reaction between anti-human serum albumin immobilized on glass beads and isoluminol isothiocyanate-labeled human serum albumin. The present polytetrafluoroethylene assembly with the capillary tube was useful as a palm-sized analysis device for separation and detection, as well as a reactor. © 2006 Elsevier B.V. All rights reserved. Keywords: Capillary electrophoresis; Chemiluminescence detection; Miniaturization; Polytetrafluoroethylene assembly
1. Introduction Capillary electrophoresis (CE) is one of the most powerful and conceptually simple separation techniques for the analysis of complex mixtures, due to its high resolution, relatively short analysis time, and low operational costs as compared with HPLC [1,2]. The ability to analyze nanoliter-scale samples makes CE ideal for extremely volume-limited biological microenvironments. In addition, chemiluminescence (CL) reactions have been characterized in a variety of species for over a century. CL detection has many advantages in various applications, as demonstrated in FIA [3] and HPLC [4]. These include: (1) high detection sensitivity; (2) a wide linear range of response signal, which is beneficial for quantitative analyte determination; (3) inexpensive reagent and apparatus as well as easy and rapid measurement; and (4) no light source or spectroscopes are needed, allowing the instrument configuration to be very simple.
∗
Corresponding author. Tel.: +81 774 65 6595; fax: +81 744 65 6803. E-mail address: [email protected] (K. Tsukagoshi).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.06.101
Over the past decade, the applicability of CL detection in CE has been demonstrated [5]. Several CL reagents, such as luminol, acridinium, peroxyoxalate, and the Ru(II) complex, have been utilized. However, compared with other detection methods that are widely incorporated in CE, CL detection is an evolving technique. Therefore, it is necessary to focus on the development of new CE–CL detection systems with simpler and more efficient instrumentation than existing systems. Several groups have used optical fibers to effectively catch CL at the tip of the capillary and transport the light to the face of the photomultiplier tube [6,7]. A combination of microchip-capillary electrophoresis with CL detection, where quarts and plastic microchips were used, was also reported [8–10]. Polytetrafluoroethylene (PTFE) is one of the most useful and known materials in chemical experiments. It is generally characterized by inertness to chemicals, insulator, easiness to process, and economic surplus. The feature was thought to suit a material for compact CE–CL detection devise. In the present study, we developed a compact polytetrafluoroethylene assembly-type CE with CL detection. The palm-sized device includes three reservoirs with electrodes and represents miniaturization of a CE–CL detection system using a fused-silica or PTFE capillary tube, and not micro-channels fabricated in a microchip.
K. Tsukagoshi et al. / J. Chromatogr. A 1125 (2006) 144–146
145
Fig. 1. Schematic diagram of a compact PTFE assembly-type CE with CL detection device.
2. Experimental All reagents used were commercially available and of analytical grade. Deionized water was distilled for use. Luminol– microperoxidase–hydrogen peroxide CL reaction was adopted. Microperoxidase and hydrogen peroxide were purchased from Nacalai Tesque. Isoluminol isothiocyanate (ILITC) were purchased from Tokyo Chemical Industry Co. Human serum albumin (HSA; MW, 66 000) and rabbit anti-human serum IgG (anti-HSA; MW, 150 000) were purchased from Sigma Chemical Co. and Wako Pure Chemical Industries Ltd., respectively. Labeling of HSA using ILITC was carried out as described previously [2]. The ILITC-labeled HSA solution was subjected to column separation for purification to remove excess ILITC. Anti-HSA was also immobilized on glass beads (1.0 mm in diameter) according to the procedure reported in our previous paper [10]. A schematic diagram of the present CE with CL detection device is shown in Fig. 1. The device constructed of PTFE is rectangular in shape (60 mm × 40 mm × 30 mm) and includes three reservoirs (each inner volume, ca. 1 ml) with platinum electrodes: i.e., sample, migration buffer, and detection reservoirs. The detection reservoir also includes an optical fiber to transport light at the capillary tip to the photomultiplier tube. The distance between the optical fiber face and the capillary outlet
was ca. 0.5 mm. A fused-silica capillary or PTFE capillary tube 10 cm in length and 100 m I.D. was used. The sample reservoir contained ILITC in 10 mM carbonate buffer (pH 10.8). The buffer reservoir contained 10 mM carbonate buffer (pH 10.8) containing 4 M microperoxidase. The detection reservoir contained 10 mM carbonate buffer (pH 10.8) containing 400 mM hydrogen peroxide. As shown in Fig. 1, sample load was carried out by applying a voltage of 1.7 kV to the sample reservoir for 10 s, with the detection reservoir at ground. Then, the inlet part of the capillary tube was moved and positioned from the sample reservoir to the buffer reservoir. Subsequently, analysis was performed by application of a voltage of 1.7 kV to the buffer reservoir, with the detection reservoir at ground. ILITC as a model sample migrated in the capillary tube and mixed with microperoxidase and hydrogen peroxide at the tip of the capillary tube to produce CL. The CL was captured by the optical fiber and transported to the photomultiplier tube. 3. Results and discussion The electropherograms obtained with fused-silica and PTFE capillary tubes are shown in Fig. 2. The components, hydrolyzed ILITC and natural ILITC, were separated on both electropherograms. We confirmed that the electropherograms were reproducibly observed at least up to the repeated fifth-measurement without changing any buffers. The first peak may have been
Fig. 2. Electropherograms obtained using fused-silica and PTFE capillaries. Conditions: capillary, 10 cm in length and 100 m I.D.; applied voltage, 1.7 kV; migration buffer, 10 mM carbonate buffer (pH 10.8) containing 4 M microperoxidase; hydrogen peroxide solution, 10 mM carbonate buffer (pH 10.8) containing 400 mM hydrogen peroxide; and sample, 5 × 10−7 M ILITC.
146
K. Tsukagoshi et al. / J. Chromatogr. A 1125 (2006) 144–146
due to hydrolyzed ILITC and the second peak due to ILITC. The isothiocyanate group must be partly decomposed through hydrolysis to an amino group, which may possess a positive charge. This positive charge caused the migration time to be shorter. Fused-silica capillary tubes feature higher electroosmotic flow than PTFE capillary tubes [11]. Even in this experiment, migration times of hydrolyzed ILITC and ILITC in fused-silica capillary tubes were about two-fold shorter than those in PTFE capillary tubes. In addition, the electropherogram obtained using PTFE capillary tubes gave better resolution than those obtained using fused-silica capillary tubes. We constructed ILITC calibration curves using fused-silica and PTFE capillary tubes. The concentration of ILITC was estimated from the sample weight ignoring the existence of hydrolyzed ILITC. In both capillary tubes, ILITC was linearly determined over the range of 5 × 10−8 –1 × 10−5 M. The correlation coefficients were 0.999 and the relative standard deviations calculated for 5 × 10−7 and 5 × 10−6 M ILITC (n = 8) were less than 5%. Next, we tentatively used the sample reservoir in the present device as a reactor. Anti-HSA immobilized or untreated glass beads (0.600 g; ca. 600 pieces) were added to the sample reservoir. Subsequently, 1.3 × 10−7 M ILITC-labeled HSA solution (0.60 ml) was added to bring about an immune reaction between the immobilized anti-HSA and the labeled HSA for 6 h. After the reaction, an aliquot of the supernatant (reactant) was injected into the capillary as a sample, and analyzed in a similar way to obtain a CL peak of ILITC-labeled HSA on the electropherogram. There was no difference in CL intensity of ILITC-labeled HSA between reaction in the presence of untreated glass beads and in the absence of glass beads. Thus, there was no nonspecific adsorption of HSA onto the untreated glass surface. On the other hand, the CL intensity of ILITC-labeled HSA decreased in the presence of anti-HSA immobilized glass beads, not changing migration times. Using the obtained CL intensity and the calibration curve of ILITC-labeled HSA (determinable range, 5 × 10−8 –5 × 10−6 M; relative standard deviations for 5 × 10−7 and 5 × 10−6 M ILITC-labeled HSA (n = 8) <7%), we estimated the amount of ILITC-labeled HSA that reacted with anti-HSA immobilized on the glass beads through immune reaction. Our observations indicated that ca. 6.6 × 10−11 mol HSA
reacted with anti-HSA on the glass beads under the present conditions, i.e., the adsorption capacity was estimated to be ca. 1.1 × 10−13 mol HSA per glass bead. We have developed a compact PTFE assembly-type CE with CL detection. The device is palm-sized and includes three reservoirs with electrodes. The device represents miniaturization of the CE–CL detection system using a fused-silica or PTFE capillary tube without using a micro-channel fabricated on a microchip. The micro-channels are often subject to problems, such as blockage. In the device described here, fused-silica and PTFE capillary tubes can be exchanged easily and rapidly. Furthermore, the inner wall of fused-silica capillary tube can easily be modified with reference to other previous reports, and the modified capillary tubes can easily be applied to the present device. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. This study was also supported by the Academic Frontier Research Project on “New Frontier of Biomedical Engineering Research” of the Ministry of Education, Culture, Sports, Science, and Technology, Japan. References [1] S.D. Mendonsa, M.T. Bowser, J. Am. Chem. Soc. 127 (2005) 9382. [2] K. Tsukagoshi, T. Nakamura, R. Nakajima, Anal. Chem. 74 (2002) 4109. [3] A. Roda, M. Guardigli, E. Michelini, M. Mirasoli, P. Pasini, Anal. Chem. 75 (2003) 462A. [4] T. Fukushima, N. Usui, T. Santa, K. Imai, J. Pharm. Biomed. Anal. 30 (2003) 1655. [5] K. Tsukagoshi, K. Nakahama, R. Nakajima, Anal. Chem. 76 (2004) 4410. [6] X.-J. Huang, Q.-S. Pu, Z.-L. Fang, Analyst 126 (2001) 281. [7] M. Hashimoto, K. Tsukagoshi, R. Nakajima, K. Riichiro, Kondo, J. Chromatogr. A 832 (1999) 191. [8] R. Su, J.-M. Lin, K. Uchiyama, M. Yamada, Talanta 64 (2004) 1024. [9] B.-F. Liu, M. Ozaki, Y. Utsumi, T. Hattori, S. Terabe, Anal. Chem. 75 (2003) 36. [10] K. Tsukagoshi, N. Jinno, R. Nakajima, Anal. Chem. 77 (2005) 1684. [11] M. Macka, W.-C. Yang, P. Zakaria, A. Shitangkoon, E.F. Hilder, P. Andersson, P. Nesterenko, P.R. Haddad, J. Chromatogr. A 1039 (2004) 193.