Development and Evaluation of a Rotary Cell for Capillary

May 14, 2010 - In this study, a novel rotary cell for CE−CL detection was fabricated and ... the capillary end as it revolves at a preset speed duri...
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Anal. Chem. 2010, 82, 5380–5383

Development and Evaluation of a Rotary Cell for Capillary Electrophoresis-Chemiluminescence Detection Junhua Wang,† Linmei Li, Weihua Huang,* and Jieke Cheng* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China Many efforts have been made toward the advancement of capillary electrophoresis chemiluminescence (CE-CL) detection and its applications through continuous development and improvement of interfaces. In this study, a novel rotary cell for CE-CL detection was fabricated and evaluated. A ring-shaped narrow channel with a quartz bottom is made in a cell body to hold CL reactants and act as the reaction chamber. The CE capillary is placed closely to the bottom of the reaction chamber where analyte is deposited into the CL reactants for reactions to occur. Detection is achieved with a photomultiplier tube below the reaction channel. An advantage of the rotary reaction cell is that it renews the reactants at the capillary end as it revolves at a preset speed during experiments. The rotary detection cell presents a new concept to the conventional coaxial-flow configuration by solving the problems of bubble formation and flow blockage that often interrupt the electrophoresis. Two standard proteins, horseradish peroxidase (HRP) and hemoglobin (Hb), were used to evaluate the interface’s performance with luminol/H2O2 CL system. Satisfactory sensitivities (LOD of 0.91 × 10-9 M for HRP, and 4.37 × 10-8 M for Hb at S/N ) 3) were obtained in this proof-of-concept experiment. This novel design provides a straightforward and robust interface for CE-CL hyphenation. Chemiluminescence (CL) is the emission of light with limited emission of heat, as the result of a chemical reaction. In the experiment, CL emission is measured against dark background and therefore avoids interference from stray light. CL detection utilizes simple and low-cost optical systems without requirement of a light source. When coupled to capillary electrophoresis (CE), CL detection provides low limits of detection (LOD) within subnanomolar to picomolar range,1 making it an attractive technique for ultrasensitive analysis. Since its conception,2-4 the * To whom correspondence should be addressed. (W.H.) E-mail: whhuang@ whu.edu.cn. Tel: +86-27-68752149. Fax: +86-27-68754067. (J.C.) E-mail: jkcheng@ whu.edu.cn. Tel: +86-27-68762291. † Current address: School of Pharmacy, University of WisconsinsMadison, 777 Highland Avenue Madison, Wisconsin, 53705-2222. (1) Liu, Y. M.; Cheng, J. K. J. Chromatogr., A 2002, 959, 1–13. (2) Hara, T.; Okamura, S.; Kato, J. Anal. Sci. 1991, 7, 261–264. (3) Dadoo, R.; Colon, L. A.; Zare, R. N. J. High Resolut. Chromatogr. 1992, 15, 133–135. (4) Ruberto, M. A.; Grayeski, M. L. Anal. Chem. 1992, 64, 2758–2762.

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coupling of CE-CL detection has attracted increasing attention in the fields of microchemical analysis.1,5-7 One of the most common chemiluminescent reactions is that of luminol in an alkaline solution with hydrogen peroxide in the presence of iron or copper ions,8 producing a blue glow. It completes in seconds to minutes depending on the reactants’ concentration. CL detection in CE is in principle achieved through two major means: (1) in-capillary reaction by immobilizing CL reagents or flowing the reactants together with analytes within the separation capillary,9-11 and (2) end-column reaction by the addition of reactants at the separation capillary outlet.12-15 To date, most CE-CL experiments have been performed using the second method, in which the analytes and reactants are mixed in a larger reaction capillary using a coaxial sheath flow. Hara and co-workers pioneered the end-column sheath flow interface for CE-CL in 1991.2 It was later on modified and improved by some research groups, such as Zare,3 Zhang,16 and our group.12-14 In this design, the electrophoretic capillary is etched in the terminal section and is inserted into a larger capillary tubing being installed slantwise across the photomultiplier tube (PMT) window. A tee joint is used to connect the CL reactants introduction tubing and the reaction capillary. The reagents run by gravity (or a pump) through the reaction tubing, forming a coaxial sheath flow around the electrophoretic capillary. The analytes and reactants meet and react at the outlet of the capillary, which is located just in front of the PMT window. While high sensitivity has been obtained, there exists some technical disadvantages for this interface. First, the flow rate of CL reactants must be well controlled to match the flow rate of CE separation to avoid signal dilution or extinction caused by ultrafast flow or peak (5) Garcı´a-Campan ˜a, A. M.; Baeyens, W. R. G.; Zhao, Y. Anal. Chem. 1997, 69, 83A–88A. (6) Huang, X. Y.; Ren, J. C. TrAC, Trends Anal. Chem. 2006, 25, 155–166. (7) Garcı´a-Campan ˜a, A. M.; Lara, F. J.; Gamiz-Gracia, L.; Huertas-Perez, J. F. TrAC, Trends Anal. Chem. 2009, 28, 973–986. (8) Liu, Y. M.; Cheng, J. K. Electrophoresis 2002, 23, 556–558. (9) Lin, J. M.; Goto, H.; Yamada, M. J. Chromatogr., A 1999, 844, 341–348. (10) Yang, W. P.; Zhang, Z. J.; Deng, W. J. Chromatogr., A 2003, 1014, 203– 214. (11) Dumke, J. C.; Nussbaum, M. A. Anal. Chem. 2007, 79, 1262–1265. (12) Huang, B.; Li, J. J.; Cheng, J. K. Chem. J. Chin. Univ. 1996, 17, 528–530. (13) Zhang, Y.; Gong, Z. L.; Zhang, H.; Cheng, J. K. Anal. Comm. 1998, 35, 293–296. (14) Ren, J. C.; Huang, X. Y. Anal. Chem. 2001, 73, 2663–2668. (15) Wang, J. H.; Huang, W. H.; Liu, Y. M.; Cheng, J. K.; Yang, J. Anal. Chem. 2004, 76, 5393–5398. (16) Yang, W. P.; Zhang, Z. J.; Deng, W. Talanta 2003, 59, 951–958. 10.1021/ac100007d  2010 American Chemical Society Published on Web 05/14/2010

broadening caused by slow flow. Failures of experiments were also often encountered as a result of bubble formation from the decomposed products of hydrogen peroxide in the limited reaction area. Second, procedures of etching and insertion of electrophoretic capillary into reaction capillary are manually intensive, which has prevented it from access to automation. Dumke and Nussbaum have recently demonstrated the feasibility of adapting a commercial CE instrument for CL detection by modifying the instrument for in-capillary luminol/horseradish peroxidase (HPR) reaction.11 Their strategy showed improved operability while sacrificed the sensitivity (with an S/N of 10 for 1.0-5.0 µM luminol) by 2 to 3 orders of magnitude compared to most CE-CL detections. Another disadvantage regarding in-capillary CL reaction is the difficulty in preparing reactant-immobilized capillary, as well as its poor reproducibility. Recently, Tsukagoshi and co-workers17-19 have devised a number of end-column reaction cell schemes (also called batchtype cells19) for CE-CL experiments. In these interfaces, the chemiluminescent reagents in electrophoresis buffer were filled into a detection cell holding the cathode and the CE capillary end in the buffer. When the CE eluents enter the solution to meet with the reactants, the CL reaction takes place and the signal is collected by the window or transferred by a fiber20 to the PMT device. The end-column reaction cell is simple in construction, easy to operate, and relatively robust in use. However, since the CL reagents are retained statically in the detection cell, the concentration of reactants near the capillary outlet will decrease as they are consumed during the experiment. This leads to irreproducible results that can only be resolved by replacing the CL reactants frequently.21 Moreover, if the CL reactions are slow, or the CL signal is intensive and lasting because of a high concentration reaction, in the case of analyzing a complex sample, their emissions will not be resolved under this static mode. It will lead to signal overlapping, peak broadening, or even signal loss for a less intense reaction. To further promote the development of CE-CL technique, especially with the aim to simplify and improve the device design toward automation and commercialization, we have fabricated a rotary detection cell for CE-CL from a polytetrafluoroethylene (PTFE) body with a ring-shaped channel. The proof-of-concept experiments with two model proteins, horseradish peroxidase (HRP) and hemoglobin (Hb) obtained satisfactory sensitivities and separation. Interestingly, the enhanced luminol/H2O2 CL reaction by Hb catalysis at picomole level was observed to yield an intensive and lasting CL emission, and the kinetic curve of the catalytic CL reaction was recorded using the rotary CE-CL detection cell. EXPERIMENTAL SECTION Chemicals. Luminol and human hemoglobin (Hb, A0-subtype, Ferrous stabilized, Mol Wt ∼64 500) were obtained from Sigma(17) Tsukagoshi, K.; Otsuka, M.; Hashimoto, M.; Nakajima, R.; Kimoto, H. Chem. Lett. 2000, 98–99. (18) Hashimoto, M.; Tsukagoshi, K.; Nakajima, R.; Kondo, K. J. Chromatogr., A 1999, 832, 191–202. (19) Tsukagoshi, K.; Nakamura, T.; Nakajima, R. Anal. Chem. 2002, 74, 4109– 4116. (20) Tsukagoshi, K.; Ishida, S.; Oda, Y.; Noda, K.; Nakajima, R. J. Chromatogr., A 2006, 1125, 144–146. (21) Lin, J. M. In ChemiluminescencesBasic Principles and Applications; Lin, J. M., Ed.; Chemical Industry Press: Beijing, China, 2004; p255.

Figure 1. Schematic diagram of the CE-CL setup with an endcolumn rotary detection cell.

Aldrich Chemical Co. (St. Louis, MO). Horseradish peroxidase (HRP, Mol Wt ∼40 000) was obtained from the Sino-American Biotechnology Co. Ltd. (Wuhan, China). A 30% hydrogen peroxide (H2O2) solution was obtained from Shanghai Chemical Co. (Shanghai, China). All other chemicals were of analyticalreagent grade and were used as received. The 18.2 MΩ/cm water used in all experiments was produced by a Water PRO PS system (Labconco, Kansas City, KS). Borate buffer was prepared from 30 mM boric acid (H3BO3) with NaOH to pH 9.0. Phosphate buffer (PBS) was prepared from 25 mM sodium dihydrogen phosphate (NaH2PO4) with NaOH to pH 8.0. Both buffers were filtered through a 0.22 µm pore-size membrane prior to use. Preparation of 10 mM luminol stock solution (remade every 2 weeks): dissolved 0.1772 g of luminol in 0.1 M NaOH, then diluted with water in a 100 mL brown flask, and stored in -4 °C. HRP at 0.1 mM and Hb at 1.0 mg/mL (∼15.5 µM) stock solution were prepared in water and refrigerated for use weekly, from which lower concentration of samples were obtained by serial dilution. Apparatus. The CE-CL arrangement in Figure 1 was built in the laboratory. It is composed of five parts: a high voltage power supply (0-30 kV, Peking University), a rotary cell (see details in the following section) for the detection was designed in laboratory also, a R928 type photomultiplier tube (Hamamatsu Photonics) for light collection, a HX-2 type signal magnifier (Institute of Chemistry, Chinese Academy of Sciences, Beijing, China), and a chromatographic workstation with computer (TL9000, Tailihua Electronic, Inc., Beijing, China). The spectral response of the PMT covers from 185 to 900 nm with the maximum response at 400 nm, which matches well with the maximum emission of luminol CL at 425 nm. The typical response time is 2 to 3 ns for rise time and 20-30 ns for transit time. Two sizes of fused-silica capillaries (50 and 75 µm i.d., Yongnian Reafine Chromatography Ltd., Hebei, China) were used for CE experiments. Fabrication of the Rotary Detection Cell. Figure 2 shows the schematic drawing (A and B) and actual photography (C and D) of the rotary detection cell composed of three parts: the cell body, lead screw (6 mm in diameter), and the step motor (having 6 mm screw matches the lead screw) with 10 levels of speed varying from 20 to 65 s/cycle with 5 s increase in each level. A Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

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Figure 2. Schematic diagram and photographs of the rotary detection cell. (A and B) Top and bottom view; (C and D) photos in close and general views. 1, Step motor and lead screw; 2, detection cell body; 3, Pt cathode; 4, slit; 5, quartz bottom; 6, PMT; 7, separation capillary; 8, offset between capillary and the slit window.

Polydimethylsiloxane (PDMS) strip was used to fix and suspend the separation capillary into the solution channel, close to the glass bottom without touching the walls. The light collecting area of the PMT was reduced by covering the entire region with black tape that had a small rectangular slit prefabricated into it (5 mm (L) × 3 mm (W)). The reduction of the light collection area minimized stray light interference that would affect the sensitivity and reproducibility of the detection of CL emission. Five cell bodies were constructed by concentrically gluing a polytetrafluoroethylene (PTFE) cylinder (o.d. ) 36 mm) and PTFE tubes (i.d.’s are from 38 to 42 mm with 1 mm increase in each level, o.d. ) i.d. + 4 mm) on to a rounded quartz glass (∼1 mm thick), forming a channel with width of 2-6 mm between the walls of the two PTFE parts. To avoid the leakage from the bottom, the edge was sealed from the outside with epoxy glue. The cell body and the step motor were connected with the lead screw. The step motor and cell body were suspended vertically on an iron support stand, with the PMT positioned directly beneath the cell channel. The rectangular slit in the black tape was placed close to the bottom, between the rotary cell’s quartz reaction chamber and the PMT to enable maximum CL emission detection (Figure 2C). The capillary was guided vertically down to the central part of the reagent channel. In initial experiments, the capillary outlet was positioned ∼1 mm from the bottom of the rotary cell’s reaction chamber directly opposite the light collecting slit on the PMT. Subsequent experiments were performed with the capillary offset 5-10 mm downstream from the light collecting slit (Figure 2 A). A PDMS strip was used to fix the Pt cathode into the cell as for the capillary (Figure 2D). For the CE-CL experiments and chemiluminescence stoppedflow measurement of hemoglobin kinetic profile, please see the Supporting Information for details. RESULTS AND DISCUSSION CE separation and CL detection conditions were optimized in initial experiments. To reduce the adsorption of protein onto the capillary wall, alkaline PBS or sodium borate (pH 8-9) was used as the electrophoresis buffer. The end-column CL reagents consist 5382

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Figure 3. Electrophorograms of Hb and HRP at both 0.25 mg/mL under static mode with rotation speed and offset at 0 (A), under rotary mode at 20 s/cycle with offsets of 5 mm (B) and 10 mm (C). Capillary, 50 µm i.d. × 60 cm (L); buffer, 25 mM PBS pH 8; sample injection, 5 kV for 5 s; separation, 20 kV. CL reactants: 10 mM H2O2, 5 mM luminol, in 30 mM sodium carbonate, pH 11.0. Peak 1, Hb; peak 2, HRP. Peaks were fitted with Gaussian model. Representative electrophorograms are shown in Figure S-1.

of 5 mM luminol and 10 mM H2O2 in sodium carbonate, pH 11, which was found to produce strong CL signals and fairly stable baseline. Five detection cells with channel width sizes of 2-6 mm were fabricated and examined. A detection cell with larger channel width (i.e., “wall-to-wall” distance) is helpful for installing the capillary and the electrode; however, we found a larger area in the reaction cell bottom facing the detection slit yielded lower response on the PMT, probably due to the greater diffusion of CL products and thus reduced CL signals. On the other hand, an overly narrow channel will pose more challenges to the suspending of the capillary and electrode into the solution without touching the walls. So we have adopted a moderate detection cell with a width of 4 mm for the ringshaped channel in the following experiments. The performance of this detection cell in separation was evaluated using two heme-protein, Hb and HRP, with the luminol/ H2O2 CL system. For comparison, we set both the rotation speed and offset at 0 to apply it as a static cell, and at 20 s/cycle with offsets of 5 and 10 mm, respectively. Hb and HRP samples both at 0.25 mg/mL were separated by CE and detected through the reaction cell under static and rotary modes (Figure S-1, Supporting Information). These results were fitted with a Gaussian model to allow direct comparison of the separation. It was observed from the electrophorograms that the resolutions (R) of the two proteins were improved from 0.54 under the static mode (Figure 3A), to 0.82 and 1.09, respectively, under rotary mode (Figure 3B,C). We propose that the observed increase in resolution during rotary mode is a result of allowing the CL reactions to occur in different spatial volumes of CL reagent. As the two proteins elute at different times from the CE capillary, they are each deposited into their own unique CL reagent reaction volumes which enables the reactions to progress independently of each other. This is in stark contrast to the CL reactions that would occur in static mode. Although the two proteins elute at

of its S/N ratio (1065) and injection concentration (15.5 µM), the LOD was estimated to be 4.37 × 10-8 M (S/N ) 3), showing near 4-fold improvement in sensitivity to the previous report on Hb and metHb (0.12 and 0.11 µM, respectively) with a static cell.22 The peaks values were extracted and plotted against time, generating a curve as seen in Figure 4 (shadow region). CL stopped-flow measurement was performed to investigate the same CL kinetic process with lower Hb concentration in another experiment, yielding a stopped-flow CL spectra that decayed much quickly (Figure S-3). Both catalytic CL reaction curves presented similar characteristics as rise-fall kinetics profile featured by a rapid increase and a slower decrease. Figure 4. Electrophorogram of 1.0 mg/mL Hb. Capillary, 75 µm i.d. × 40 cm (L); buffer, sodium 30 mM borate, pH 9.0; other conditions are the same as in Figure 3B, with 20 s/cycle rotation and 5 mm offset.

different times, the second protein may elute before the first protein’s CL reaction is completed. Depending on the time frame, this could lead the investigator to conclude that only one protein reacted with the CL reagent or that both proteins eluted at the same time. The rotary mode allows proteins that elute at similar times to be detected separate from each other and thereby enhances the resolution of these types of analyses. Our experience indicated that the 20 s/cycle speed with 5 mm offset produced reliable signals under current conditions. This setting was used in the following experiments. We should admit that improved fabrication of the rotary cell is necessary to improve the device’s performance for future applications. Since CE-CL is most widely used for ultratrace analysis, the sensitivity of this interface was evaluated by using femtomole amounts of HPR standard first. Results (Figure S-2) showed that injection of 15 nL (5 kV for 5 s) sample of HRP at 10 nM yielded signal-to-noise ratio (S/N) of 33; the limit of detection (LOD) for HRP has thus been calculated to be ∼0.91 nM (S/N ) 3). It is 2 to 3 orders of magnitude lower than that with in-capillary luminol/ HPR reaction.11 A 75 µm i.d. capillary was used to run concentrated Hb sample at 1.0 mg/mL (15.5 µM) with larger injection (60 nL at 5 kV for 5 s). Interestingly, the resultant electrophoretogram presented multiple (13) peaks in about 4 min from 3.5 to 7.3 min (Figure 4). It was found that these peaks appeared periodically in about every 20 s, a interval which matched well with a full rotation of the reaction cell (20 s). This indicates that the Hb CL reaction was of long duration; it lasted for about 240 s and was recorded 13 times when the rotary cell passed the PMT window. The most intensive signal (peak 3) was selected to calculate the LOD. On the basis (22) Tsukagoshi, K.; Nakahama, K.; Nakajima, R. Anal. Chem. 2004, 76, 4410– 4415.

CONCLUSION A novel rotary cell for the CE-CL end-column detection has been developed in this study. With the use of the HRP and Hb for the proof-of-concept experiment evaluation, satisfactory separation and sensitivity was obtained with the new interface. While interesting results can be obtained in this preliminary study, advanced fabrication technique and material (such as thinner bottom with excellent optical transparency, step motor with linearly adjustable speed) are needed for the rotary cell to achieve improved performance. One potential that could make it even more attractive is the possibility to put it into a commercialized CE instrument for CE-CL automation. ACKNOWLEDGMENT The authors wish to thank Professor Ruxiu Cai and Dr. Zhihong Liu group (Department of Chemistry, Wuhan University) for providing the stopped-flow spectra analysis and discussion. J.W. thanks Jiaxiang Xu at Peking University, Physical Chemistry facility for help in making the rotary cell, thanks Dr. Lingjun Li for support as a postdoctoral research fellow at UW-Madison and Robert Sturm for critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China (No. 20975077), the Science Fund for Creative Research Groups (No. 20921062), and the National Basic Research Program of China (973 Program, No. 2007CB714507). SUPPORTING INFORMATION AVAILABLE Additional experimental section; electrophoretogram of 10.0 nM HRP; representative electrophorogram of Hb and HRP; peak values from electrophorogram for 1.0 mg/mL Hb and the simulated CL kinetic profile; stopped-flow kinetic trace for the reactions of 3.75 mM luminol and 5 mM H2O2 catalyzed by 0.75 µM Hb. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 3, 2010. Accepted May 6, 2010. AC100007D

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