Direct Detection of Biomolecules in a Capillary Electrophoresis

Direct detection of biomolecules, such as r-amino acids, peptides, and proteins, was accomplished using a capil- lary electrophoresis-chemiluminescenc...
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Anal. Chem. 2004, 76, 4410-4415

Direct Detection of Biomolecules in a Capillary Electrophoresis-Chemiluminescence Detection System Kazuhiko Tsukagoshi,* Koji Nakahama, and Riichiro Nakajima

Department of Chemical Engineering and Materials Science, Faculty of Engineering,Doshisha University, Kyotanabe, Kyoto 610-0321, Japan

Direct detection of biomolecules, such as r-amino acids, peptides, and proteins, was accomplished using a capillary electrophoresis-chemiluminescence detection system, in which a luminol-hydrogen peroxide-Cu(II)-catalyzed chemiluminescence reaction was utilized. Biomolecules migrated in the capillary, where they mixed with luminol and the Cu(II) catalyst included in the running buffer. The capillary outlet was inserted into a batch-type chemiluminescence detection cell with hydrogen peroxidesupplemented electrolyte solution. Chemiluminescence was observed at the tip of the capillary outlet. The chemiluminescence peak from biomolecules appeared due to the enhancement of Cu(II) catalytic activity for luminol-hydrogen peroxide chemiluminescence. The Cu(II) was more catalytically active when it interacted with biomolecules forming Cu(II)-biomolecule complexes. In this study, biomolecules were directly separated and detected in a capillary electrophoresis-chemiluminescence detection system. Twenty r-amino acids, 4 peptides, and 11 proteins were examined. Most of them were detected with satisfactory CL intensity response. Glutamic acid, an r-amino acid, was detected at concentrations ranging from 2.0 × 10-7 to 1.2 × 10-5 M with a detection limit (S/N ) 3) of 1.0 × 10-7 M (0.6 fmol). Glycylglycine, a peptide, was detected at concentrations ranging from 1.7 × 10-7 to 1.2 × 10-5 M with a detection limit (S/N ) 3) of 1.7 × 10-7 M (0.9 fmol). Hemoglobin, a heme protein, in which the heme structure was independently catalytically active, was detected at concentrations ranging from 1.2 × 10-7 to 1.0 × 10-5 M with a detection limit (S/N ) 3) of 1.2 × 10-7 M (0.6 fmol). Representative mixtures of r-amino acids and peptides were well detected with superior separation. Absorbance and fluorescence have been fundamental methods of detection for instrumental analysis. The principles of chemiluminescence (CL) are correlated to those of both absorbance and fluorescence detection. Recently, CL has received a great amount of attention as an attractive detection technique due to the following characteristics: high sensitivity, determinable over a wide range of sample, easy operation, and an inexpensive ap* To whom correspondence should be addressed. Tel: +81-774-65-6595. Fax: +81-774-65-6803. E-mail: [email protected].

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paratus and reagents. It has been used in flow injection analysis (FIA), high-performance liquid chromatography (HPLC),1-4 and capillary electrophoresis (CE).5-9 Various types of CE coupled with CL detection have been developed by utilizing different CL reagents (luminol, 1,10phenanthroline, ruthenium(II) complex, peroxyoxalate). Many kinds of samples could be separated and detected using this technique, including metal ions, metal compounds, amino acids, peptides, proteins, saccharides, nucleic acids, phenolic compounds, fluorescence compounds, and liposomes.10 CL detection is also becoming one of the detection methods most matched to the micro total analysis system, because it does not require a light source or spectroscope. Combination of microchip CE with a CL detector using luminol and peroxyoxalate CL reagents was successfully demonstrated.11,12 However, compared with absorption and fluorescence detection, it is possible that CL detection lacks generality of application. To solve this problem, many scientists have tried to synthesize novel CL reagents and explore more effective analytical conditions in various fields including medicine, pharmacology, and chemistry.2,13,14 Derivatization of non-CL analytes, which is generally required prior to detection, has been the most applicable of these methods.2 One drawback is that derivatizing processes are often tedious and time-consuming. Quantitative labeling of a lowconcentration analyte is also difficult. Thus, a direct detection method, which does not require a complex labeling procedure, has been a continued area of interest in CL analysis.15-17 (1) Hara, T.; Tsukagoshi, K. Anal. Sci. 1990, 6, 797-806. (2) Yamaguchi, M.; Yoshida, H.; Nohta, H. J. Chromatogr., A 2002, 950, 1-19. (3) Garcia-Campana, A. M.; Baeyens, W. R. G. Analusis 2000, 28, 686-698. (4) Nakashima, K. Bunseki Kagaku 2000, 49, 135-159. (5) Tsukagoshi, K.; Nakamura, T.; Nakajima, R. Anal. Chem. 2002, 74, 41094116. (6) Huang, X.-J.; Fang, Z.-L. Anal. Chim. Acta 2000, 414, 1-14. (7) Timerbaev, A. R.; Shpigun, O. A. Electrophoresis 2000, 21, 4179-4191. (8) Kuyper, C.; Milofsky, R. Trends Anal. Chem. 2001, 5, 232-240. (9) J.-M. Lin, H. Goto, M. Yamada: J. Chromatogr., A 1999, 844, 341-348. (10) Tsukagoshi, K. Bunseki Kagaku 2003, 52, 1-13 and the references therein. (11) Hashimoto, M.; Tsukagoshi, K.; Nakajima, R.; Kondo, K.; Arai, A. J. Chromatogr., A 2000, 867, 271-279. (12) Tsukagoshi, K.; Hashimoto, M.; Nakajima, R.; Arai, A. Anal. Sci. 2000, 16, 111-1112. (13) Hinze, W. L.; Srinivassan, N.; Smith, T. K.; Igarashi, S.; Hoshino, H. In Organized Assemblies in Analytical Chemiluminescence Spectroscopy: An Overview. Advances in Multidimensional Luminescence; Warner, I. M., et al., Eds.; JAI: London, 1990; Chapter 8. (14) Nakagawa, T.; Yamada, M.; Hobo, T. Anal. Chim. Acta 1990, 231, 7-12. 10.1021/ac030344i CCC: $27.50

© 2004 American Chemical Society Published on Web 06/30/2004

Herein, a novel principle for the detection of biomolecules in a CL reaction has been described and a direct detection method of biomolecules in a CE-CL detection system has been proposed. We briefly described the preliminary results of this investigation in a previous communication.18 Biomolecules, such as R-amino acids, peptides, and proteins, were directly detected using CE coupled with CL detection; pretreatments such as labeling were not required. The CL peak due to biomolecules was detected because of enhanced Cu(II) catalytic activity for luminolhydrogen peroxide CL. The Cu(II) catalyst was more active when it interacted with biomolecules to form Cu (II)-biomolecule complexes. Twenty R-amino acids, 4 peptides, and 11 proteins were examined in detail. EXPERIMENTAL SECTION Materials. All reagents used for this study were commercially available, analytical grade reagents. The water used for ion exchange was distilled before use. Luminol and all R-amino acids (L-isomers except for glycine) used in this study were purchased from Nacalai Tesque. All peptides and proteins used were purchased from Sigma Co. A hydrogen peroxide solution (30 wt %) was purchased from Wako Pure Chemical Industries, Ltd. CL Detection Cell. A batch-type cell was used in the present CE-CL detection system. The concept for this cell was proposed as previously described.19 The detection cell was composed of Teflon (outer diameter, 4 cm; height, 2.5 cm; inner volume, 8 mL). The cell was fixed with an optical fiber (1.2-mm core diameter; ST 1200I-SY, Mitsubishi Cable Industries, Ltd.), a fused-silica capillary (50- or 75-µm i.d.; GL Sciences Inc.), and a platinum wire used as a grounding electrode. The cell served as an outlet reservoir including an electrolyte solution. As the analytes emerged from the capillary, they reacted with the reagent at the capillary outlet thereby emitting visible light. This CL light was subsequently captured by the optical fiber. Analytical Procedure and Apparatus. The luminol and hydrogen peroxide CL system was used in conjunction with a Cu(II) catalyst; 10 mM phosphate buffer (pH 10.8) was prepared. All samples were prepared in phosphate buffer. Phosphate buffer (pH 10.8) containing 5.0 × 10-5 M luminol, 5.0 × 10-6 Cu(II), and 5.0 × 10-5 M potassium sodium tartrate was used as a running buffer; potassium sodium tartrate worked as a masking agent for producing an interaction between Cu(II) and biomolecules in an alkaline solution without olation. And phosphate buffer (pH 10.8) containing 0.4 M hydrogen peroxide solution was added to the outlet reservoir or the detection cell. Sample injections (∼5.6 nL for the 50-µm-i.d. capillary and ∼29 nL for the 75-µm-i.d. capillary; capillary length, 50 cm) were performed by gravity over a period of 10 s from a height of 20 cm. High voltage (12 kV) was applied to electrodes using a dc power supplier (model HCZE-30PNO. 25, Matsusada Precision Devices Co. Ltd.). Samples migrated in the running buffer toward the CL detection cell where they reacted with the reagents. The CL that resulted at the capillary outlet was (15) Hara, T.; Toriyama, M.; Tsukagoshi, K. Bull. Chem. Soc. Jpn. 1983, 56, 1382-1387. (16) Tsai, H.-C.; Whang, C. W. Electrophoresis 1999, 20, 2533-2538. (17) Yang, W.; Zhang, Z. Talanta 2003, 59, 951-958. (18) Tsukagoshi, K.; Nakahama, K.; Nakajima, R. Chem. Lett. 2003, 32, 634635. (19) Hashimoto, M.; Tsukagoshi, K.; Nakajima, R.; Kondo, K. J. Chromatogr., A 1999, 832, 191-202.

Figure 1. Typical electropherogram of a biomolecule obtained using the present CE-CL detection system. Conditions: fused-silica capillary, length 50 cm and i.d. 50 µm; applied voltage, 12 kV; reagent, 10 mM phosphate buffer (pH 10.8) containing 5.0 × 10-5 M luminol, 5.0 × 10-6 M Cu(II), and 5.0 × 10-5 M potassium sodium tartrate in the inlet reservoir and 10 mM phosphate buffer (pH 10.8) containing 0.4 M hydrogen peroxide in the outlet reservoir; sample, 1.0 × 10-5 M glutamic acid.

then transported to the photosenser module through the optical fiber in the CL detector (model EN-21, Kimoto Electric, Inc.). The output from the detector was fed to an integrator (Chromatopac C-R6A, Shimadzu) producing electropherograms. RESULTS AND DISCUSSION Sensitive Detection of r-Amino Acid and the Detection Principle. A CL peak from the biomolecules was observed in the CE-CL detection system. A typical CL peak (glutamic acid) is shown in Figure 1. The luminol and Cu(II) catalyst-supplemented phosphate buffer traveled from the capillary inlet to the outlet by electroosmotic flow. At the tip of capillary outlet, luminol and Cu(II) reacted with hydrogen peroxide in the outlet reservoir or CL detection cell producing CL light. This CL intensity value was the baseline CL. When a sample (glutamic acid) was introduced, it migrated in the capillary where it mixed with luminol and the Cu(II) catalyst and then emerged at the tip of the capillary, where it mixed with the hydrogen peroxide, producing an increased CL intensity signified by a positive peak on the electropherogram (Figure 1). This peak was created due to the magnified CL intensity of the luminol-hydrogen peroxide-Cu(II) catalyst in the presence of sample (glutamic acid). The increased catalytic activity of Cu(II) upon interaction with biomolecules was thought to be due to the formation of a Cu(II)-biomolecule complex. The catalytic activity of a Cu(II) complex is at its maximum when two of its four coordination sites are occupied by a ligand;20,21 thus, this Cu(II)-biomolecule complex is formed upon the occupation (20) Ojima, H. Nippon Kagaku Zasshi 1958, 79, 1076-1081. (21) Hara, T.; Arai, A.; Iharada, T. Bull. Chem. Soc. Jpn. 1986, 59, 3684-3686.

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of two coordination sites by a biomolecule. A specific microspace of the capillary inside, which was kept at relative high temperature with Joule heat, may cause such a peculiar coordination form to enhance the catalytic activity of Cu(II). Chemistry of CL Reaction in the Cell. Several reports have been published on optimizing conditions (reagent concentrations, mixing order, methods of mixing, etc.) to establish a sensitive and reproducible system of CL detection in conjunction with FIA and HPLC. However, there have been very few studies on CL detection in CE. Generally, with luminol CL, luminol must be mixed with hydrogen peroxide and catalyst. If the three components are not introduced as separate streams, then the peroxide is destroyed by the catalyst before reacting with luminol or its derivatives, and no CL is observed. To optimize the connection of the CE system with the CL detector, two methods of mixing the hydrogen peroxide and the catalyst were evaluated (methods A and B). In method A, the hydrogen peroxide was added to the inlet reservoir (5 × 10-5 M luminol, 400 mM hydrogen peroxide, and 5 × 10-5 M potassium sodium tartrate), and the catalyst was added to the outlet reservoir (5 × 10-6 M Cu(II)). Under these conditions, the glutamic acid signal gave a relatively high response, similar to the electropherogram shown in Figure 1. However, the CL signal produced by glutamic acid decreased markedly after continuous injection analysis and finally disappeared. To investigate the cause of this decreased signal, a 10repeated injection test was conducted without exchange of reagent in the cell or washing in the capillary (the first series measurements). The CL intensity gradually decreased and dropped to less than 10% of its original value after the 10-repeated injection test (data not shown). The decomposition of hydrogen peroxide due to electrolytic oxidation at the anodic electrode was considered as a cause of the decreased CL intensity. This possibility was discussed in detail in a previous report.22 Upon completion of the first series measurements, the electrolyte solution in the outlet was replaced, while the electrolyte solution in the inlet reservoir was not exchanged. A second series of repeated measurements was subsequently performed. Each of the series consisted of 10 measurements of glutamic acid, and 1 measurement took ∼15 min. The data of the second series was fitted to that of the first. This evaluation indicated that the decreased CL intensity after repeated injection experiment was not likely to be due to the decomposition of hydrogen peroxide. Next, the CL intensity for repeated injection experiments at various hydrogen peroxide concentrations (1, 5, 100, and 400 mM) in the inlet reservoir was examined while maintaining a constant Cu(II) catalyst concentration (5 × 10-6 M) (Figure 2). Although the rate of decrease of the CL intensity became less significant at lower hydrogen peroxide concentrations, the decrease of CL intensity was present, even at the lowest hydrogen peroxide concentration (1 mM). When using 1 mM hydrogen peroxide in the inlet reservoir, the CL intensity for the tenth injection was within ∼55% of the first injection. The volume of electrolyte solution eluted from the capillary to the outlet reservoir for one measurement was only ∼0.15% of volume of the outlet reservoir. In addition, the volume of electrolyte solution eluted from the capillary to the outlet reservoir (22) Liao, S.-Y.; Whang, C.-W. J. Chromatogr., A 1996, 736, 247-254.

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Figure 2. CL intensity for the repeated injection experiment at various hydrogen peroxide concentrations. Conditions: fused-silica capillary, length 50 cm and i.d. 50 µm; applied voltage, 12 kV; reagent, 10 mM phosphate buffer (pH 10.8) containing 5.0 × 10-5 M luminol, 1 (×), 5 (4), 100 (0), or 400 (O) mM hydrogen peroxide, and 5.0 × 10-5 M potassium sodium tartrate in the inlet reservoir and 10 mM phosphate buffer (pH 10.8) containing 5.0 × 10-6 M Cu(II) in the outlet reservoir; sample, 1.0 × 10-5 M glutamic acid.

Figure 3. CL intensity for the repeated injection experiment performed by gravity flow at a height of 20 cm. Conditions: fusedsilica capillary, length 50 cm and i.d. 50 µm; reagent, 10 mM phosphate buffer (pH 10.8) containing 5.0 × 10-5 M luminol, 0.4 M hydrogen peroxide, and 5.0 × 10-5 M potassium sodium tartrate in the inlet reservoir and 10 mM phosphate buffer (pH 10.8) containing 5.0 × 10-6 M Cu(II) in the outlet reservoir; sample, 1.0 × 10-5 M glutamic acid.

for one measurement was examined when using gravity flow with no electrophoresis and was found to be was only ∼0.07% of the volume of the outlet reservoir. However, the intensity of the CL signal for gravity analysis of a 10-repeated injection test decreased markedly, finally almost disappearing (Figure 3). These observations collectively indicate that the small electrolyte volume eluted from the capillary resulted in the decreased luminol CL signal, although evident cause of the decreased signal has not been clear yet. When hydrogen peroxide was added to the inlet reservoir as described above, the CL intensity gradually decreased over time. In addition, it is well known that hydrogen peroxide solutions are difficult to degas, and bubble formation in the separation capillary is problematic. Furthermore, a relatively high concentration of hydrogen peroxide would perturb separations. Due to the additional complications involved in using hydrogen peroxide in the inlet reservoir, coupled with the profound decrease in luminol CL intensity, method A was rendered an impractical analytical method. Therefore, the author examined method B in which the hydrogen peroxide was added to the outlet reservoir and the catalyst was added to the inlet.

In method B, Cu(II) catalyst (2.5 × 10-5 M) was added to the inlet reservoir and allowed to migrate with luminol through the capillary and 0.4 M hydrogen peroxide was added to the outlet reservoir. The CL intensity of glutamic acid did not change after 10 repeated injections; the relative standard deviation of the CL intensity was within 2.7% (n ) 10). Thus, method B was used to conduct the experiments herein. Preparation of Sample Solution. When the sample was prepared in phosphate buffer, the resultant electropherogram had an additional peak at ∼5.5 min (Figure 1). To produce a clean electrophorogram (without a solvent peak) but maintain a high CL intensity, the samples were prepared in the running buffer (phosphate buffer with luminol, Cu(II), and potassium sodium tartrate) and the resultant electrophorograms were examined. The interaction between the sample and the Cu(II) in the sample solution may cause the reaction to proceed effectively before being injected into the capillary, which would lead to a higher intensity response. The relationship between the time a sample solution was allowed to stand (after sample preparation) before injection and the CL intensity was examined (data not shown). The CL intensity decreased with time after sample preparation. This decease in intensity of response may be because as sample reacts with the Cu(II) in solution, it may proceed to a complex with four occupied sites before injection. To prevent samples from reacting with the Cu(II) in the running buffer and yield optimal CL intensity and reproducibility, all samples were prepared in phosphate buffer. Optimization of Reagent Concentrations. The concentration of the reagents is also a very important factor for CL detection in CE, FIA, and HPLC. The effects of varying the luminol (5 × 10-61 × 10-4 M) and Cu(II) catalyst (1 × 10-6-1 × 10-4 M) concentrations on CL intensity at a constant hydrogen peroxide concentration were examined (data not shown). The 5.0 × 10-5 M luminol and 5.0 × 10-6 M Cu(II) catalyst resulted in a maximum CL intensity response. Next, we evaluated the effect of varying the hydrogen peroxide concentration (0.050-1.0 M) in the outlet reservoir on the CL intensity while maintaining the optimal concentrations of luminol (5.0 × 10-5 M) and Cu(II) catalyst (5.0 × 10-6 M) (data not shown). Maximum CL intensity was achieved at a concentration of ∼0.40 M hydrogen peroxide. The following experiments were conducted at these optimized reagent concentrations. The relative standard deviation of 1.0 × 10-5 M glutamic acid was 3.2% (n ) 7). Analysis of r-Amino Acids. Calibration curves were constructed for glutamic acid using 50- and 75-µm-i.d. capillaries under optimized reagent concentrations. Glutamic acid was determined over the range from 2.0 × 10-7 to 1.2 × 10-5 M. It had a detection limit of 1.0 × 10-7 M (0.6 fmol) (S/N ) 3), correlation coefficient of 0.999, and theoretical plate number of 10 000-15 000 when a 50-µm-i.d. capillary was used. In addition, it was evaluated with a 75-µm-i.d. capillary over the range of 8.0 × 10-8-3.0 × 10-6 M and had a detection limit of 5.0 × 10-8 M (1.4 fmol) (S/N ) 3), correlation coefficient of 0.999, and theoretical plate number of 5000-10 000. The following experiments were carried using a 50µm-i.d. capillary, because of the superior separation obtained with the capillary. The migration times and CL intensities were examined for 20 R-amino acids. The results are shown in Table 1. The relative CL intensities were estimated when the CL intensity of glycine was

Table 1. Migration Times and Relative Peak Heights of r-Amino Acidsa R-amino acid

migration time (min)

rel peak heightb

glycine alanine valine leucine isoleucine asparagine glutamine serine threonine cysteine methionine phenylalanine throsine tryptophan proline

4.9 5.0 5.1 5.0 5.0 5.2 5.1 6.8 5.3 5.5 5.2 5.4 5.4 5.0 5.3

1.0 2.3 1.1 4.0 2.3 1.2 1.3 1.3 3.7 2.9 0.8 3.5 2.3 7.2 2.4

lysine arginine histidine

7.1 7.2 7.5

2.6 1.9 8.2

aspartic acid glutamic acid

8.0 8.8

4.0 3.9

a Sample concentration, 1.0 × 10-5 M. b Relative peak heights were estimated when the CL intensity of glycine was to be 1.0.

1.0. All R-amino acids yielded positive CL response peaks. The migration times of amino acids increased from neutral to basic and were longest for acidic amino acids. The negative charge of acidic amino acids at pH 10.8 must increase their electrophoretic mobility toward the capillary inlet. Histidine, which had the largest stability constant with Cu(II),23 produced the largest CL intensity. However, a correlation explaining the relationship between the stability constants of Cu(II)-R-amino acid complexes and the CL intensities and that of the stability constants of Cu(II)-R-amino acid complexes and the migration times has not been clear yet. We will continue to consider the correlation among the stability constants, the migration times, and the CL intensities, together with the isoelectric points, the experimental pH values, etc. The calibration curves of glycine, serine, lysine, aspartic acid, and glutamic acid were constructed (data not shown). They were constructed according to the following parameters: glycine, over a range of 2.0 × 10-7-1.5 × 10-5 M with a detection limit of 2.0 × 10-7 M (1.1 fmol); serine, 1.8 × 10-7-1.8 × 10-5 M with 1.8 × 10-7 M (1.1 fmol); lysine, 1.7 × 10-7-1.3 × 10-5 M with 1.7 × 10-7 M (0.9 fmol); aspartic acid, 1.1 × 10-7-1.8 × 10-5 M with 1.1 × 10-7 M (0.6 fmol); and glutamic acid 2.0 × 10-7-1.8 × 10-5 M with 1.0 × 10-7 M (0.6 fmol). The correlation coefficients were 0.999. Figure 4 shows the electropherogram of a sample containing a mixture of these five amino acids. The mixture of these R-amino acids was well separated under the given reaction conditions. Analysis of Peptides. The migration times and CL intensities for glycine and four peptides were examined. The results are shown in Table 2. Both the migration times and CL intensities increased with increasing molecular weight. At pH 10.8, glycine and the peptides are negatively charged due to the dissociation (23) Stability Constants of Metal-Ion Complexes; The Chemical Society, Burlington House: London, 1971; p 451.

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Figure 4. Electropherogram of a mixture of R-amino acids: 1, glycine; 2, serine; 3, lycine; 4, aspartic acid; and 5, glutamic acid. Conditions: fused-silica capillary, length 50 cm and i.d. 50 µm; applied voltage, 12 kV; reagent, 10 mM phosphate buffer (pH 10.8) containing 5.0 × 10-5 M luminol, 5.0 × 10-6 M Cu(II), and 5.0 × 10-5 M potassium sodium tartrate in the inlet reservoir and 10 mM phosphate buffer (pH 10.8) containing 0.4 M hydrogen peroxide in the outlet reservoir; sample, 1.0 × 10-5 M. Table 2. Molecular Weights, Migration Times, and Peak Heights of Glycine and Peptidesa sample

MW

migration time (min)

rel peak heightb (mV)

glycine glycylglycine glycylglycylglycine glycylglycylglycylglycine glycylglycylglyclyglycilglycine

75.07 132.1 189.2 246.2 303.3

4.9 6.7 7.6 8.3 9.1

1.0 2.1 2.4 2.5 2.8

a Sample concentration, 1.0 × 10-5 M. b Relative peak heights were estimated when the CL intensity of glycine was to be 1.0.

of the carboxyl group. If they do not interact with Cu(II) through complex formation, their electrophoretic mobilities turn to the capillary inlet because of their negative charges; the magnitude of electrophoretic mobility toward the inlet was glycine > glycylglycine > glycylglycylglycine > glycylglycylglycylglycine > glycylglycylglycylglycylglycine. Consequently, as the molecules finally move to the capillary outlet with electroosmotic flow, glycylglycylglycylglycylglycine, glycylglycylglycylglycine, glycylglycylglycine, glycylglycine, and glycine would be eluted at the capillary outlet in this order. However, in fact, the reverse elution order was observed as shown in Table 2. The obtained results undoubtedly clarified the existent of an interaction between Cu(II) and the samples under these running conditions. The interaction put the positive charge on the Cu(II)sglycine orspeptide com4414 Analytical Chemistry, Vol. 76, No. 15, August 1, 2004

Figure 5. Electropherogram of a mixture of glycine and peptides: 1, glycine; 2, glycylglycine; 3, glycylglycylglycine; 4, glycylglycylglycylglycine; and 5, glycylglycylglycylglycylglycine. Conditions: fusedsilica capillary, length 50 cm and i.d. 50 µm; applied voltage, 12 kV; reagent, 10 mM phosphate buffer (pH 10.8) containing 5.0 × 10-5 M luminol, 5.0 × 10-6 M Cu(II), and 5.0 × 10-5 M potassium sodium tartrate in the inlet reservoir and 10 mM phosphate buffer (pH 10.8) containing 0.4 M hydrogen peroxide in the outlet reservoir; sample, 1.0 × 10-5 M.

plex. The positive charge made the electrophoretic mobility toward the outlet and led to the experimental results in Table 2. We had examined a mixture sample of glycine, glycylglycine, and glycylglycylglycine, which were labeled with isoluminol isothiocyanate in the previous paper,5 where the running buffer did not include Cu(II). Glycylglycylglycine, glycylglycine, and glycine were eluted in this order in the previous CE-CL detection method. The order obtained by the previous method without using Cu(II) was the reverse of that obtained in the present method. The difference in the elution order between the previous and present methods also supported the existence of an interaction between Cu(II) and biomolecules in the present method. The peptide calibration curves were constructed. They were constructed according to the following parameters: glycine, over a range of 2.0 × 10-7-1.5 × 10-5 M with a detection limit of 2.0 × 10-7 M (1.1 fmol) (S/N ) 3); glycylglycine, 1.7 × 10-7-1.2 × 10-5 M with 1.7 × 10-7 M (0.9 fmol); glycylglycylglycine, 2.0 × 10-7-1.6 × 10-5 M with 2.0 × 10-7 M (1.1 fmol); glycylglycylglycylglycine, 1.8 × 10-7-1.6 × 10-5 M with 1.8 × 10-7 M (1.0 fmol); and glycylglycylglycylglycylglycine, 2.0 × 10-7-1.8 × 10-5 M with 2.0 × 10-7 M (1.1 fmol). The correlation coefficients were 0.999. Figure 5 shows the electropherogram of a sample containing a mixture of glycine and these peptides. The mixture sample of glycine and these peptides was well separated under the given reaction conditions without any additional labeling procedure.

Table 3. Migratin Times and Peak Heights of Proteins samplea albumin, bovine albumin, chicken albumin, rabitt albumin, human albumin, chicken egg γ-globulin, human hemoglobin methemoglobin ribonuclease lysozyme cytochrome c

migration time (min)

peak height (mV)

7.4 8.0 8.2 7.3 (6.0) 6.1 10.0 9.5

30 10 14 4.5 (1)b 5 230 128 ndc nd nd

a Sample concentration, 0.66 g L-1. b Sample concentration, 6.6 g L-1. c Not detected.

Analysis of Proteins. The migration times and CL intensities for 11 proteins were examined and the results are shown in Table 3. Hemeproteins, such as hemoglobin and methemoglobin, showed much higher CL intensities than albumins and globulin. This may be because the heme structure in these proteins is a catalyst activity for luminol and hydrogen peroxide CL. Ribonuclease, lysozyme, and cytochrome c, which possess relatively high isoelectric points of 9.5-11, yield no CL peaks. This may be explained because their amino groups are protonated at pH 10.8, thus repelling the positively charged Cu(II) catalyst or strongly being adsorbed to the capillary surface. Calibration curves for hemoglobin and methemoglobin were constructed. They were constructed over the range of 1.2 × 10-7-1.0 × 10-5 M with a detection limit of 1.2 × 10-7 M (0.6 fmol) and 1.1 × 10-7-1.0 × 10-5 M with a detection limit of 1.1 × 10-7 M (0.6 fmol), respectively. The correlation coefficients were 0.999.

problem, derivatization of non-CL analytes prior to detection is useful and generally required. The derivatizing processes, however, are often tedious and time-consuming. One of the most interesting and useful applications of current CL analysis has been focused on a direct mode of detection of biomolecules that does not require a tedious labeling procedure. In this study, a novel method of detection for biomolecules in a CL reaction was examined, and a proposal to develop the direct detection of biomolecules in a CE-CL detection system was introduced. Biomolecules, such as R-amino acids, peptides, and proteins, were directly detected by CE with a CL detection system, and tedious pretreatments and labeling procedures were not required. The CL peak from biomolecules appeared by enhancing the Cu(II) catalytic activity of luminol-hydrogen peroxide CL. The Cu(II) was more catalytically active when it interacted with biomolecules, forming Cu(II)-biomolecule complexes. Recently, a very significant feature of the CE-CL detector was recognized; naturally, while absorption and fluorescence detection is accomplished in an oncapillary manner, CL detection is accomplished by end-capillary (postcolumn) detection. That is, the CE-CL detector possesses a very interesting and useful “microspace area” for reaction/ detection at the tip of the capillary outlet. The microspace area was utilized for simultaneous analysis of multiple samples in the CE-CL detector.24 This specific microspace area for reaction/ detection may enable direct detection, with the positive peaks for biomolecules, in the present system. ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors also acknowledge financial support for this research from Doshisha University’s Research Promotion Fund.

CONCLUSIONS Compared with absorption and fluorescence methods of detection, CL detection lacks general practicality. To solve this

Received for review October 1, 2003. Accepted April 20, 2004.

(24) Tsukagoshi, K.; Ikegami, K.; Nakajima, R. Anal. Sci. 2003, 19, 1339-1340.

AC030344I

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