Anal. Chem. 2006, 78, 3815-3819
Technical Notes
Isotachophoresis in Free-Flow Using a Miniaturized Device Dirk Janasek,* Michael Schilling, Joachim Franzke, and Andreas Manz
ISAS-Institute for Analytical Sciences Dortmund and Berlin, Bunsen-Kirchhoff-Strasse 11, D-44139 Dortmund, Germany
For the first time, we report a miniaturized approach for isotachophoresis employing the technique of free-flow electrophoresis. Using a micromachined separation chamber with a volume of 200 nL, a sample mixture of fluorescein, eosin G, and acetylsalicylic acid was separated, stacked, and concentrated in less than a minute. Additionally, an isotachophoretic separation of a reaction mixture of myoglobin and fluoresceinisothiocyanate as a fluorescence label has shown the potential of this method for on-line sample preparation. Isotachophoresis (ITP) is a well-known and established technique in capillary electrophoresis (CE) both for separation and for preconcentration purposes. In ITP, a large volume of sample is placed between two electrolytes that generate a gradient of electric field strength. Thereby, the mobility of all analyte ions is lower than the mobility of the ions of the leading electrolyte (LE) but also higher than the mobility of the terminating electrolyte (TE) ions. During the electrophoretic separation, analyte ions arrange and stack into zones along this gradient. According to Kohlrausch’s regulating function,1 the concentration of the analyte ions is adapted to the concentration of the leading electrolyte. This means that there is a dilution effect for high concentrated samples and a concentration effect for low concentrated solutions, respectively. The original concentration of the analyte can be determined by the length of the zones that are proportional to the concentration. ITP is performed in conventional capillary electrophoresis2 as well as in miniaturized approaches.3 In CE, the electric field is applied along the capillary. Consequently, the separation is executed in a one-dimensional manner, which results in a discontinuous technique alternating between injection and separation steps (Figure 1A). In contrast, in free-flow electrophoresis (FFE), the sample is fed continuously into a separation chamber, and an electric field is applied perpendicular to a hydrodynamic flow through this chamber (Figure 1B). The two forces (hydrodynamic flow and electric field) create two orthogonally acting velocity vectors. One vector is equal to the flow velocity, and the other is proportional to the electro* Corresponding author. E-mail:
[email protected]. Tel.: +49 (0)231 1392202. Fax: +49 (0)231 1392-120. (1) Kohlrausch, F. W. G. Ann. Phys. Chem. 1897, 62, 209-239. (2) Gebauer, P.; Bocek, P. Electrophoresis 2002, 23, 3858-3864. (3) Chen, L.; Prest, J. E.; Fielden, P. R.; Goddard, N. J.; Manz, A.; Day, P. J. R. Lab Chip 2006, 6, 474-487. 10.1021/ac060063l CCC: $33.50 Published on Web 04/26/2006
© 2006 American Chemical Society
Figure 1. Comparison between the separation principle in CE (A) and FFE (B).
phoretic mobility µ. The sum vector is at an angle δ to the flow direction. Having different electrophoretic mobilities, the sum vectors for two analytes are at different angles and thus separated continuously in a two-dimensional manner at the outlet of the separation chamber. Mostly used in conventional benchtop devices with volumes in the range of tens of milliliters,4 also miniaturized versions of FFE are reported employing zone-electrophoresis5-9 and isoelectric focusing9-11 modes. (4) Canut, H.; Bauer, J.; Weber, G. J. Chromatogr. B 1999, 722, 121-139. (5) Mazereeuw, M.; de Best, C. M.; Tjaden, U. R.; Irth, H.; van der Greef, J. Anal. Chem. 2000, 72, 3881-3886. (6) Kobayashi, H.; Shimamura, K.; Akaida, T.; Sakano, K.; Tajima, N.; Funazaki, J.; Suzuki, H.; Shinohara, E. J. Chromatogr. A 2003, 990, 169-178. (7) Zhang, C. X.; Manz, A. Anal. Chem. 2003, 75, 5759-5766. (8) Fonslow, B. R.; Bowser, M. T. Anal. Chem. 2005, 77, 5706-5710. (9) Kohlheyer, D.; Besselink, G. A. J.; Schlautmann, S.; Schasfoort, R. B. M. Lab Chip 2006, 6, 374-380. (10) Albrecht, J.; Gaudet, S.; Jensen, K. F. Rapid Free Flow Isoelectric Focusing via Novel Electrode Structures. 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences: Boston, MA, 2005; pp 15371539. (11) Xu, Y.; Zhang, C. X.; Janasek, D.; Manz, A. Lab Chip 2003, 3, 224-227.
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Figure 2. Drawing of the chip layout. The size of the inlet reservoirs for LE, TE, and sample was 4 mm × 15 mm, 4 mm × 15 mm, and ca. 2 mm × 15 mm, respectively. Both side reservoirs for the electrodes had a size of 13 mm × 6 mm.
Figure 3. Isotachophoresis of fluorescein if (A) no voltage and (B) an electric field of 350 V cm-1 and (C) 525 V cm-1, respectively, was applied. Conditions: LE: 10 mM chloride, adjusted to pH 9.0 using bis-tris-propane and 0.1% MHEC; TE: 5 mM HEPES, adjusted to pH 9.0 with TRIS; cfluorescein ) 5 µM; v ) 10 µL min-1; pictures in false color (blue indicates no fluorescence, red highest fluorescence intensity), and the dashed rectangle indicates the size of the separation compartment.
EXPERIMENTAL PROCEDURES Chemicals. Deionized water was used in all experiments. All other chemicals were of analytical grade. Fluorescein sodium salt, fluorescein isothiocyanate, myoglobin, tris(hydroxymethyl)ami3816 Analytical Chemistry, Vol. 78, No. 11, June 1, 2006
nomethane, bis-tris-propane, morpholino-ethanesulfonic acid (MES), ethanol amine, and acetylsalicylic acid (ASS) were purchased from Sigma-Aldrich-Fluka (Taufkirchen, Germany). Serine, β-alanine, methylhydroxyethyl cellulose (MHEC), 2-[4-(2-hydroxyethyl)-1-
Figure 4. Separation and concentration of fluorescein, ASS, and eosin G displayed as an assembly of single micrographs over the whole chamber in false colors (panel A; same color code as in Figure 2). The comparison of a fluorescence intensity profile at the outlet cross-section of the chamber (panel B; data obtained from raw data of picture A) and an ITP-gram of the same separation in capillary (C) has enabled the recognition of the zones. For easier comparability, the time axis of the ITP-gram is from the right to the left. Both signals of the conductivity detector (s) and UV absorbance detector (- - -) are depicted in the ITP-gram. Conditions: LE: 10 mM chloride, adjusted to pH 6.0 using histidine and 0.1% MHEC; TE: 10 mM MES, adjusted to pH 6.0 using histidine; cfluorescein ) ceosin G ) 5 µM; cASS ) 250 µM; FF-ITP: E ) 210 V cm-1; v ) 10 µL min-1; and capillary ITP: electric current was program controlled, see Experimental Procedures.
piperazinyl]-ethanesulfonic acid (HEPES) buffer, hydrochloric acid, barium hydroxide, and sodium phosphate salts were obtained from Merck (Darmstadt, Germany). The procedure of labeling proteins with FITC was similar to the previously reported method.7 Briefly, 200 µL of the FITC stock solution (60 mM in ethanol) was added to 800 µL of the protein solution (12.5 mM in phosphate buffer pH 9.0). After reaction overnight at room temperature, the reaction mixture was stored in a refrigerator until usage and diluted in LE or TE, respectively, to obtain the concentration needed in the experiments. Microchip Fabrication and Layout. The chip used was made from poly(dimethylsiloxane) (PDMS) by soft-lithography. The master was fabricated by means of photolithography and electroplating described by Neyer et al.12 Briefly, the structures of the chip layout were transferred to a SU-8 photoresist and then replicated in nickel. The height of the structures of the nickel master was 10 µm, which was defined later as the height of the structures in the PDMS chip. Next, the PDMS monomer and curing agent were mixed in a ratio of 10:1, poured onto the master, and peeled off after curing. After cutting reservoirs and access holes, the structured PDMS layer was bonded to a plain PDMS layer using oxygen plasma treatment. To have mechanically stable conditions, the PDMS chip was placed on a glass plate. No additional surface treatment was carried out. (12) Neyer, A.; Knoche, T.; Mu ¨ ller, L. Electron. Lett. 1993, 29, 399-400.
The general layout of the chip structures (Figure 2) was the same as described by Xu et al.11 Briefly, the separation chamber of a width of 4.41 mm and a length of 12.18 mm consists of 31 104 diamond shaped posts. These posts (30 µm × 30 µm) were tilted in an angle of 45° and assembled in such a way that 10 µm wide channels were formed between them (see Figure 2, Z2). To meet the demands of ITP, reservoirs for leading and terminating electrolyte, the sample as well as the side reservoirs for the electrodes were cut at the appropriate locations and could hold 112, 112, 22, and 117 µL each, respectively. The lengths for inlet, outlet, and connection channels were 2, 0.35, and 15 mm, respectively. For simplicity reasons, the 64 outlet channels were reassembled to one single outlet channel, which was connected via a tube to a syringe. The reservoirs were filled with the appropriate buffers, and a hydrodynamic flow through the separation chamber was generated by the application of a vacuum at the outlet channel by means of a syringe pump. For measurements, the chip was placed on the stage of an inverted confocal microscope (Leica, Milton Keynes, UK) equipped with a 50 W mercury lamp for fluorescence imaging. The fluorescence emission was recorded by a CCD color camera (Sony). Since the dimensions of the chamber were too large to record it in one picture, the single micrographs corresponding to 3.2 mm × 2.4 mm and 5.3 mm × 3.9 mm, respectively, on the chip were assembled. Platinum wires placed in the Analytical Chemistry, Vol. 78, No. 11, June 1, 2006
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appropriate reservoirs served as electrodes that were connected to a power supply (F.u.G. Elektronik GmbH, Rosenheim, Germany). The power supply was also connected to a multimeter for current monitoring. For capillary ITP experiments, an ItaChrom EA 101 device (J&M, Aalen, Germany) was used. The device was equipped with a preseparation column (FEP capillary 0.8 mm i.d., 90 mm long; conductivity detection) and an analytical column (FEP capillary 0.3 mm i.d., 90 mm long; conductivity and UV absorption detection). The current program of the device was set as follows: step 1: 1000 s at 250 µA; step 2: 80 s at 250 µA; step 3: 1000 s at 50 µA; and step 4: 200 s at 50 µA. Safety Considerations. For the experiments, a high voltage was applied. High power poses a health danger and thus demands special caution. RESULTS AND DISCUSSION As mentioned in the Experimental Procedures, fluorescence detection was the only method that was readily available for the FF-ITP experiments. Therefore, a system had to be found where two fluorescent compounds could be isotachophoretically separated. Since the zones in ITP are stacked and adjacent, two separated compound zones can be only distinguished optically if the emission wavelengths are quite different or if a nonfluorescent spacer is stacked between them. The first option has been found to be difficult because the fluorescence dyes in the appropriate range of electrophoretic mobility had close emission spectras. For instance, fluorescein and eosin G finally used in the experiments have emission maxima of 572 and 564 nm, respectively. A commercial capillary ITP device was utilized to find an ITP system that meets the second option. The advantage of this device was the availability of both an optical and a conductivity detector that was convenient for the search of a system that separated completely and met the demands for the optical detection regime in FFE. Three systems were found to be applicable to FF-ITP: fluorescein for focusing experiments, fluorescein, eosin G, and ASS; as well as FITC-labeled myoglobin; and serine for separation and stacking. A vacuum was applied at the outlet of the FF-ITP device so that the fluorescent sample flowed through the chamber as shown in Figure 3. Each single figure shows an assembly of single micrographs in false color mode where blue indicates no fluorescence and red stands for the highest fluorescence. When no voltage was applied across the chamber, neither a focusing nor a concentrating effect was obtained as indicated by the homogeneous color of the sample stream through the chamber in Figure 3A. The narrowing of the stream toward the outlet was caused by some influx of buffer through the connection channels. The buffer influx from the electrode reservoirs also affected the overall flow conditions. The linear velocity of the sample was measured to be 131 µm s-1 at an applied withdrawal flow rate of 10 µL s-1. In theory, the linear velocity should be 10.15 mm s-1 at that flow rate. Figure 3B depicts the situation when a voltage was applied. A focusing effect began within seconds. The shift of the color to red toward the outlet of the chamber implies an increase of the fluorescence intensity. Since the fluorescence intensity is proportional to the concentration of the sample, a concentration effect can be derived from this picture. When a voltage of 1400 V was applied (electric field: 350 V cm-1), 3818 Analytical Chemistry, Vol. 78, No. 11, June 1, 2006
Figure 5. Intensity profiles across the separation chamber just before the inlet (A), at the inlet (B), and at the outlet (C) of the chamber for a FF-ITP of a reaction mixture of myoglobin and FITC. Serine had been added after the labeling reaction as a nonfluorescent spacer. SIC indicates the position of the inlet channels for the sample, and the spike (*) points indicate the beginning of focusing. Conditions: LE: 10 mM chloride, adjusted to pH 9.3 using ethanol amine and 0.1% MHEC; TE: 6 mM β-alanine, adjusted to pH 10.2 using barium hydroxide; cmyoglobin ) 0.1 mM; cserine ) 5 mM; E ) 480 V cm-1; and v ) 20 µL min-1.
the focusing was completed shortly before the outlet of the separation chamber. The residence time in the chamber was 93.7
s (Figure 3B). If higher voltages were applied, the sample was focused within an even shorter time (Figure 3C). However, if the applied voltage was too high, outgassing as well as convection occurred inside the chamber since the Joule’s heating could not be dissipated efficiently. The limit for the shown system has been found at 2180 V cm-1. Another limitation due to higher voltages is the isotachophoretic movement of the zones toward the anode, which results in a movement of the zones into the side channels. Thus, the two orthogonal forces (hydrodynamical flow and electric field) had to be balanced. As the introductory section pointed out, in capillary electrophoresis, the initial sample concentration is determined by the width of the zone. By varying the concentration between 5 and 20 µM, it could be proven that in continuous free-flow ITP, the width of the stream is proportional to the concentration (data not shown). Figure 4 shows the separation of a mixture of fluorescein, ASS, and eosin G. The focusing and separation begins as soon as the sample mixture enters the separation chamber where the electric field takes effect. Comparing the intensity profile at the chamber outlet (Figure 4B) to the isotachopherogram (ITP-gram) recorded with the capillary ITP (Figure 4C), the two fluorescence peaks could be recognized as the two dyes with the nonfluorescent ASS between them. However, at the moment, we have no explanation as to why the fluorescence signal is not baseline-separated both in cITP and in FF-ITP and why the separation resolution in the optical manner is inferior to the conductivity one. Nevertheless, the signal of the conductivity detector in Figure 4C indicates clearly the complete separation and stacking of the sample mixture. A two-dimensional contactless conductivity detector will be employed in the future. Therefore, a better comparability of the results in cITP and FF-ITP should be possible. This would obsolete both the need for analyte labeling prior detection and the need for a nonfluorescent spacer between the analyte zones. Furthermore, the resolution already obtained in cITP by conductivity detection should be achieved. In contradiction to the first, focusing experiment, an increase of the voltage did not result in a better performance of the separation since for the chosen system, the movement into the side channels occurred already at 219 V cm-1. To partly overcome the side channel problem, which is connected to the normal isotachophoretic movement toward the anode, the sample inlet reservoir had been cut almost at the cathodic side of the separation chamber. Figure 5 depicts fluorescence intensity profiles across the width of the separation chamber at different longitudinal positions, which are for panel A: 50 µm ahead of the position where the sample enters the separation chamber; panel B: 50 µm after the entering point; and panel C: at the outlet of the chamber. For panel A, a location 50 µm ahead of the chamber entrance position means that the sample mixture is still confined inside the inlet channels. Therefore, even if an electric field was applied, no transverse flow
could take place. The sample mixture contained myoglobin labeled with FITC, serine as a nonfluorescent spacer, and an excess of nonreacted, nonbound FITC. The residence time of the sample in the chamber was 50 s. It can be easily seen from the profile at the chamber outlet (Figure 5C) that separation, stacking, and concentration of the sample mixture were achieved. The appearance of the spike (*) in Figure 5B suggests that a focusing effect had already started within the first 50 µm, which corresponds to 0.4 s. That result confirms the result of the experiment shown in Figure 4. The focusing begins immediately after the sample enters the separation chamber, and it is then influenced by the electric field. The reason the focusing began on the border between sample and LE was due to the fact that the sample had been diluted in TE. In experiments where the sample was diluted in LE, the focusing began at the border between sample zone and TE. CONCLUSION It could be shown that the isotachophoretic separations can be performed in miniaturized free-flow devices of a volume in the submicroliter range within seconds. Since the separated zones contain only the analyte ion and the counterion, ITP in free-flow can serve as an ideal on-line sample preparation and purification for subsequent reaction processes or as an interface to mass spectrometry. However, to meet the requirements for readily applicable devices and techniques, the miniaturized procedure reported here as a proof of principle has to be optimized furthermore. The optical detection employed in this work has been proven as a strong limitation factor for the monitoring of the separation process due to the quality of the detector and the need for nonfluorescent spacers. Second, the labeling of the analytes presupposes an additional sample preparation step and furthermore makes it difficult to use the separated compounds in subsequent reaction processes. Therefore, in future experiments, a detector based on contactless conductivity shall be employed. ACKNOWLEDGMENT The authors thank Mr. Ulrich Marggraf for his help during the micromachining process and Prof. A. Neyer from University of Dortmund for providing the special cleanroom facilities of his department. The financial support by the Ministerium fu ¨ r Innovation, Wissenschaft, Forschung und Technologie des Landes Nordrhein-Westfalen and by the Bundesministerium fu¨r Bildung und Forschung and by the European Community (CellPROM project, contract no. NMP4-CT-2004-500039 under the 6th Framework Programme for Research and Technological Development) is gratefully acknowledged.
Received for review January 10, 2006. Accepted March 28, 2006. AC060063L
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