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Separation of proteins using a novel two-depth miniaturized free-flow electrophoresis device with multiple outlet fractionation channels
Journal of Chromatography A, 1216 (2009) 8265–8269
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Separation of proteins using a novel two-depth miniaturized free-flow electrophoresis device with multiple outlet fractionation channels Marco Becker, Ulrich Marggraf, Dirk Janasek ∗ ISAS – Institute for Analytical Sciences, Bunsen-Kirchhoff-Strasse 11, 44139 Dortmund, Germany
a r t i c l e
i n f o
Article history: Available online 5 July 2009 Keywords: Microfluidic Free-flow electrophoresis Microfabrication
a b s t r a c t A novel free-flow electrophoresis glass chip design with two-depth etched structures for the separation and fractionation of proteins is presented. The microfluidic structures etched in two depths enhance the flow characteristics inside the miniaturized device. A novel nine-port outlet interface enables the fractionation of the separated analytes. The separation and focussing of a protein sample mixture demonstrated the ability of the new chip. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The technique of free-flow electrophoresis (FFE) is based on the action of two orthogonally operating forces inside a separation compartment. Usually, these forces are generated by firstly a hydrodynamic flow of a buffer and the sample through the compartment and secondly the perpendicularly applied electrical field. The two created orthogonally acting velocity vectors are equal to the flow velocity and proportional to the electrophoretic mobility, respectively. The resulting sum vector is at a defined angle to the flow direction. For two analytes which differ in size and charge and therefore have different electrophoretic mobilities, the deflection angles are different and thus the analytes are separated continuously to different outlet channels. If the applied hydrodynamic pressure and the voltage are constant the two forces’ respective velocities are in a steady-state, and thus the analytes will always be deflected to the same outlet channels. The time domain of separation is transferred to a local domain, and that is the reason for the possibility of continuous sample separation in preparative, large scale employing FFE. Conventionally, bench top devices with volumes in the range of hundreds of millilitres [1,2] are used, but also miniaturized versions of FFE were developed in the last years [3,17]. All operational modes known from capillary electrophoresis can be performed in FFE like zone-electrophoresis [4–6], isoelectric focussing [7,8] and isotachophoresis [9,10]. In order to benefit from the continuous separation abilities and the employment of miniaturized FFE devices
∗ Corresponding author. E-mail address: [email protected] (D. Janasek). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.06.079
as up-stream and down-stream process tools, as well as for collecting the separated analytes for further analyses or storage a “micro-to-macro” interface with multiple outlet channels is necessary. Kohlheyer et al. fabricated a chip with five outlet channels in order to collect the separated analytes [11]. For simplicity reasons, in “proof-of-principle” experiments multiple channels at the outlet of the separation chamber were often re-assembled to finally only one outlet channel [4,9]. The greatest challenge in FFE chip fabrication is the introduction of the electrical field into the separation compartment. A disturbance of the separation by gas bubbles generated at the electrodes due to electrolysis has to be avoided. A commonly used method is the introduction of acrylamide membranes to isolate the separation compartment from the electrode, respectively electrode reservoirs [12,13]. The membranes are only permeable for ions, the generated gas bubbles cannot pass the membranes and thus the separation is not disturbed. The disadvantages of acrylamide membranes are the complex fabrication process, the high toxicity of the monomers and the fact that the membranes are instable at flow rates higher than 8.4 L min−1 [11]. The membranes break at this flow rate and a separation is not longer possible. Fonslow et al. implemented special electrode channels to avoid gas bubbles entering the separation chamber during FFE separation [14,18]. The electrode channels isolating the separation compartment from the electrodes were deeper than the chamber and are flown by buffer at higher flow rate than inside the chamber in order to flush the generated air bubbles away. Side channels were also employed to mimic membranes and to avoid gas bubbles entering the separation chamber [4,6,7,9,10,15]. However, often the flow resistance of these side channel arrays is not high enough to avoid in-flux or out-flux of buffer from the electrode reservoir or from the separation compartment, respectively.
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Fig. 1. Design overview of the two-depth FFE chips.
Electrochemical approaches to suppress the electrolysis of water molecules were also investigated [19]. In this paper the fabrication of a novel two-depth glass FFE chip and its application for the separation and focussing of proteins is described. Shallow side channels were used to mimic membranes to avoid gas bubble disturbance of the separation and in-flux or out-flux through these channels. We also integrated nine outlet channels and developed an outlet fractionation interface to collect the separated analytes from the chip. 2. Experimental
Fig. 2. Overview of the chip fabrication process: (a) pre-coated glass slide, (b) structures were transferred into the chrome layer by photolitography, (c) removal of the photoresist, (d) gold sputtering to cover the side channels, (e) first wet etching step, (f) gold removal, (g) second wet etching step, (h) drilling of connection holes and side reservoirs, (i) removal of chrome layer, and (j) thermal bonding of structured glass and plain glass slide.
the separation chamber 222 parallel side channels connect the separation compartment with the electrode buffer reservoir. In these buffer reservoirs the electrodes are placed which apply the voltage generating the electrical field inside the separation chamber. Each side channel is 10 m wide and 1.2 cm long. 2.3. Fabrication of the glass chip and the outlet fractionation interface
2.1. Materials Hydrofluoric acid, acetone, dimethylformamide (DMF), methanol, sulfuric acid, hydrogen peroxide, fluoresceinisothiocyanate (FITC) and the proteins myoglobin from horse heart and trypsin inhibitor from soybean were purchased from Sigma–Aldrich Chemie (Steinheim, Germany). Nitric acid, and tris(hydoxymethyl)amino methane (Tris) were obtained from Carl Roth (Karlsruhe, Germany). Phenol, iodine and potassium iodide were purchased from Merck (Darmstadt, Germany). Chrome etchant 18 was delivered from Micro Resist Technology (Berlin, Germany), polydimethylsiloxane (PDMS, Sylgard 184) was from Dow Corning (Midland, MI, USA). All chemicals were used as received without further purification. The procedure of labelling proteins with FITC was similar to the previously reported method [6]. Deionized water was employed for the preparation of all solutions and dilutions. 2.2. Chip design For the two-depth FFE chips the design shown in Fig. 1 was used. The separation chamber is 4 mm wide and 12 mm long. It consists of 31,104 diamond-shaped posts [9,10], which avoid a collapse of the chamber during the bonding √ process and extend the separation path length by a factor of 2. Each post has an edge length of 40 m; the distance between the centre points of two adjacent posts is 50 m, resulting in 10 m wide channels between the posts. There are 67 inlet channels, each 10 m wide, where buffer and sample enter the chamber. Channels 2–33 and 35–66 are reassembled in a branch-like design to two main inlet channels. These main inlet channels are connected to syringes pumping running buffer. Both most outer channels as well as the middle channel are single inlet connected to boundary buffer pumps and the sample pump, respectively. The outlet of the chip consists of nine outlet channels; each one is 70 m wide and 7 mm long. On each side of
Pre-coated soda-lime glass slides (Nanofilm, CA, USA) were used to fabricate the two-depth microfluidic chips. The glass slides were coated with a 100 nm layer of low reflective chrome. On top of the chrome is a 530 nm thick layer of AZ1518 photoresist (Fig. 2a). The chip design is transferred into the photoresist applying standard photolithography techniques. After exposure and development of the resist, the chrome is wet-etched resulting in a transfer of the chip design into the chrome layer (Fig. 2b). After removing the remaining photoresist with DMF (Fig. 2c) the areas of side channels were sputtered with gold to avoid etching (Fig. 2d). A polycarbonate (PC) cover was used as a mask for the gold sputtering. The gold sputtered glass slide was placed in a solution of 4% hydrofluoric acid (HF) and 4% nitric acid (HNO3 ) in order to wet-etch the non-covered parts of the glass slide (etch step 1). After etching for about 20 min, the non-covered structures were etched about 15.5 m deep (Fig. 2e). Now, the side channels were uncovered again by removing the gold layer using iodine/potassium iodide (Fig. 2f). After this, the glass slide was wet-etched again in HF/HNO3 solution for about 100 s (etch step 2). This process resulted in a side channel depth of around 1.5 m. The previously etched structures were also etched for additional 1.5 m, resulting in an overall depth of about 17 m (Fig. 2g). After etch step 2, the connection holes for the inlet and outlet channels as well as the side reservoirs were drilled into the glass slide (Fig. 2h). Finally, the remains of the chrome layer were removed using chrome etchant (Fig. 2i), and the structured glass slide is cleaned for 5 min in an ultrasonic water bath. The following cleaning steps for preparing the glass for thermal bonding were carried out with both the structured glass slide and a blank, unstructured glass slide which was to serve as a sealing cover for the microfluidic chip. Acetone, DMF and methanol were used consecutively for the cleaning. In the last step the glass slides were placed in permonosulfuric acid for 30 min to get a hydrophilic surface. After finishing the bonding preparation, the two glass slides
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Fig. 3. Schematic of the outlet fractionation interface. For illustration purposes only one chip-tubing connection is shown.
were assembled and put into a high temperature oven (Heraeus, Hanau, Germany) for thermal glass bonding (Fig. 2j). After bonding the outlet fractionation interface was fabricated. First, a 3 mm thick PDMS fixture was cast which was able to cover all outlet holes of the chip. In order to connect the outlet holes of the glass chip through the PDMS, holes were punched into the fixture. Then the surface of the PDMS fixture is activated by an oxygen plasma, aligned to the outlet holes and bonded to the glass chip. Capillaries were used to provide a connection between glass chip and rubber tubing. The capillaries were inserted into the punched holes in the PDMS which sealed the capillaries avoiding leakage. Fig. 3 shows a schematic drawing of the fractionation interface. The inlet channels were connected to five syringe pumps by fused silica capillaries and rubber tubes which were glued onto the glass surface. 2.4. Experimental setup The chip was mounted on an inverse microscope (Leica, Wetzlar, Germany) using a laboratory-made chip holder. The sample and buffer solutions were pumped into the chip by a computer controlled syringe pump system (neMESYS, Cetoni, Korbußen, Germany). Electrodes were placed into the side reservoirs and connected to a high voltage power supply (F.u.G. Elektronik, Rosenheim, Germany). A mercury lamp was employed to excite the FITC-labeled proteins. A filter (H3, Leica) in the optical path was used to set the excitation and emission wavelength to 435 and 510 nm, respectively. The fluorescence signal was detected by a charged-coupled device (CCD) camera (EHDkamPro02DN, EHD Imaging, Damme, Germany) and recorded with a grabber card and the WinTV 2000 software (Hauppauge Computer Works, Mönchengladbach, Germany). The scanning electron microscope images were taken with a SEM from FEI Company (Hillsboro, OR, USA). The interferometric measurements were done using a NewView 5000 surface profiler (Zygo Corporation, Middlefield, CT, USA). 3. Results and discussion The chip was operated by a pressure driven flow generated by syringe pumps pushing the buffer and sample into the chip. If the flow resistance of the side channels is not high enough, as in the case of same depth of side channels and separation chamber, a part of the fluid entering the chamber will leave the chamber through the side channels. A computer simulation utilizing Cosmos Floworks (SolidWorks Corporation, Concord, MA, USA) shows an out-flux through
Fig. 4. Simulation of fluid velocity and direction of a pressure driven flow inside a separation chamber where the side channels have the same depth as the chamber.
the side channels, a broadening of the streams and a lower flow velocity at the outlet of the chamber (Fig. 4). These non-optimal conditions lead to a poor or even impossible separation. In order to avoid a leakage through the side channels the hydrodynamic flow resistance of the side channels has to be much higher than the one of the separation compartment. The flow resistance of a rectangular shaped channel can be calculated using the Hagen–Poiseuille equation: 3
V˙ =
K × min (w, d) × max(w, d) × p 12l
(1)
V˙ is the volume of the liquid poured, w is the width and d is the depth of the channel, is the dynamic flow viscosity of the liquid flowing through the channel, l is the length of the channel, and p is the pressure difference between the two ends of the channel. K is a factor calculated according to Eq. (2). K = 1−
∞ n=1
×
1 (2n − 1)
5
×
192 5
min(w, d) max(w, d) tan h (2n − 1) 2 min(w, d) max(w, d)
(2)
where n is the number of iterations. Applying Eq. (1), the ratio between the flow resistance of the side channel array and that of the separation chamber had been calculated. A rectangular channel profile was assumed. For simplification, the chamber was considered without posts, inlet and outlet channels. The graph in Fig. 5 shows that the flow resistance of the side channels increases rapidly when the depth of the side channels decreases. If the side channels and the chamber are both 17 m deep the flow resistance of the side channels is about two times higher than the resistance of the chamber. For a depth of the side channels of 1.5 m and a chamber depth of 17 m, the ratio between the flow resistance of all side channels and the flow resistance of the separation chamber increases to 9879. Since the ratio seemed high enough these depths of chamber and side channels were used in the experiments to avoid leakage through the side channels during pressure driven flow.
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Fig. 5. Flow resistance ratios of shallow side channel and 17 m deep side channel as well as of all side channels and chamber.
As already mentioned in the section above, gold was used to cover the side channels during the etch step 1. For the very first experiments, a 200 nm gold layer was sputtered to cover the side channels. But after etch step 1 the gold layer was perforated and the side channels under the gold were also etched in some spots (Fig. 6a). This resulted in a lower flow resistance as expected because the deep etched parts of the side channels reduce the effective length of the shallow channels and therefore decrease the flow resistance of the channels. In order to avoid this unwanted spotty etching we increased the thickness of the gold layer to 550 nm. Fig. 6b shows such side channels after the etch step 2. No deeperetched spots inside the side channels are visible, hence the 550 nm thick gold layer is able to cover the side channel area from etching during etch step 1 without perforation. An interferometric measurement was carried out to check the surface profile of the intersection between chamber and side channels (Fig. 7). The picture demonstrates a smooth depth increase in the side channels towards the chamber compartment indicating a
Fig. 6. (a) SEM picture of spotty etched side channels due to a perforated gold layer, and (b) SEM picture of side channels which were covered with a 550 nm gold layer during wet etching.
Fig. 7. Surface profile of the intersection between chamber and side channels.
Fig. 8. Picture of separation chamber when no voltage was applied to the electrodes (left picture), and associated intensity spectrum shows the light intensity over the chamber width at the indicated location. The right part of the figure shows the separation chamber and associated intensity spectrum when a voltage (3.25 kV) was applied to the electrodes. The proteins were separated and focussed to different outlet channel.
M. Becker et al. / J. Chromatogr. A 1216 (2009) 8265–8269
slight under-etching of the gold layer fringes in etch step 1. The black dots are the posts which are distributed over the chamber. In order to test the separation capability of the new chip design a two-depths glass chip was fabricated and used for the separation and focussing of proteins in temperature gradient focussing mode [16,20–22]. The protein sample mixture consisted of 10 M myoglobin and 10 M trypsin inhibitor, both labelled with fluorescein isothiocyanate (FITC) for fluorescence detection. 0.5 M Tris–phenol buffer was used as the running buffer. The overall flow rate in the chip was 4.3 L min−1 including a sample flow rate of 1.5 L min−1 . According to Ohmic law the electrical resistance of each side channel array (222 side channels; each side channel 1.5 m deep, 13 m wide and 1.2 cm long) was around 50 G. Thus, for separation a voltage of 3.15 kV had to be applied to the side reservoirs resulting in an electrical field strength of 119 V cm−1 inside the separation compartment. The left picture in Fig. 8 shows the sample stream inside the separation compartment when no voltage was applied. A broadening of the sample stream and a leakage through the side channels were not observed. The sample stream just flows through the chamber since no electrophoretic velocity was present. The associated intensity spectrum over the chamber width at the indicated location shows no intensity change over the width of the sample stream. When the voltage of 3.15 kV was applied to the electrodes, in the intensity spectrum two peaks appeared indicating higher fluorescence intensity due to a higher concentration of labelled proteins at these locations (Fig. 8, right picture). The peaks represented streams of concentrated myoglobin and trypsin inhibitor, respectively, which were clearly separated and focussed to different outlet channels. At the end of the separation chamber the focussed analyte streams broaden before entering the outlet channels. The reason is the unfavourable flow conditions due to the fact of just nine outlet channels. In order to check the influence of the electroosmotic flow (EOF) on the separation, the velocity of the EOF was measured using the uncharged dye rhodamine B. The measured EOF velocity is 0.3 mm s−1 at a voltage of 3.15 kV. The calculated deflection of the sample stream by EOF is approx. 74 m towards the cathode over the entire length of the chamber (12 mm). For the calculation an overall flow rate of 4.3 L min−1 was used. Considering the chamber width of 4 mm, the deflection of the sample stream caused by EOF is insignificantly small. 4. Conclusion A new free-flow electrophoresis two-depth chip design was presented using shallow side channels to mimic membranes. Because of a more than 9800-fold higher hydrodynamic flow resistance of the side channel array compared to the deeper-etched separation chamber of the chip a leakage through the side channels could not be observed. At the same time the electrical resistance increased by a factor of 38. A voltage of more than 3 kV had to be applied to the electrode reservoirs resulting in an electrical field strength of 119 V cm−1 inside the separation compartment. A clearly detectable separation and focussing of a protein mixture demonstrated the
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performance of the new chip design. Even after one hour of operation no electrolysis products (gas bubbles) disturbed the separation nor were observed inside the separation compartment. The new outlet fractionation interface offered the possibility of collecting the separated analytes from the chip. In order to increase resolution of the separation a higher electrical field strength inside the separation chamber is needed. By optimizing the parameters of the side channels (channel depth, channel length) the electrical resistance of the side channels can be reduced in order to increase the separation field strength. A lower flow rate and therefore a longer residence time of the analyte inside the electric field should also increase the resolution of the separation. In future the number of outlet channels will be increased up to 64 in order to avoid band broadening due to unfavourable flow conditions at the chamber–outlet channel intersection. A combination of this chip with label-free online detection methods like surface enhanced Raman spectroscopy (SERS) [10] will also strengthen the ability of miniaturized free-flow electrophoresis as chemical processing tools. Acknowledgements The authors thank Professor A. Neyer from Laboratory of Micro Structure Technology at Technische Universität Dortmund for providing the special cleanroom facilities of his department. In addition, funding from the Ministerium für Innovation, Wissenschaft, Forschung und Technologie des Landes NordrheinWestfalen and from the Bundesministerium für Bildung und Forschung is also greatly appreciated. References [1] P. Glukhovskij, G. Vigh, Electrophoresis 13 (2001) 2639. [2] P. Hoffmann, H. Wagner, M. Lanz, J. Caslavska, W. Thormann, Anal. Chem. 9 (1999) 1840. [3] D. Kohlheyer, J.C.T. Eijkel, A. van den Berg, R.B.M. Schasfoort, Electrophoresis 29 (2008) 977. [4] D.E. Raymond, A. Manz, H.M. Eidmer, Anal. Chem. 66 (1994) 2858. [5] A. Chartogne, U.R. Tjaden, J. van der Greef, Rapid Commun. Mass Spectrom. 14 (2000) 1269. [6] C.-X. Zhang, A. Manz, Anal. Chem. 75 (2003) 5759. [7] Y. Xu, C.-X. Zhang, D. Janasek, A. Manz, Lab. Chip 3 (2003) 231. [8] J.W. Albrecht, J. El-Ali, K.F. Jensen, Anal. Chem. 24 (2007) 9364. [9] D. Janasek, M. Schilling, J. Franzke, A. Manz, Anal. Chem. 11 (2006) 3815. [10] M. Becker, C. Budich, V. Deckert, D. Janasek, Analyst 1 (2009) 38. [11] D. Kohlheyer, J.C.T. Eijkel, S. Schlautmann, A. van den Berg, R.B.M. Schasfoort, Anal. Chem. 79 (2007) 8190. [12] D. Kohlheyer, G.A.J. Besselink, S. Schlautmann, R.B.M. Schasfoort, Lab. Chip 6 (2006) 374. [13] J.W. Albrecht, K.F. Jensen, Electrophoresis 24 (2006) 4960. [14] B.R. Fonslow, V.H. Barocas, M.T. Bowser, Anal. Chem. 15 (2006) 5369. [15] B.R. Fonslow, M.T. Bowser, Anal. Chem. 17 (2005) 5706. [16] M. Becker, A. Mansouri, C. Beilein, D. Janasek, Electrophoresis (accepted for publication). [17] R.T. Turgeon, M.T. Bowser, Anal. Bioanal. Chem. 394 (2009) 187. [18] B.R. Fonslow, M.T. Bowser, Anal. Chem. 80 (2008) 3182. [19] D. Kohlheyer, J.C.T. Eijkel, S. Schlautmann, A. van den Berg, R.B.M. Schasfoort, Anal. Chem. 80 (2008) 4111. [20] D. Ross, L.E. Locascio, Anal. Chem. 11 (2002) 2556. [21] J.G. Shackman, M.S. Munson, D. Ross, Anal. Bioanal. Chem. 1 (2007) 155. [22] T. Matsui, J. Franzke, A. Manz, D. Janasek, Electrophoresis 24 (2007) 4606.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Separation of proteins using a novel two-depth miniaturized free-flow electrophoresis device with multiple outlet fractionation channels Marco Becker, Ulrich Marggraf, Dirk Janasek ∗ ISAS – Institute for Analytical Sciences, Bunsen-Kirchhoff-Strasse 11, 44139 Dortmund, Germany
a r t i c l e
i n f o
Article history: Available online 5 July 2009 Keywords: Microfluidic Free-flow electrophoresis Microfabrication
a b s t r a c t A novel free-flow electrophoresis glass chip design with two-depth etched structures for the separation and fractionation of proteins is presented. The microfluidic structures etched in two depths enhance the flow characteristics inside the miniaturized device. A novel nine-port outlet interface enables the fractionation of the separated analytes. The separation and focussing of a protein sample mixture demonstrated the ability of the new chip. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The technique of free-flow electrophoresis (FFE) is based on the action of two orthogonally operating forces inside a separation compartment. Usually, these forces are generated by firstly a hydrodynamic flow of a buffer and the sample through the compartment and secondly the perpendicularly applied electrical field. The two created orthogonally acting velocity vectors are equal to the flow velocity and proportional to the electrophoretic mobility, respectively. The resulting sum vector is at a defined angle to the flow direction. For two analytes which differ in size and charge and therefore have different electrophoretic mobilities, the deflection angles are different and thus the analytes are separated continuously to different outlet channels. If the applied hydrodynamic pressure and the voltage are constant the two forces’ respective velocities are in a steady-state, and thus the analytes will always be deflected to the same outlet channels. The time domain of separation is transferred to a local domain, and that is the reason for the possibility of continuous sample separation in preparative, large scale employing FFE. Conventionally, bench top devices with volumes in the range of hundreds of millilitres [1,2] are used, but also miniaturized versions of FFE were developed in the last years [3,17]. All operational modes known from capillary electrophoresis can be performed in FFE like zone-electrophoresis [4–6], isoelectric focussing [7,8] and isotachophoresis [9,10]. In order to benefit from the continuous separation abilities and the employment of miniaturized FFE devices
∗ Corresponding author. E-mail address: [email protected] (D. Janasek). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.06.079
as up-stream and down-stream process tools, as well as for collecting the separated analytes for further analyses or storage a “micro-to-macro” interface with multiple outlet channels is necessary. Kohlheyer et al. fabricated a chip with five outlet channels in order to collect the separated analytes [11]. For simplicity reasons, in “proof-of-principle” experiments multiple channels at the outlet of the separation chamber were often re-assembled to finally only one outlet channel [4,9]. The greatest challenge in FFE chip fabrication is the introduction of the electrical field into the separation compartment. A disturbance of the separation by gas bubbles generated at the electrodes due to electrolysis has to be avoided. A commonly used method is the introduction of acrylamide membranes to isolate the separation compartment from the electrode, respectively electrode reservoirs [12,13]. The membranes are only permeable for ions, the generated gas bubbles cannot pass the membranes and thus the separation is not disturbed. The disadvantages of acrylamide membranes are the complex fabrication process, the high toxicity of the monomers and the fact that the membranes are instable at flow rates higher than 8.4 L min−1 [11]. The membranes break at this flow rate and a separation is not longer possible. Fonslow et al. implemented special electrode channels to avoid gas bubbles entering the separation chamber during FFE separation [14,18]. The electrode channels isolating the separation compartment from the electrodes were deeper than the chamber and are flown by buffer at higher flow rate than inside the chamber in order to flush the generated air bubbles away. Side channels were also employed to mimic membranes and to avoid gas bubbles entering the separation chamber [4,6,7,9,10,15]. However, often the flow resistance of these side channel arrays is not high enough to avoid in-flux or out-flux of buffer from the electrode reservoir or from the separation compartment, respectively.
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Fig. 1. Design overview of the two-depth FFE chips.
Electrochemical approaches to suppress the electrolysis of water molecules were also investigated [19]. In this paper the fabrication of a novel two-depth glass FFE chip and its application for the separation and focussing of proteins is described. Shallow side channels were used to mimic membranes to avoid gas bubble disturbance of the separation and in-flux or out-flux through these channels. We also integrated nine outlet channels and developed an outlet fractionation interface to collect the separated analytes from the chip. 2. Experimental
Fig. 2. Overview of the chip fabrication process: (a) pre-coated glass slide, (b) structures were transferred into the chrome layer by photolitography, (c) removal of the photoresist, (d) gold sputtering to cover the side channels, (e) first wet etching step, (f) gold removal, (g) second wet etching step, (h) drilling of connection holes and side reservoirs, (i) removal of chrome layer, and (j) thermal bonding of structured glass and plain glass slide.
the separation chamber 222 parallel side channels connect the separation compartment with the electrode buffer reservoir. In these buffer reservoirs the electrodes are placed which apply the voltage generating the electrical field inside the separation chamber. Each side channel is 10 m wide and 1.2 cm long. 2.3. Fabrication of the glass chip and the outlet fractionation interface
2.1. Materials Hydrofluoric acid, acetone, dimethylformamide (DMF), methanol, sulfuric acid, hydrogen peroxide, fluoresceinisothiocyanate (FITC) and the proteins myoglobin from horse heart and trypsin inhibitor from soybean were purchased from Sigma–Aldrich Chemie (Steinheim, Germany). Nitric acid, and tris(hydoxymethyl)amino methane (Tris) were obtained from Carl Roth (Karlsruhe, Germany). Phenol, iodine and potassium iodide were purchased from Merck (Darmstadt, Germany). Chrome etchant 18 was delivered from Micro Resist Technology (Berlin, Germany), polydimethylsiloxane (PDMS, Sylgard 184) was from Dow Corning (Midland, MI, USA). All chemicals were used as received without further purification. The procedure of labelling proteins with FITC was similar to the previously reported method [6]. Deionized water was employed for the preparation of all solutions and dilutions. 2.2. Chip design For the two-depth FFE chips the design shown in Fig. 1 was used. The separation chamber is 4 mm wide and 12 mm long. It consists of 31,104 diamond-shaped posts [9,10], which avoid a collapse of the chamber during the bonding √ process and extend the separation path length by a factor of 2. Each post has an edge length of 40 m; the distance between the centre points of two adjacent posts is 50 m, resulting in 10 m wide channels between the posts. There are 67 inlet channels, each 10 m wide, where buffer and sample enter the chamber. Channels 2–33 and 35–66 are reassembled in a branch-like design to two main inlet channels. These main inlet channels are connected to syringes pumping running buffer. Both most outer channels as well as the middle channel are single inlet connected to boundary buffer pumps and the sample pump, respectively. The outlet of the chip consists of nine outlet channels; each one is 70 m wide and 7 mm long. On each side of
Pre-coated soda-lime glass slides (Nanofilm, CA, USA) were used to fabricate the two-depth microfluidic chips. The glass slides were coated with a 100 nm layer of low reflective chrome. On top of the chrome is a 530 nm thick layer of AZ1518 photoresist (Fig. 2a). The chip design is transferred into the photoresist applying standard photolithography techniques. After exposure and development of the resist, the chrome is wet-etched resulting in a transfer of the chip design into the chrome layer (Fig. 2b). After removing the remaining photoresist with DMF (Fig. 2c) the areas of side channels were sputtered with gold to avoid etching (Fig. 2d). A polycarbonate (PC) cover was used as a mask for the gold sputtering. The gold sputtered glass slide was placed in a solution of 4% hydrofluoric acid (HF) and 4% nitric acid (HNO3 ) in order to wet-etch the non-covered parts of the glass slide (etch step 1). After etching for about 20 min, the non-covered structures were etched about 15.5 m deep (Fig. 2e). Now, the side channels were uncovered again by removing the gold layer using iodine/potassium iodide (Fig. 2f). After this, the glass slide was wet-etched again in HF/HNO3 solution for about 100 s (etch step 2). This process resulted in a side channel depth of around 1.5 m. The previously etched structures were also etched for additional 1.5 m, resulting in an overall depth of about 17 m (Fig. 2g). After etch step 2, the connection holes for the inlet and outlet channels as well as the side reservoirs were drilled into the glass slide (Fig. 2h). Finally, the remains of the chrome layer were removed using chrome etchant (Fig. 2i), and the structured glass slide is cleaned for 5 min in an ultrasonic water bath. The following cleaning steps for preparing the glass for thermal bonding were carried out with both the structured glass slide and a blank, unstructured glass slide which was to serve as a sealing cover for the microfluidic chip. Acetone, DMF and methanol were used consecutively for the cleaning. In the last step the glass slides were placed in permonosulfuric acid for 30 min to get a hydrophilic surface. After finishing the bonding preparation, the two glass slides
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Fig. 3. Schematic of the outlet fractionation interface. For illustration purposes only one chip-tubing connection is shown.
were assembled and put into a high temperature oven (Heraeus, Hanau, Germany) for thermal glass bonding (Fig. 2j). After bonding the outlet fractionation interface was fabricated. First, a 3 mm thick PDMS fixture was cast which was able to cover all outlet holes of the chip. In order to connect the outlet holes of the glass chip through the PDMS, holes were punched into the fixture. Then the surface of the PDMS fixture is activated by an oxygen plasma, aligned to the outlet holes and bonded to the glass chip. Capillaries were used to provide a connection between glass chip and rubber tubing. The capillaries were inserted into the punched holes in the PDMS which sealed the capillaries avoiding leakage. Fig. 3 shows a schematic drawing of the fractionation interface. The inlet channels were connected to five syringe pumps by fused silica capillaries and rubber tubes which were glued onto the glass surface. 2.4. Experimental setup The chip was mounted on an inverse microscope (Leica, Wetzlar, Germany) using a laboratory-made chip holder. The sample and buffer solutions were pumped into the chip by a computer controlled syringe pump system (neMESYS, Cetoni, Korbußen, Germany). Electrodes were placed into the side reservoirs and connected to a high voltage power supply (F.u.G. Elektronik, Rosenheim, Germany). A mercury lamp was employed to excite the FITC-labeled proteins. A filter (H3, Leica) in the optical path was used to set the excitation and emission wavelength to 435 and 510 nm, respectively. The fluorescence signal was detected by a charged-coupled device (CCD) camera (EHDkamPro02DN, EHD Imaging, Damme, Germany) and recorded with a grabber card and the WinTV 2000 software (Hauppauge Computer Works, Mönchengladbach, Germany). The scanning electron microscope images were taken with a SEM from FEI Company (Hillsboro, OR, USA). The interferometric measurements were done using a NewView 5000 surface profiler (Zygo Corporation, Middlefield, CT, USA). 3. Results and discussion The chip was operated by a pressure driven flow generated by syringe pumps pushing the buffer and sample into the chip. If the flow resistance of the side channels is not high enough, as in the case of same depth of side channels and separation chamber, a part of the fluid entering the chamber will leave the chamber through the side channels. A computer simulation utilizing Cosmos Floworks (SolidWorks Corporation, Concord, MA, USA) shows an out-flux through
Fig. 4. Simulation of fluid velocity and direction of a pressure driven flow inside a separation chamber where the side channels have the same depth as the chamber.
the side channels, a broadening of the streams and a lower flow velocity at the outlet of the chamber (Fig. 4). These non-optimal conditions lead to a poor or even impossible separation. In order to avoid a leakage through the side channels the hydrodynamic flow resistance of the side channels has to be much higher than the one of the separation compartment. The flow resistance of a rectangular shaped channel can be calculated using the Hagen–Poiseuille equation: 3
V˙ =
K × min (w, d) × max(w, d) × p 12l
(1)
V˙ is the volume of the liquid poured, w is the width and d is the depth of the channel, is the dynamic flow viscosity of the liquid flowing through the channel, l is the length of the channel, and p is the pressure difference between the two ends of the channel. K is a factor calculated according to Eq. (2). K = 1−
∞ n=1
×
1 (2n − 1)
5
×
192 5
min(w, d) max(w, d) tan h (2n − 1) 2 min(w, d) max(w, d)
(2)
where n is the number of iterations. Applying Eq. (1), the ratio between the flow resistance of the side channel array and that of the separation chamber had been calculated. A rectangular channel profile was assumed. For simplification, the chamber was considered without posts, inlet and outlet channels. The graph in Fig. 5 shows that the flow resistance of the side channels increases rapidly when the depth of the side channels decreases. If the side channels and the chamber are both 17 m deep the flow resistance of the side channels is about two times higher than the resistance of the chamber. For a depth of the side channels of 1.5 m and a chamber depth of 17 m, the ratio between the flow resistance of all side channels and the flow resistance of the separation chamber increases to 9879. Since the ratio seemed high enough these depths of chamber and side channels were used in the experiments to avoid leakage through the side channels during pressure driven flow.
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Fig. 5. Flow resistance ratios of shallow side channel and 17 m deep side channel as well as of all side channels and chamber.
As already mentioned in the section above, gold was used to cover the side channels during the etch step 1. For the very first experiments, a 200 nm gold layer was sputtered to cover the side channels. But after etch step 1 the gold layer was perforated and the side channels under the gold were also etched in some spots (Fig. 6a). This resulted in a lower flow resistance as expected because the deep etched parts of the side channels reduce the effective length of the shallow channels and therefore decrease the flow resistance of the channels. In order to avoid this unwanted spotty etching we increased the thickness of the gold layer to 550 nm. Fig. 6b shows such side channels after the etch step 2. No deeperetched spots inside the side channels are visible, hence the 550 nm thick gold layer is able to cover the side channel area from etching during etch step 1 without perforation. An interferometric measurement was carried out to check the surface profile of the intersection between chamber and side channels (Fig. 7). The picture demonstrates a smooth depth increase in the side channels towards the chamber compartment indicating a
Fig. 6. (a) SEM picture of spotty etched side channels due to a perforated gold layer, and (b) SEM picture of side channels which were covered with a 550 nm gold layer during wet etching.
Fig. 7. Surface profile of the intersection between chamber and side channels.
Fig. 8. Picture of separation chamber when no voltage was applied to the electrodes (left picture), and associated intensity spectrum shows the light intensity over the chamber width at the indicated location. The right part of the figure shows the separation chamber and associated intensity spectrum when a voltage (3.25 kV) was applied to the electrodes. The proteins were separated and focussed to different outlet channel.
M. Becker et al. / J. Chromatogr. A 1216 (2009) 8265–8269
slight under-etching of the gold layer fringes in etch step 1. The black dots are the posts which are distributed over the chamber. In order to test the separation capability of the new chip design a two-depths glass chip was fabricated and used for the separation and focussing of proteins in temperature gradient focussing mode [16,20–22]. The protein sample mixture consisted of 10 M myoglobin and 10 M trypsin inhibitor, both labelled with fluorescein isothiocyanate (FITC) for fluorescence detection. 0.5 M Tris–phenol buffer was used as the running buffer. The overall flow rate in the chip was 4.3 L min−1 including a sample flow rate of 1.5 L min−1 . According to Ohmic law the electrical resistance of each side channel array (222 side channels; each side channel 1.5 m deep, 13 m wide and 1.2 cm long) was around 50 G. Thus, for separation a voltage of 3.15 kV had to be applied to the side reservoirs resulting in an electrical field strength of 119 V cm−1 inside the separation compartment. The left picture in Fig. 8 shows the sample stream inside the separation compartment when no voltage was applied. A broadening of the sample stream and a leakage through the side channels were not observed. The sample stream just flows through the chamber since no electrophoretic velocity was present. The associated intensity spectrum over the chamber width at the indicated location shows no intensity change over the width of the sample stream. When the voltage of 3.15 kV was applied to the electrodes, in the intensity spectrum two peaks appeared indicating higher fluorescence intensity due to a higher concentration of labelled proteins at these locations (Fig. 8, right picture). The peaks represented streams of concentrated myoglobin and trypsin inhibitor, respectively, which were clearly separated and focussed to different outlet channels. At the end of the separation chamber the focussed analyte streams broaden before entering the outlet channels. The reason is the unfavourable flow conditions due to the fact of just nine outlet channels. In order to check the influence of the electroosmotic flow (EOF) on the separation, the velocity of the EOF was measured using the uncharged dye rhodamine B. The measured EOF velocity is 0.3 mm s−1 at a voltage of 3.15 kV. The calculated deflection of the sample stream by EOF is approx. 74 m towards the cathode over the entire length of the chamber (12 mm). For the calculation an overall flow rate of 4.3 L min−1 was used. Considering the chamber width of 4 mm, the deflection of the sample stream caused by EOF is insignificantly small. 4. Conclusion A new free-flow electrophoresis two-depth chip design was presented using shallow side channels to mimic membranes. Because of a more than 9800-fold higher hydrodynamic flow resistance of the side channel array compared to the deeper-etched separation chamber of the chip a leakage through the side channels could not be observed. At the same time the electrical resistance increased by a factor of 38. A voltage of more than 3 kV had to be applied to the electrode reservoirs resulting in an electrical field strength of 119 V cm−1 inside the separation compartment. A clearly detectable separation and focussing of a protein mixture demonstrated the
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performance of the new chip design. Even after one hour of operation no electrolysis products (gas bubbles) disturbed the separation nor were observed inside the separation compartment. The new outlet fractionation interface offered the possibility of collecting the separated analytes from the chip. In order to increase resolution of the separation a higher electrical field strength inside the separation chamber is needed. By optimizing the parameters of the side channels (channel depth, channel length) the electrical resistance of the side channels can be reduced in order to increase the separation field strength. A lower flow rate and therefore a longer residence time of the analyte inside the electric field should also increase the resolution of the separation. In future the number of outlet channels will be increased up to 64 in order to avoid band broadening due to unfavourable flow conditions at the chamber–outlet channel intersection. A combination of this chip with label-free online detection methods like surface enhanced Raman spectroscopy (SERS) [10] will also strengthen the ability of miniaturized free-flow electrophoresis as chemical processing tools. Acknowledgements The authors thank Professor A. Neyer from Laboratory of Micro Structure Technology at Technische Universität Dortmund for providing the special cleanroom facilities of his department. In addition, funding from the Ministerium für Innovation, Wissenschaft, Forschung und Technologie des Landes NordrheinWestfalen and from the Bundesministerium für Bildung und Forschung is also greatly appreciated. References [1] P. Glukhovskij, G. Vigh, Electrophoresis 13 (2001) 2639. [2] P. Hoffmann, H. Wagner, M. Lanz, J. Caslavska, W. Thormann, Anal. Chem. 9 (1999) 1840. [3] D. Kohlheyer, J.C.T. Eijkel, A. van den Berg, R.B.M. Schasfoort, Electrophoresis 29 (2008) 977. [4] D.E. Raymond, A. Manz, H.M. Eidmer, Anal. Chem. 66 (1994) 2858. [5] A. Chartogne, U.R. Tjaden, J. van der Greef, Rapid Commun. Mass Spectrom. 14 (2000) 1269. [6] C.-X. Zhang, A. Manz, Anal. Chem. 75 (2003) 5759. [7] Y. Xu, C.-X. Zhang, D. Janasek, A. Manz, Lab. Chip 3 (2003) 231. [8] J.W. Albrecht, J. El-Ali, K.F. Jensen, Anal. Chem. 24 (2007) 9364. [9] D. Janasek, M. Schilling, J. Franzke, A. Manz, Anal. Chem. 11 (2006) 3815. [10] M. Becker, C. Budich, V. Deckert, D. Janasek, Analyst 1 (2009) 38. [11] D. Kohlheyer, J.C.T. Eijkel, S. Schlautmann, A. van den Berg, R.B.M. Schasfoort, Anal. Chem. 79 (2007) 8190. [12] D. Kohlheyer, G.A.J. Besselink, S. Schlautmann, R.B.M. Schasfoort, Lab. Chip 6 (2006) 374. [13] J.W. Albrecht, K.F. Jensen, Electrophoresis 24 (2006) 4960. [14] B.R. Fonslow, V.H. Barocas, M.T. Bowser, Anal. Chem. 15 (2006) 5369. [15] B.R. Fonslow, M.T. Bowser, Anal. Chem. 17 (2005) 5706. [16] M. Becker, A. Mansouri, C. Beilein, D. Janasek, Electrophoresis (accepted for publication). [17] R.T. Turgeon, M.T. Bowser, Anal. Bioanal. Chem. 394 (2009) 187. [18] B.R. Fonslow, M.T. Bowser, Anal. Chem. 80 (2008) 3182. [19] D. Kohlheyer, J.C.T. Eijkel, S. Schlautmann, A. van den Berg, R.B.M. Schasfoort, Anal. Chem. 80 (2008) 4111. [20] D. Ross, L.E. Locascio, Anal. Chem. 11 (2002) 2556. [21] J.G. Shackman, M.S. Munson, D. Ross, Anal. Bioanal. Chem. 1 (2007) 155. [22] T. Matsui, J. Franzke, A. Manz, D. Janasek, Electrophoresis 24 (2007) 4606.