Anal. Chem. 2007, 79, 1057-1063
Formation of Stable Stacking Zones in a Flow Stream for Sample Immobilization in Microfluidic Systems Juan Astorga-Wells, Susanne Vollmer, Tomas Bergman, and Hans Jo 1 rnvall*
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden
Electrocapture is a multifunctional microfluidic tool that can be used for concentration, sample cleanup, multistep reactions, and separation of biomolecules. Herein, we investigate the mechanisms underlying the electrocapture principle. A microfluidic electrocapture device was found to be capable of generating regions of different electric field, which are maintained in the flow by electric and hydrodynamic forces, with the zones of lower electric field strength upstream of those with higher strength. In addition to detection of the local electric fields by direct measurements, the existence of the zones was observed by the capture of a solution containing Coomassie and myoglobin. The two molecules were captured at different spots in a steady-state manner and were released (separated) at different electric fields. Considering these observations and the experimental values for the electric field strengths, flow velocities, and electrophoretic mobilities of DNA, proteins, and peptides, it is concluded that the macromolecules are captured between the field zones by a stacking mechanism. Miniaturization has been visualized as the next generation of analytical instrumentation.1-3 The technological challenge to incorporate into a microdevice all analytical procedures carried out with normal instrumentation requires in part novel strategies, because of significant mismatches between the macro- and microworlds. In protein and peptide analysis, several approaches have demonstrated the potential of microfluidic devices to perform fast and integrated analysis. For example, detection of cytokines from cerebrospinal fluid has been achieved by combination of microimmunoaffinity capture4 and chip-based capillary electro* Corresponding author. Tel: +46-8-524 8 7702. Fax: +46-8-337 462. E-mail:
[email protected]. (1) Kricka, L. J. Clin. Chim. Acta 2001, 307, 219-223. (2) Jo ¨rnvall, H. Comprehensive Biochem. 2003, 42, 53-102. (3) Dittrich, P. S.; Manz, A. Nat. Rev. Drug Discovery 2006, 5, 210-218. (4) Guzman, N. A.; Phillips, T. M. Anal. Chem. 2005, 77, 61A-67A. (5) Phillips, T. M. Electrophoresis 2004, 25, 1652-1659. (6) Wolters, D. A.; Washburn, M. P.; Yates, J. R., 3rd. Anal. Chem. 2001, 73, 5683-5690. (7) Lee, H.; Yi, E. C.; Wen, B.; Reily, T. P.; Pohl, L.; Nelson, S.; Aebersold, R.; Goodlett, D. R. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2004, 803, 101-110. (8) Pelzing, M.; Neususs, C. Electrophoresis 2005, 26, 2717-2728. (9) Hernandez-Borges, J.; Neususs, C.; Cifuentes, A.; Pelzing, M. Electrophoresis 2004, 25, 2257-2281. 10.1021/ac061699f CCC: $37.00 Published on Web 01/05/2007
© 2007 American Chemical Society
phoresis;5 and microLC6,7 and capillary electrophoresis8-10 have been coupled to tandem mass spectrometry. In addition to downscaling existing analytical tools (LC to microLC and electrophoresis to capillary electrophoresis), microfluidics have permitted the development of novel methodologies based on the inherent characteristics of micrometer-size structures. In this context, microfluidic electrocapture was developed in order to immobilize charged molecules in a flow stream without use of solid-phase materials or chemical binding. This is achieved through the counteracting effects of hydrodynamic and electric forces exerted upon target molecules captured in a nanoliter zone of a flow stream.11 Using the electrocapture principle, desalting, concentration, buffer exchange, removal of detergents, on-line multistep microreactions (protein reduction, alkylation, and tryptic digestion), and peptide separations can be achieved.12-16 Although immobilization in this system is the result of interaction of hydrodynamic and electric forces, experimental evidence suggests that ionic interactions between the fluidic channel and the electrode chambers play a key role in the capture process. From analysis of plots of current versus flow rate, and from the cation-selective properties of Nafion membranes, it has been postulated that, at certain flow rates and voltages, zones with different ionic strengths (and therefore different local electric fields) are created, and these zones have been suggested to be responsible for slight discrepancies between theoretical and experimental conditions of capture.12 Similarly, it has been suggested that molecules are captured between low electric field (high concentration of ions) and high electric field (low concentration of ions) zones by stacking,13 the zones interpreted to be the result of cation-selective properties of the Nafion membrane separating the fluidic channel from the electrode chambers. According to this interpretation, cations could freely move in and out of the channel, but anions in the channel would be concen(10) Stroink, T.; Paarlberg, E.; Waterval, J. C.; Bult, A.; Underberg, W. J. Electrophoresis 2001, 22, 2375-2383. (11) Astorga-Wells, J.; Vollmer, S.; Bergman, T.; Jo ¨rnvall, H. Anal. Biochem. 2005, 345, 10-17. (12) Park, S. R.; Swerdlow, H. Anal. Chem. 2003, 75, 4467-4474. (13) Astorga-Wells, J.; Swerdlow, H. Anal. Chem. 2003, 75, 5207-5212. (14) Astorga-Wells, J.; Jo ¨rnvall, H.; Bergman, T. Anal. Chem. 2003, 75, 52135219. (15) Astorga-Wells, J.; Bergman, T.; Jo ¨rnvall, H. Anal. Chem. 2004, 76, 24252429. (16) Astorga-Wells, J.; Vollmer, S.; Tryggvason, S.; Bergman, T.; Jornvall, H. Anal. Chem. 2005, 77, 7131-7136.
Analytical Chemistry, Vol. 79, No. 3, February 1, 2007 1057
trated at the upstream gap (when the anode is upstream), because they cannot pass through the membrane. On the other hand, anions situated outsidesin the downstream cathode buffer chamberscannot enter into the channel to replace those that moved upstream. This would produce an ion depletion zone at the downstream junction, likely responsible for the high electric resistance observed at low or no flow rates. According to Ohm’s law, the zone of higher electric resistanceslower ionic concentration and conductivityswould have a higher electric field strength than the zone of higher ionic concentration and conductivity, located upstream. Under this principle, certain anions situated at the depletion zone (downstream) will be subjected to an electric force of higher magnitude than the hydrodynamic force, thus pushing them upstream toward the lower electric field zone. At some point of this movement, the magnitude of the electric force will become weaker than the hydrodynamic force, upon which the ions will be pushed downstream, where the process is repeated. In this report, we present experimental data supporting this stacking theory. Zones with different electric field strengths were detected a few seconds after application of the electric field. EXPERIMENTAL SECTION Reagents and Chemicals. All buffers and solutions were degassed prior to use. Water was obtained from a Milli-Q waterpurification system (Millipore Corp., Billerica, MA). Tris-HCl (1 M), pH 8.0, was purchased from United States Biochemicals (Cleveland, OH). Horse muscle myoglobin, NH4HCOO, and NH4Cl were purchased from Sigma Chemical Co. (St. Louis, MO). Coomassie (Brilliant Blue G-250) was obtained from Bio-Rad Laboratories (Hercules, CA). Capture Device. In order to measure the electric field of the upstream and downstream regions, the design of the capture device was modified from that in previous studies.12-15 The system now has four electric junctions, where the electric field is applied between the first and fourth junctions. The junctions in between are electrically floating and are used to measure local electric fields between junctions. Four openings were made with a razor blade to PEEK tubing (125-µm i.d., 635-µm o.d., Upchurch Scientific, Oak Harbor, WA) at a distance of 1 cm from each other. The first three openings were half-thickness cuts of the PEEK tubing, while the gap at the end of the device was created by completely cutting the PEEK tubing and separating the two pieces by 0.3 cm. Each gap was covered with a piece of Nafion tubing (360-µm i.d., 510-µm o.d., Permapure Inc, Toms River, NY), placed into separate electrode chambers made of 0.5-mL plastic microcentrifuge tubes (Eppendorff, Hamburg, Germany), and fixed in place with an epoxy glue (Figure 1). Depending on the experiments, the electrode chambers were filled with 100 mM Tris-HCl, pH 8.0, 100 mM NH4Cl, pH 8.0, or 100 mM NH4HCOO, pH 5.5. The electrode at the first electric junction was connected to a high-voltage power supply (CZE1000PN30, Spellman, Plainview, NY), and the last junction was grounded. In order not to disturb the operation of the electrocapture device, the voltages in these regions were measured using a homemade, high-impedance, voltage-reducer circuit connected to digital voltmeters. The resistance of this circuit was built in such a manner that it was much higher than the device; therefore, only a small amount of current was used to measure the voltage drop between the 1058 Analytical Chemistry, Vol. 79, No. 3, February 1, 2007
Figure 1. Modified version of the electrocapture device with the junctions between the anode and cathode electrically floating, allowing measurement of the voltage drop between the junctions via a highimpedance circuit.
junctions. The circuit was electrically floating. In this manner, the electrocapture device operation was not influenced by the measurements. Depending on the flow rate, 200-400 V was applied between the first and the last junctions, such that the electrode located upstream (first junction) was positive relative to the last one (fourth junction). A syringe pump (model 33, Harvard Apparatus, Holliston, MA), equipped with a 500-µL gastight syringe (Hamilton Corp., Reno, NV) produced the flow stream in the PEEK channel. The capture device was connected to the syringe using microfluidic connectors obtained from Upchurch. Flow rates were adjusted in the range 0-5 µL/min according to the manufacturer’s pump settings without further calibration or measurement. RESULTS AND DISCUSSION Validation of the Measurements. An electrocapture device, designed to allow direct measurements of local electric fields along the microfluidic channel, was first validated to test the performance of this modified version. Plots of current versus flow rate showed the same electric behavior as the original design. Myoglobin was captured at the same conditions of voltage and flow rate. Voltage determinations were carried out by measurements of the voltage drops between adjacent electric junctions at a flow rate of 2 µL/ min under total voltage drops of 200, 300, and 400 V over the 3-cm length of the device (Figure 1). Given these potential drops and the length of the device, measurements between adjacent junctions must result in values of 66, 100, and 133 V/cm for 200, 300, and 400 V, respectively. The data obtained gave an average value of 65, 96, and 128 V/cm, respectively, proving the validity of the measurements with an overall error of only 1-5 V per junction. To some extent, minor disturbing effects might be present, but comparison of the theoretical values with the data obtained show that the configuration can be used to accurately measure the voltage drops between the junctions. Using three different electrolytes, all local electric field versus flow rate graphs showed the same characteristics: a low electric field (upstream) and a high electric field (downstream) at lower flow rates, and a steady electric field (upstream and downstream) that corresponds to homogeneous distributions of the electric field at higher flow rates. This behavior was observed using different voltages and electrolytes. Voltage Measurements. After this system validation, the electric field strengths were measured between adjacent electric
Table 1. Electric Field Values of Upstream and Downstream Regions under Different Flow Rates and Voltages Using 10 mM Tris-HCl, pH 8.0a applied voltage 200 V
300 V
400 V
flow rate (µL/min)
linear velocity (cm/s)
E up (V/cm)
E down (V/cm)
E up (V/cm)
E down (V/cm)
E up (V/cm)
E down (V/cm)
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.9 1.0 2.0
0.013 0.026 0.039 0.052 0.065 0.078 0.091 0.117 0.131 0.262
14 ( 1 34 ( 7 48 ( 4 61 ( 2 65 ( 2 63 ( 2 63 ( 2 63 ( 2 63 ( 2 63 ( 2
165 ( 1 124 ( 33 100 ( 9 74 ( 4 67 ( 2 67 ( 2 67 ( 2 67 ( 2 67 ( 2 67 ( 2
15 ( 3 34 ( 3 49 ( 5 69 ( 9 75 ( 8 97 ( 7 95 ( 2 95 ( 2 91 ( 2 91 ( 2
255 ( 5 222 ( 13 196 ( 11 164 ( 12 136 ( 4 104 ( 4 102 ( 2 100 ( 2 102 ( 2 102 ( 2
17 ( 1 32 ( 3 54 ( 10 67 ( 11 79 ( 10 95 ( 12 111 ( 11 130 ( 4 122 ( 5 122 ( 2
348 ( 1 320 ( 11 279 ( 19 257 ( 15 238 ( 17 207 ( 22 130 ( 20 137 ( 2 137 ( 4 135 ( 3
a Values between the first electric junction (anode gap) and the next junction 1-cm downstream (E up), as well as between the last electric junction (cathode gap) and the one situated 1-cm upstream (E down). The measurements were made 15-20 min after the application of the voltage. Italic data points are upstream voltages, where zones of different electric fields are created.
Table 2. Minimum Electrophoretic Mobilities µmin under Different Conditions of Flow Rate and Voltagea applied voltage 200 V
300 V
400 V
flow rate (µL/min)
linear velocity (cm/s)
µmin up (× 10-4 cm2 -1 V s-1)
µmin down (× 10-4 cm2 V-1 s-1)
µmin up (× 10-4 cm2 -1 V s-1)
µmin down (× 10-4 cm2 V-1 s-1)
µmin up (× 10-4 cm2 -1 V s-1)
µmin down (× 10-4 cm2 V-1 s-1)
0.1 0.2 0.3 0.4 0.5 0.6 0.7
0.013 0.026 0.039 0.052 0.065 0.078 0.091
10.0 7.6 8.1 nab na na na
0.8 2.1 3.9 na na na na
8.7 7.6 8.0 7.5 8.7 na na
0,5 1.2 2.0 3.2 4.8 na na
7.6 8.1 7.2 7.8 8.2 8.2 8.2
0.3 0.8 1.4 2.0 2.7 3.8 7.0
a Each value corresponds to the ratio between flow velocity and electric field, eq 2. b n/a, not applicable since zones of different electric field were not observed.
junctions under different flow rates. As seen in Table 1, differences in the voltage up to 20-fold were measured between the upstream and downstream regions. The maximum flow rate to produce zones of different electric field was found to be proportional to the voltage applied (0.3 µL/min for 200 V, 0.5 µL/min for 300 V, and 0.7 µL/min for 400 V). Below these flow rates, the electric field difference between upstream and downstream zones was found to be inversely proportional to the flow rate. Mechanism of Formation of Zones of Different Electric Field. An unexpected observation from the analysis of Table 1 is that once the zones of different electric field are built, the upstream electric field becomes independent of the applied voltage but dependent on the flow rate. At a given flow rate, the electric field measured at the upstream zonesbut not the downstream zones is similar for the three different voltages applied (200, 300, and 400 V). This suggests that once the zones with different electric field are created, the interaction between the flow rate and the ionic composition of the electrolyte rather than the voltage is responsible for the upstream electric field value. To explain this phenomenon, we have to consider the characteristics of conventional stacking and isotachophoresis. Ions with different electrophoretic mobilities have been utilized to concentrate and separate (isotachophoresis) charged molecules.
In electrokinetic stacking with discontinuous electrolyte systems, target molecules are stacked between a leading (low electric field strength) and a terminating (high electric field strength) zone. The leading ions create a zone of low electric field in front of the target molecules, resulting in a considerable slowdown of these molecules, concentrating them at the interface of the zones. At the same time, the terminating zone (the one located behind the molecules of interest) has a high electric field, pushing said molecules toward the leading ions, and in this manner stacking the target molecules between the two zones. In isotachophoresis, a steady-state stacking is produced by using a two-buffer system with a common cation and a different anion: a fast-mobility leading anion at the anode buffer and a low-mobility anion at the cathode buffer. The result is that all anions move in the electric field with the same velocity, thus they are arranged in discrete bands in the order of decreasing electrophoretic mobilities. The first indication that molecules were electrocaptured by similar mechanisms was the observation of the capture of two differently colored molecules (Figure 2). A solution of myoglobin (2 mg/mL) and Coomassie stain in 50 mM Tris-HCl was injected and captured at 300 V/cm. Myoglobin and Coomassie blue were then found to have been captured in a steady-state manner at separated spots, with the Coomassie band upstream from that of Analytical Chemistry, Vol. 79, No. 3, February 1, 2007
1059
Figure 2. Photograph of the downstream cathode junction during operation. Since Nafion membrane is transparent when hydrated, it is possible to observe the capture of colored compounds located at the junction. (A) A solution of myoglobin (2 mg/mL) and Coomassie stain in 50 mM Tris-HCl was injected (1 µL) and captured at 300 V and 0.3 µL/min. At this voltage and flow rate, a myoglobin band is visible. (B). When the voltage was decreased to 200 V, separate bands of Coomassie and myoglobin are visible. (C) When the voltage was decreased to 168 V, the myoglobin band was released, but not the Coomassie band.
myoglobin (Figure 2B). Since both ions move at the same velocity (the velocity of the flow stream), a theoretical frame work similar to isotachophoresis can be applied, related in particular to counterflow isotachophoresis.17 To immobilize target molecules in the myoglobin and Coomassie stain system, the electrophoretic velocity created by the electric field must counteract the velocity of the flow. Consequently, the electrophoretic velocity must be of equal or higher magnitude (and opposite direction) than the velocity of the flow stream. Ions with electrophoretic velocities lower than the velocity of the flow stream will be swept out from the system. The electrophoretic velocity of an ion in an electrolyte solution is given by
ve ) µE
(1)
where νe is the electrophoretic velocity (cm/s), µ is the electrophoretic mobility (cm2 V-1 s-1), and E is the electric field strength (V/cm). During capture, νe must be of equal or higher magnitude (and opposite direction) than the velocity of the flow stream (νh, cm/s). Therefore, eq 1 can be rearranged to
E ) vh/µ
(2)
when νe ) νh. Now, since all captured anions are moving at the same velocity, each individual ion will have a constant vh/µ ratio. In other words, it will be captured at an E given by its µ value (vh is constant for all the captured ions). This is in agreement with the observation that myoglobin was released at a higher voltage since it is expected to have much lower µsand therefore captured at a higher Esthan Coomassie stain (Figure 2C and eq 2). Two issues remain to analyze: how are the zones of different electric fields created in a device filled with a continuous electrolyte? And why does the electric field upstream become independent of the applied field when the zones are created? In the present electrocapture experiments, the anion with highest concentration in the electrolyte solution is chloride, and because of its high mobility and availability, chloride is commonly used as a leading ion in stacking and isotachophoresis. Since the fluidic channel is closed to anions, chloride ions tend to accumulate at (17) Abelev, G. I.; Karamova, E. R. Anal. Biochem. 1984, 142, 437-444.
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Figure 3. Experimental versus the theoretic values of the upstream electric field. Experimental values extracted from Table 1 and the theoretic values obtained by eq 3.
the upstream anode junction. Thus, it is likely that the chloride is the ion responsible for the generation of the low electric field observed at the upstream zone of the electrocapture device. In order to see whther chloride can be captured at the upstream junction, we can calculate the minimum electrophoretic mobility (µmin) that a molecule must have in order to comply with the conditions of capture at any electric field in the upstream and downstream regions. Thus, using eq 1 and under the condition that νe is at least equal to νh, µmin can be expressed as
µmin) vh/E
(3)
when νe ) νh. From the upstream and downstream µmin values (Table 2), it is clear that when the zones of different electric field strengths are created, chloride ions will be located at the upstream region, and indeed, the µmin at the upstream region, (8.1 ( 0.6) × 10-4 cm2 V-1 s-1, n ) 15, is similar to that of chloride, 7.91 × 10-4 cm2 V-1 s-1. Under these circumstances, the same theoretical model as that proposed for myoglobin and the Coomassie stain can be applied to the chloride ions. Therefore, the electric field strength at the position where the chloride ions are located can be calculated from eq 2, meaning that the chloride ions during capture will be under the influence of an electric field (ECl) given by
ECl ) νh/µCl
(4)
Table 3. Electric Field Values of Upstream and Downstream Regions under Different Flow Rates and Voltages Using 5 mM NH4Cl, pH 5.5a applied voltage 200 V
a
300 V
400 V
flow rate (µL/min)
linear velocity (cm/s)
E up (V/cm)
E down (V/cm)
E up (V/cm)
E down (V/cm)
E up (V/cm)
E down (V/cm)
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.9 1.0 2.0
0.013 0.026 0.039 0.052 0.065 0.078 0.091 0.117 0.13 0.26
17 ( 1 37 ( 4 50 ( 8 65 ( 4 65 ( 2 63 ( 2 67 ( 2 65 ( 2 65 ( 2 65 ( 2
161 ( 1 124 ( 7 96 ( 10 65 ( 3 65 ( 2 65 ( 2 65 ( 2 65 ( 2 65 ( 2 67 ( 2
20 ( 5 37 ( 5 51 ( 8 65 ( 5 85 ( 2 100 ( 4 100 ( 4 98 ( 4 98 ( 4 98 ( 4
255 ( 7 219 ( 12 190 ( 15 162 ( 12 130 ( 14 100 ( 4 100 ( 2 102 ( 2 102 ( 2 102 ( 2
18 ( 1 29 ( 6 54 ( 7 65 ( 5 75 ( 12 91 ( 7 117 ( 5 130 ( 7 133 ( 5 130 ( 5
355 ( 1 307 ( 2 283 ( 15 262 ( 13 256 ( 16 207 ( 15 177 ( 8 137 ( 4 136 ( 5 134 ( 5
Designations as in Table 1.
Table 4. Electric Field Values of Upstream and Downstream Regions under Different Flow Rates and Voltages Using 5 mM NH4HCOO, pH 5.5a applied voltage 200 V
a
300 V
400 V
flow rate (µL/min)
linear velocity (cm/s)
E up (V/cm)
E down (V/cm)
E up (V/cm)
E down (V/cm)
E up (V/cm)
E down (V/cm)
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.9 1.0 2.0
0.013 0.026 0.039 0.052 0.065 0.078 0.091 0.117 0.131 0.262
24 ( 1 45 ( 14 60 ( 5 65 ( 3 63 ( 2 64 ( 2 65 ( 2 65 ( 2 65 ( 2 65 ( 2
148 ( 2 105 ( 22 76 ( 8 65 ( 3 69 ( 2 67 ( 2 67 ( 2 67 ( 2 67 ( 2 67 ( 2
27 ( 5 50 ( 10 69 ( 11 85 ( 10 98 ( 9 95 ( 2 98 ( 2 98 ( 2 98 ( 2 98 ( 2
241 ( 10 198 ( 16 137 ( 20 126 ( 18 100 ( 8 102 ( 2 102 ( 2 100 ( 2 102 ( 2 102 ( 2
29 ( 2 47 ( 11 69 ( 9 98 ( 11 111 ( 15 132 ( 9 132 ( 2 132 ( 2 130 ( 2 126 ( 2
338 ( 4 292 ( 14 255 ( 15 205 ( 21 176 ( 28 135 ( 8 135 ( 7 130 ( 7 132 ( 7 137 ( 7
Designations as in Table 1.
where ECl is the electric field strength at the chloride ions and µCl is the electrophoretic mobility of the chloride ion. The equation is an approximation since chloride ions must be accompanied by counterions in order to maintain overall ionic electroneutrality. Figure 3 shows the theoretic ECl values plotted against the empirical data (ECl values are presented in Supporting Information, Table S-1). As soon as E upstream decreases (because of formation of a region of different electric fields), the measured upstream E is similar to the theoretical (ECl) value under a given flow rate, consistent with the hypothesis that chloride ions are responsible for the creation of the upstream zone of low electric field. According to this model, the use of different electrolytes containing chloride must give a similar E value at the upstream zone (like NH4Cl), while an electrolyte in which chloride is replaced by an anion with a lower electrophoretic mobility (like formate) must result in a higher E value at the upstream zone for a given vh (eq 2). To confirm this model, E values at the upstream and downstream positions were measured using NH4Cl and NH4HCOO solutions at different voltages and flow rates (Tables 3 and 4). The two electrolytes gave the same overall electric behavior as using Tris-HCl. First, when the zones of different electric field
Table 5. Equation of the Curve, Correlation Factor (r2) and Inverse Value of the Slope (1/m) of the Graph Relating the Upstream Voltage to the Flow Velocity of Tris-HCl, NH4Cl, and NH4HCOOa E (V/cm)
equation of the curve
r2
1/m (cm2 V-1 s-1)
200 300 400
Tris-HCl y ) 1307x - 3.3 y ) 1192x + 1.9 y ) 1189x + 3.1
0.99 0.98 0.99
7.7 × 10-4 8.4 × 10-4 8.4 × 10-4
200 300 400
NH4Cl y ) 1269x + 1.6 y ) 1215x + 4.2 y ) 1214x + 1
0.98 0.99 0.98
7.9 × 10-4 8.2 × 10-4 8.2 × 10-4
NH4HCOO 200 300 400 a
y ) 1484x + 9.5 y ) 1653x + 6.3
only two data points 0.99 0.99
6.7 × 10-4 6.0 × 10-4
The value of µ corresponds to 1/m. The graphs are given in Figure
S-1.
were observed, the upstream voltagesbut not the downstreams became independent of the voltage applied but dependent on the Analytical Chemistry, Vol. 79, No. 3, February 1, 2007
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Figure 4. Representation of the stacking mechanism of the electrocapture device. The scheme shows a section of the device under operation. To simplify the figure, membranes are not shown.
flow rate. Second, the electric field difference between upstream and downstream zones was found to be inversely proportional to the flow rate. In accordance with the model proposed, the electrolyte containing ammonium chloride gave local electric field values similar to those containing Tris-HCl. Upstream electric fields utilizing NH4HCOO were higher than those with Tris-HCl and NH4Cl, which is in agreement with eq 2. According to the equation, the electrophoretic mobility (in cm2 V-1 s-1) of the ions located at the upstream position can be obtained by calculation of the slope of the graph of the upstream voltage (in V/cm) versus flow velocity (in cm/s). The µ value corresponds to the inverse value of the slope (1/m), since in this case, the velocity is the independent variable and the voltage the dependent one. The 1/m values of the NH4HCOO buffer were found to be considerably lower than those of the Tris-HCl, NH4Cl, and µCl value (Table 5 and Supporting Information Figure S-1). In agreement with eq 3, the 1/m values of Tris-HCl and NH4Cl were similar to the µCl (Table 5 and Supporting Information in Figure S-1). Stacking. To understand the consequences of the generation of different electric field zones along the channel, the minimum theoretic value of µ (Table 2) that a molecule must have in order to comply with the conditions of capture at the upstream and downstream zones can be analyzed. For a flow rate of 0.2 µL/ min at 400 V, the electric field upstream is 32 V/cm, which shows that only molecules with µ higher than 8.1 × 10-4 cm2 V-1 s-1 will produce an electrophoretic velocity sufficiently high to counteract the velocity of the flow. Thus, at the upstream region, molecules with µ values lower than 8.1 × 10-4 cm2 V-1 s-1 will be swept downstream of a zone with higher electric field. Under the same flow rate and voltage, the electric field downstream is 320 V/cm, which gives a µmin of 0.8 × 10-4 cm2 V-1 s-1. Therefore, in this region, molecules with µ values higher than 0.8 × 10-4 cm2 V-1 s-1 will move upstream, and molecules with lower µ values will be swept out from the system. Considering the µmin values upstream and downstream, we can conclude that molecules with µ values lower than 8.1 × 10-4 cm2 V-1 s-1 and higher than 0.8 × 10-4 cm2 V-1 s-1 will be captured or “trapped” between these zones 1062
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(Table 2), because their electrophoretic velocities in the downstream zone are sufficiently high to move them upstream but insufficiently high to make them enter into the upstream zone. The same conclusion can be made for any pair of µmin values at the same voltage and flow rate, as shown by a diagram of the capture mechanism (Figure 4). Although the range of µmin values does not appear to be broad, the larger diameter of the downstream cathode gap (the very last portion of the electrocapture device) facilitates the capture of molecules of lower electrophoretic mobilities. The downstream electric junction consists of a 0.3-cm-long Nafion tubing with an internal diameter of 635 µm (versus 125 µm of the PEEK tubing), which will produce an ∼16-fold decrease in the velocity of the flow stream (Figure 2). From eq 2, we can see that the hydrodynamic trap, produces a 16-fold decrease of µmin downstream, thus increasing the range of molecules that can be captured. Since DNA has µ values between 2 × 10-4 and 4 × 10-4 cm2 -1 V s-1,18,19 and proteins and peptides have µ values not higher than 2 × 10-4 cm2 V-1 s-1 (usually in the order of 1 × 10-4 cm2 V-1 s-1 or lower),20-23 the experimental data for E values in the upstream and downstream regions provide strong support that these molecules are “stacked” or immobilized between the zones. For example, literature searches and experimental21 µ values for 51 polypeptides over a molecular weight range of 178-140.000 show no protein with a µ value high enough to locate it at the upstream region. In contrast, all µ values are sufficiently high to localize the biomolecules at the downstream region. Only small (18) Stellwagen, N. C.; Gelfi, C.; Righetti, P. G. Biopolymers 1997, 42, 687-703. (19) Nkodo, A. E.; Garnier, J. M.; Tinland, B.; Ren, H.; Desruisseaux, C.; McCormick, L. C.; Drouin, G.; Slater, G. W. Electrophoresis 2001, 22, 24242432. (20) Rickard, E. C.; Strohl, M. M.; Nielsen, R. G. Anal. Biochem. 1991, 197, 197-207. (21) Basak, S. K.; Ladisch, M. R. Anal. Biochem. 1995, 226, 51-58. (22) Adamson, N. J.; Reynolds, E. C. J. Chromatogr., B: Biomed. Sci. Appl. 1997, 699, 133-147. (23) Janini, G. M.; Metral, C. J.; Issaq, H. J. J. Chromatogr., A 2001, 924, 291306.
ions such as bromide, chloride, or iodide with µ values of ∼7.9 × 10-4 cm2 V-1 s-1 would be captured at the upstream region. CONCLUSION We give experimental evidence that the electrocapture device generates stable zones of different electric field in a microflow stream. The electric field upstream is low enough to allow capture only of ions with high electrophoretic mobility, such as chloride ions; the downstream region has a significantly higher electric field allowing molecules with low electrophoretic mobility, DNA, proteins, and peptides, to remain in the system. A similar phenomenon can be observed by modification of the shape of the channel24 (electric field gradient focusing) or by use of multipleelectrode arrangements25,26 (dynamic isoelectric focusing). The electrocapture device therefore constitutes a system that utilizes stacking to immobilize charged molecules in a flow stream, (24) Humble, P. H.; Kelly, R. T.; Woolley, A. T.; Tolley, H. D.; Lee, M. L. Anal. Chem. 2004, 76, 5641-5648. (25) Montgomery, R.; Jia, X.; Tolley, L. Anal. Chem. 2006, 78, 6511-6518. (26) Huang, H.; Ivory, C. F. Anal. Chem. 1999, 71, 1628-1632. (27) Khandurina, J.; Jacobson, S. C.; Waters, L. C.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1999, 71, 1815-1819. (28) Foote, R. S.; Khandurina, J.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 2005, 77, 57-63. (29) Wu, X.-Z.; Hosaka, A.; Hobo, T. Anal. Chem. 1998, 70, 2081-2084. (30) Wang, Q.; Yue, B.; Lee, M. L. J. Chromatogr., A 2004, 1025, 139-146.
which can be utilized to perform multiple analytical tasks. A significant advantage is that the molecules are immobilized at the interface between zones of different electric fields rather than onto the surface of porous membranes,27-30 making it possible to work with small biological molecules (peptides). The “contactless” nature of the immobilization process decreases the risk of crosscontamination between samples. It simplifies the release of the captured molecules such that the method can be used as a separation tool by a voltage gradient elution of the captured molecules.16 We visualize that this mode of stacking will make possible further designs and applications of microfluidic systems. ACKNOWLEDGMENT This work was supported by grants from the Swedish Research Council, the Swedish Cancer Society, Vinnova, the European Commission (LSHC-CT-2003-503297) and Karolinska Institutet. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text (Table S-1 and Figure S-1). This material is available free of charge via the Internet at http://pubs.acs.org. Received for review September 8, 2006. Accepted October 20, 2006. AC061699F
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