Contactless Conductivity Detector for Microchip Capillary

Anal. Chem. 2002, 74, 1968-1971

Contactless Conductivity Detector for Microchip Capillary Electrophoresis Martin Pumera,† Joseph Wang,*,† Frantisˇek Opekar,‡ Ivan Jelı´nek,‡ Jason Feldman,§ Holger Lo 1 we,| and Steffen Hardt|

Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003, Department of Analytical Chemistry, Charles University, 12043 Prague 2, Czech Republic, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California 91109, and Institut fu¨r Mikrotechnik Mainz GmbH, Carl-Zeiss Strasse, 18-20, D-55129 Mainz, Germany

A microfabricated electrophoresis chip with an integrated contactless conductivity detection system is described. The new contactless conductivity microchip detector is based on placing two planar sensing aluminum film electrodes on the outer side of a poly(methyl methacrylate) (PMMA) microchip (without contacting the solution) and measuring the impedance of the solution in the separation channel. The contactless route obviates problems (e.g., fouling, unwanted reactions) associated with the electrodesolution contact, offers isolation of the detection system from high separation fields, does not compromise the separation efficiency, and greatly simplifies the detector fabrication. Relevant experimental variables, such as the frequency and amplitude of the applied ac voltage or the separation voltage, were examined and optimized. The detector performance was illustrated by the separation of potassium, sodium, barium, and lithium cations and the chloride, sulfate, fluoride, acetate, and phosphate anions. The response was linear (over the 20 µM-7 mM range) and reproducible (RSD ) 3.4-4.9%; n ) 10), with detection limits of 2.8 and 6.4 µM (for potassium and chloride, respectively). The advantages associated with the contactless conductivity detection, along with the low cost of the integrated PMMA chip/detection system, should enhance the power and scope of microfluidic analytical devices. Microfabricated analytical systems, known also as “lab-on-achip” devices, can dramatically change the way (bio)chemical assays are performed.1,2 Particularly attractive are micromachined capillary electrophoresis (CE) chips because of their fast and efficient separation capabilities.3,4 As the field of chip-based separation microsystems continues its rapid growth, there are * Corresponding author. E-mail: [email protected]. † New Mexico State University. ‡ Charles University. § California Institute of Technology. | Institut fu ¨ r Mikrotechnik Mainz GmbH. (1) Figeys, D.; Pinto, D. Anal. Chem. 2000, 71, 330A. (2) Kutter, J. P. Trends Anal. Chem. 2000, 19, 352. (3) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C.; Manz, A. Science 1993, 261, 895. (4) Hadd, A.; Raymond, D.; Halliwell, J.; Jacobson, S.; Ramsey, M. Anal. Chem. 1997, 69, 3407.

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urgent needs for developing compatible detection modes. While laser-induced fluorescence (LIF) has dominated the detection for microfluidic devices, the past five years have witnessed the development of alternative and effective detection methods.5 These new on-chip detection methods include optical modes such as absorbance, chemiluminescence, or refraction and the electrochemical methods of amperometry, potentiometry, and conductometry. This report focuses on the development of a contactless conductivity detector for a micromachined CE microchip system. Conductivity detection is a simple and universal detection technique that has seen a renaissance in conventional CE systems.6 Such detection involves measurement of the conductance between two electrodes (through which an alternating current is passed) and allows convenient detection of ionic species (not readily detected by other techniques) down to the submicromolar level. Conductivity detection can be accomplished either by a direct galvanic contact of the run buffer and the sensing electrodes (using on-column or end-column configurations) or by a contactless mode in which the electrodes do not contact the solution. The contactless detection route has several advantages over the contact mode, including the absence of problems (e.g., passivation, bubble formation) associated with the electrode-solution contact, effective isolation from high separation voltages, a simplified construction and alignment of the detector (including placement of the detector or multiple ones at different locations), and the use of narrow capillaries/microchannels.7 Contactless conductivity detectors for conventional CE systems are based on a two-electrode detection cell with tubular or semitubular electrodes placed over the separation capillary.7-10 In contrast, conductivity detection for CE microchips usually relies on a galvanic contact of the run buffer and the electrode.11-13 Guijt (5) Schwartz, M. A.; Hauser, P. Lab Chip 2001, 1, 1. (6) Zemann, A. J. Trends Anal. Chem. 2001, 20, 346. (7) Mayrhofer, K.; Zemann, A. J.; Schnell, E.; Bonn, G. K. Anal. Chem. 1999, 71, 3828. (8) Zemann, A. J.; Schnell, E.; Volger, D.; Bonn, G. K. Anal. Chem. 1998, 70, 563. (9) Fracassi da Silva, J. A.; do Lago, C. L. Anal. Chem. 1998, 70, 4339. (10) Tu˚ma, P.; Opekar, F.; Jelı´nek, I. Electroanalysis 2001, 13, 989. (11) Prest, J. E.; Baldock, S. J.; Bektas, N.; Fielden, P. R.; Treves Brown, B. J. J. Chromatogr., A 1999, 838, 59. (12) Grass, B.; Neyer, A.; Johnck, M.; Siepe, D.; Eisenbeiss, F.; Weber, G.; Hergenroder, R. Sens. Actuators, B 2001, 72, 249. 10.1021/ac011219e CCC: $22.00

© 2002 American Chemical Society Published on Web 03/22/2002

Figure 1. Microchip electrophoretic system with contactless conductivity detection. (A) Key: (a) PMMA microfluidic device, (b) separation channel, (c) run buffer reservoir, (d) unused reservoir, (e) sample reservoir, (f) outlet reservoir, and (g) aluminum sensing electrodes. (B) Enlarged view of the detector; dimensions: (a) 800, (b) 700, and (c) 570 µm. (C) Scheme of electronic circuit: (a) function generator, (b) sensing electrodes, (c) electronics (based on ref 9), (d) filter (R ) 10 kΩ, C ) 1 µF), (e) voltage follower, and (f) output to a data acquisition system.

et al.14 proposed recently to minimize problems associated with such direct contact through coverage of in-channel sensing electrodes with a dielectric (silicon-carbide) layer. However, such coating with an insulating layer is limited to low separation fields (and hence to long analysis times) and requires a cumbersome design and fabrication effort, along with a widened channel. The present contactless conductivity detector for microfabricated CE chips is based on placing two external metallic film electrodes on the cover of a plastic (poly(methyl methacrylate), PMMA) microchip to act as planar capacitor. Such coupling of low-cost PMMA separation chips and easily constructed contactless detectors holds great promise for creating effective and disposable CEconductivity microchips. The attractive performance characteristics of such contactless conductivity microchip detector are reported in the following sections. EXPERIMENTAL SECTION Apparatus. The plastic microchip, shown in Figure 1A, consisted of two sealed PMMA plates (70 × 24 mm) with a 60mm-long separation channel (between the run buffer reservoir and the outlet reservoir) and an 18-mm-long injection channel (between the sample reservoir and the unused reservoir). The two channels crossed each other halfway between the sample and the unused reservoir and 9 mm from the run buffer reservoir. The channels had a 50 µm × 50 µm squared cross section. The PMMA microchips were manufactured at the Institut fu¨r Mikrotechnik Mainz (IMM, Mainz, Germany) and were described earlier.15 The homemade high-voltage power supply had an adjustable voltage range between 0 and +5000 V. A Plexiglas holder was (13) Prest, J. E.; Baldock, S. J.; Fielden, P. R.; Treves Brown, B. J. Analyst 2001, 126, 433. (14) Guijt, R. M.; Baltussen, E.; van der Steen, G.; Frank, H.; Billiet, H.; Schalkhammer, T.; Laugere, F.; Vellekoop, M.; Berthold, A.; Sarro, L.; van Dedem, G. W. K. Electrophoresis 2001, 22, 2537. (15) Wang, J.; Pumera, M.; Chatrathi, M. P.; Escarpa, A.; Konrad, R.; Griebel, A.; Do ¨rner, W.; Lo ¨we, H. Electrophoresis 2002, 23, 596.

fabricated for accommodating the separation chip. Short pipet tips were inserted into each of the four holes on the PMMA chip for providing solution contact between the channel on the chip and corresponding reservoir on the chip holder. The electronic circuitry of the conductivity detector was placed on the top of the chip. Electrode Fabrication. The rectangular-shaped electrodes (0.8 mm × 10 mm) were fabricated from two 10 µm-thick aluminum foil strips. The end side of the electrode was widened to 4 mm to facilitate the electrical connection. These electrodes were fixed to the top of the 125-µm-thick PMMA cover plate of the microchip using a common epoxy, at a distance of 570 µm from the end of microchannel and with a distance of 700 µm between them (see Figure 1B). The thin copper wires were attached to the electrodes using a conducing epoxy (Chemtronics, Kennesaw, GA) and were tin-soldered to the detector electronics. The length of these wires was minimized to prevent induction of electric noise. The electrodes were placed in an “antiparallel” orientation to minimize the stray capacitance between them. Note that such fabrication process is extremely simple and that the cost of the electrodes is negligible. Electronic Circuit. The electronic circuit of the contactless detector was designed in accordance with a previously reported scheme.9 The circuit was completed by adding a passive RC filter (time constant, 0.01 s) followed by a voltage follower (LF 356) to the circuit output (the third amplifier in ref 9) (Figure 1C). This modification allows convenient interface to data acquisition systems (chart recorder or a computer DAQ). The electronic components were purchased from local suppliers. A HP 8116A function generator (Hewlett-Packard, Palo Alto, CA) was used for generating the sinusoidal signal (usually with a frequency of 200 kHz with peak-to-peak amplitude of 5 V). The electronic circuit was placed in a shielding box to protect the electronics from external electric fields. One side of the box was open; this side was placed as close as possible to the sensing electrodes (on the chip), so that the box also acts as a shield for the electrodes. To further minimize the noise, the chip (along with the printed electronic board) was secured from possible mechanical vibrations. Reagents. Histidine (His), 2-(N-morpholino)ethanesulfonic acid (MES), potassium nitrate, barium nitrate, lithium nitrate, sodium phosphate monobasic, sodium sulfate, sodium fluoride, and sodium chloride were purchased from Sigma. Sodium nitrate and sodium acetate were obtained from Aldrich. The run buffer (20 mM, pH 6.1) was prepared by dissolving MES and His in deionized water. Stock solutions of the target cations (potassium, sodium, lithium, and barium, at 10 mM) and anions (acetate, phosphate, sulfate, chloride, and fluoride; at 10 mM) were prepared by dissolving the corresponding nitrate salts (for cations) and sodium salts (for anions) in the run buffer. All chemicals were used without any further purification. Electrophoresis Procedure. The channels of the plastic chip were treated before use by rinsing with deionized water for 10 min. Reservoirs c, d, and f (Figure 1A) were filled with the electrophoretic run buffer solution, while reservoir e was filled with the sample mixture (target ions dissolved in the run buffer). After an initial loading of the sample in the injection channel, the sample was injected by applying potentials of +500 (cations) or Analytical Chemistry, Vol. 74, No. 9, May 1, 2002

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Figure 2. Electrophoregrams showing the separation of cations (A) and anions (B) using the contactless conductivity detection. (A) Mixture containing 450 µM potassium (a), 800 µM barium (b), 650 µM sodium (c), and 550 µM lithium (d). (B) Mixture of chloride (a), sulfate (b), fluoride (c), acetate (d), and phosphate (e), at 800 (a-c) and 1600 µM (d, e). Conditions: (A) separation voltage, +1500 V; injection voltage, +500 V; frequency, 200 kHz; peak-to-peak amplitude, 5 V; run buffer, MES/His buffer (20 mM, pH 6.1). (B) Separation voltage, -1500 V; injection for 3 s at -500 V; other conditions, as in (A).

-500 V (anions) for 3 s between the sample reservoir e and the grounded outlet reservoir f. This drove the sample “plug” into the separation channel through the intersection. The analytical separation was proceeded by switching the high-voltage contacts; the separation potential was subsequently applied to the run buffer reservoir c with the outlet reservoir f grounded and all other reservoirs floating. Safety Considerations. The high-voltage power supply should be handled with extreme care to avoid electrical shock. Sodium fluoride is toxic and other used chemicals are irritants, and skin and eye contact and accidental inhalation or ingestion should be avoided. RESULTS AND DISCUSSION The new CE microchip detection couples the distinct advantages of contactless detection schemes with an attractive analytical performance. Such performance characteristics compare favorably with those common to chip-based direct-contact conductivity detectors.11-13 For example, Figure 2 displays typical electropherograms recorded with the contactless CE-microchip conductivity detector for model mixtures of inorganic cations [A: potassium (a), barium (b), sodium (c), and lithium (d)] and anions [B: chloride (a), sulfate (b), fluoride (c), acetate (d), and phosphate (e)]. Well-defined and resolved peaks are observed for all four cations and five anions. The half-peak width ranges from 0.8 (potassium) to 4.1 s (phosphate). Such a defined response, coupled to the low noise level, offers convenient quantification of these submillimolar and millimolar levels of cations and anions within short periods, 40 and 90 s, respectively. (Faster assays of lower concentrations are described below.) The anion separation was carried out in the anodic mode, using the same run buffer, without adding an electroosmotic flow reservoir (due to the favorably low electroosmotic flow of the PMMA microfluidic device; µEOF ) 1.96 × 10-4 cm2/V‚s, in our case). This may facilitate the switching between analyses of cations and anions 1970 Analytical Chemistry, Vol. 74, No. 9, May 1, 2002

Figure 3. Influence of the separation voltage upon the response for 800 µM potassium (a), sodium (b) and lithium (c). Separation voltage, +1000 (A), +2000 (B); +3000 (C), +4000 (D), and +5000 (E) V. Also shown (as inset), the influence of separation voltage upon the resulting migration time. Other conditions, as in Figure 2A.

using the same microchannel (to allow rapid monitoring of both groups). The absence of an EOF modifier also leads to a lower background conductivity. Such anodic operation results in a slight initial baseline slope that reflects the proximity of the electrodes to the outlet reservoir (onto which the separation potential was applied). In contrast, a highly flat baseline is observed for the cations, where the high separation potential is applied to the runbuffer reservoir. Various experimental parameters affecting the response were examined and optimized. Figure 3 shows the influence of the separation voltage upon the detector output. As expected, increasing the separation voltage from +500 to +5000 V dramatically decreases the migration time for potassium, sodium, and lithium from 71.5 to 5.6 s, from 98.5 to 7.5 s, and from 118.5 to 8.8 s, respectively. Also shown are the resulting electrophoregrams for separation voltages of +1000 (A), +2000 (B), +3000 (C), +4000 (D), and +5000 (E) V. The shorter migration times observed at higher fields are coupled to substantially sharper peaks. The plate number for the three cations thus increases from 2237 to 2722 (potassium), from 2741 to 3202 (sodium), and from 4058 to 5226 (lithium) V over +500 and +5000 V range. Such values reflect the high separation efficiency and compare favorably with those reported for other conductivity detectors for capillary column and microchip systems (e.g., plate number per meter (N/m) for sodium of 46 460-54 210 here versus 33 300-70 0007 and 645015 80016). Obviously, placement of the detector on the cover of the chip ensures detection of the target ionic analytes directly in the separation channel, hence circumventing detector zone broadening and related loss of separation efficiency. The separation voltage has a negligible effect upon the peak-to-peak background noise level for voltages ranging from +500 to +3000 V. Separation voltages higher than +3000 V result in higher background and noise levels (attributed to Joule heating effects). Yet, a flat baseline is observed even at +5000 V (E), reflecting the effective isolation of the detector. Note that this voltage allows the separation of the three cations within 10 s. As expected, the response of the contactless conductivity detector is strongly dependent upon the frequency and amplitude (16) Guijt, R. M.; Baltussen, E.; van der Steen, G.; Schasfoort, R. B.; Schlautmann, S.; Billiet, H. A.; Frank, J.; van Dedem, G. W.; van den Berg, V. Electrophoresis 2001, 22, 235.

Figure 4. Influence of the applied ac voltage frequency upon the detector output. Sample contained 800 µM potassium (a), sodium (b), and lithium (c). Also shown (as inset), the corresponding electrophoregrams at operation frequencies of 200 (A) and 600 kHz (B). Other conditions, as in Figure 2A.

of the applied voltage. The influence of the applied ac voltage frequency is shown in Figure 4. The curves were recorded pointwise over the 25-1000 kHz range for a sample containing potassium (a), sodium (b), and lithium (c) ions. All three cations exhibit a similar trend. The response increases slowly between 25 and 350-400 kHz, then more rapidly (reaching the maximum at 600 kHz), and decreases sharply thereafter. Yet, an unstable response and distorted peaks were observed over the 400-800 kHz range (note the larger error bars). A negative signal was also observed in front of each cation peak at frequencies higher than 350 (potassium), 250 (sodium), and 200 kHz (lithium). For example, the inset of Figure 4 displays the corresponding electrophoregrams at frequencies 200 (A) and 600 kHz (B). It is clear that low frequencies lead to more favorable signal-to-noise characteristics and to a well-defined and stable response. The irreproducible and distorted response observed over the 350800 kHz range makes this frequency region not useful for reliable detection work. The exact reason for the peak distortion and fluctuations observed at high frequencies is not fully understood in view of the complex nature of the registered high-frequency impedance signal. It can be attributed to the complicated equivalence circuit of the detection cell.9 Some peak distortion and fluctuations were reported for other contactless-conductivity CE detection systems.7,17 All subsequent work employed a frequency of 200 kHz that offered the most favorable response characteristics. The response for potassium, sodium, and lithium increased linearly with the peak-to-peak amplitude of the ac voltage (Vp-p) over the entire range (from 0 to 15 Vp-p; not shown). Such amplitude change resulted also in a nearly linear increase of the noise level and in a reduced baseline stability. The most favorable signal-to-backround characteristics were obtained at a voltage of 5 Vp-p. (17) Chvojka, T.; Jelinek, I.; Opekar, F.; Stulik, K. Anal. Chim. Acta 2001, 433, 13.

The contactless conductivity microchip detector displays a welldefined concentration dependence for both cationic and anionic species. A linear range of almost 3 orders of magnitude (from 20 µM to 7 mM; correlation coefficients, >0.999) was obtained for lithium and fluoride. Limits of detection of 2.8, 5.8, 5.1, 6.4, and 8.1 µM were estimated for potassium, sodium, sulfate, chloride, and fluoride, respectively [based on the signal-to-noise characteristics (S/N ) 3) of the response of a mixture containing 20 µM ions; conditions, as in Figure 2]. Further lowering of the detection limits, to 1.2 and 2.0 µM for potassium and sodium, respectively, was observed upon lowering the buffer concentration from 20 to 10 mM. The high sensitivity and speed of the microchip contactless conductivity detection system is coupled to very good reproducibility and stability (associated with the absence of unwanted reactions). For example, series of 10 repetitive injections of a mixture containing 200 µM potassium, sodium, and lithium ions yielded standard deviations of 3.4%, 4.2%, and 4.9%, respectively. In conclusion, the results presented here clearly demonstrate that the combination of a contactless conductivity detection with a microchip CE system results in a versatile analytical device. As a result of such coupling, separation microchips can greatly benefit from the distinct advantages of the contactless detection, including the elimination of surface fouling or bubble formation, effective isolation from high separation voltages, a simplified construction and alignment of the detector, and the use of narrow microchannels (without compromising the separation efficiency). The contactless microchip avenue allows convenient placement of the detector (or multiple ones) at any location along the separation microchannel, as desired for optimizing the separation length or studying kinetic phenomena. Such coupling of low-cost PMMA separation chips and easily constructed contactless conductivity detectors holds great promise for creating single-use microfluidic analytical devices (that eliminate problems such as carry-over or cross contamination). Current efforts are aimed at expanding the contactless CE-conductivity microchip to different chip materials and toward the separation and detection of organic ions (e.g., fatty acids) and ionic explosives. Such integrated CE-conductivity microsystems are expected to find a wide range of applications. ACKNOWLEDGMENT This research was supported by grants from NASA Jet Propulsion Lab (Award AS02-0042) and Office of Naval Research (Award N00014-02-1-0213).

Received for review November 27, 2001. Accepted February 6, 2002. AC011219E

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