Anal. Chem. 2000, 72, 2875-2878
Integrated Potentiometric Detector for Use in Chip-Based Flow Cells Ratna Tantra and Andreas Manz*
Zeneca/Smithkline Beecham Centre for Analytical Science, Department of Chemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AY, U.K.
A new kind of potentiometric chip sensor for ion-selective electrodes (ISE) based on a solvent polymeric membrane is described. The chip sensor is designed to trap the organic cocktail inside the chip and to permit sample solution to flow past the membrane. The design allows the sensor to overcome technical problems of ruggedness and would therefore be ideal for industrial processes. The sensor performance for a Ba2+-ISE membrane based on a Vogtle ionophore showed electrochemical behavior similar to that observed in conventional electrodes and microelectrode arrangements. Ion-selective electrodes (ISEs) work in a potentiometric manner. The most common potentiometric cell arrangement includes two reference electrodes in their respective solutions separated by an ion-selective membrane. This membrane often contains an appropriate lipophilic ionophore, which is dissolved in an organic phase. The membrane is responsive to a particular ion of interest and the membrane potential (mV) is proportional to the logarithm of the ion’s concentration in the sample.1 In the 1970s, measurements of ionic concentration were often made by ISE microelectrodes.2 These microelectrodes are based on a glass micropipet construction, which requires a special pipet drawing instrument. Past studies3,4 have shown the feasibility of these electrodes in the detection of inorganic anions and cations, but the ruggedness of these detection systems is still not satisfying. The glass electrode is thin and fragile. Therefore, it cannot withstand exposure to movements, mechanical shocks, moisture, dust, and temperature changes which arise when such electrodes are employed in industrial process instrumentation.5 As work with these electrodes proceeded, it was observed that leaching out of membrane components during sensor operation could be a major problem. To solve this problem, it was found that more durable supports, e.g., poly(vinyl chloride) (PVC), can be added to the organic cocktail mixture. Hence, the emergence of a new type of ISE electrode, i.e., the coated wire electrode, which is more rigid than the conventional microelectrode.6 In this case, the membrane has been coated directly onto a metal wire. (1) Prantis, D. M.; Telting, D.; Meyerhoff, M. E. Crit Rev. Anal. Chem. 1992, 23 (3), 163-186. (2) Simon, W.; Morf, W. E.; Ammann, C. In Calcium Binding Proteins and Calcium Function; Wassermann, R. H., Ed.; Elsevier North-Holland: New York, 1977; p 50. (3) Kruse-Jarres, J. D. Med. Prog. Technol. 1988, 13, 107-130. (4) Oesch, U.; Ammann, D.; Simon, W. Clin. Chem. 1986, 32, 1448-1459. (5) Buhrer, T.; Gehrig, P.; Simon, W. Anal. Sci. 1988, 4, 547-557. 10.1021/ac000036+ CCC: $19.00 Published on Web 05/31/2000
© 2000 American Chemical Society
These electrodes impart a longer lifetime and a higher mechanical stability. However, the internal filling solution of the conventional design is missing from this type of electrode, as the internal reference wire contacts the ISE membrane directly. When measurements are made with such coated electrodes, a certain amount of drift in the electrode potential from one set of analysis to the next can be expected, as conditions at the membrane/wire interface change. Therefore, the success of such electrodes often requires frequent calibration. Besides coated electrodes, rigidity can also be achieved by means of silicon chip technology. An ISE design, which places an ion-selective membrane in contact with a gate region of a solidstate field effect transistor, is often known as a CHEMFET.7,8 These devices operate on the principle that the outer membrane phase-boundary potential affects the voltage experienced by the FET gate. Although these devices can be claimed of being very small, low-noise, self-contained rugged probes, drawbacks do exist. Most prominent among these is the same problem mentioned in the context of the coated wire ISE, because there may be no welldefined potential at the gate/membrane interface. Therefore, measurements are susceptible to drift caused by various species diffusing through the membrane from the sample. More recently, Uhlig and co-workers9,10 proposed a sensor that involves depositing the ISE membrane into a pyramidal-shaped metallized opening. Such an opening was formed by etching the backside of [100] orientated silicon. For measurement, the analyte was brought in contact with the front side of the chip. The main advantage for this type of sensor is that the mechanical stability of the sensor is considerably improved, as the membrane is protected against damage by the containment. One of the goals in the biomedical field is to be able to measure instantly and at the bedside, thus avoiding time-consuming centralized laboratory analysis.11 For this purpose, a chip-based potentiometric detector would be particularly useful. The resulting system could be denoted as a miniaturized total analysis system (µTAS). Accordingly, a principle object of this paper is to (6) Janata, J.; Huber, R. J. Ion-Selective Electrodes in Analytical Chemistry; Fresiser H., Ed.; Plenum Press: New York, 1980; Vol. 2, 107-174. (7) Heliey, R. G.; Owen, A. E. J. Chem. Soc., Faraday Trans. 1 1986, 82, 1195. (8) Covington, A. H.; Whalley, P. D. J. Chem. Soc., Faraday Trans. 1 1986, 82, 1209. (9) Uhlig, A.; Lindner, E.; Teutloff, C.; Schnakenberg, U.; Hintsche, R. Anal. Chem. 1997, 69, 4032-4038. (10) Schnakenberg, U.; Lisec, T.; Hintsche, R.; Kuna, I.; Uhlig, A.; Wagner, B. Sens. Actuators B 1996, 34, 476-480. (11) Arquint, P.; Koudelka-Hep, M.; van der Schoot, B. H.; van der Wal, P.; de Rooij, N. F. Clin. Chem. 1994, 40 (9), 1805-1809.
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demonstrate a novel ISE construction that employs chip technology. The rationale behind the development of µTAS lies in the number of advantages associated with miniaturization.12 By incorporating the sensors into a total chemical analysis system based on the use of the continuous injection analysis technique, a continuous monitoring of electrolyte is possible. In a recent paper,13 a similar system was described in which a flow-through channel was integrated on the chip to allow the use of a continuous-flow injection technique. Over the last several years, since the introduction of the microchip, a number of classical electrophoretic separation strategies have been adapted in such devices.14 The ability to combine and connect micromachined channels with virtually no dead volume or other band-broadening influences allows the integration of sample manipulations and separations that are not as easily or efficiently realized with capillary systems.15,16 Furthermore, in the field of inorganic analysis, it is possible to couple a potentiometric detection technique with a capillary electrophoretic separation system.17 The possibility of integrating the separation system with on-chip potentiometric detection will produce a very powerful instrument for inorganic ion analysis. EXPERIMENTAL SECTION Chip Dimension and Design. Figure 1 shows a layout of the micromachined glass ISE chip. A schematic diagram of the structure (Figure 1a) shows that the chip (12 mm × 6 mm) is described to comprise two distinct channels: (a) a sample channel, 10 mm in length (200 µm in width, 10 µm depth), holding the flowing sample solution; (b) a U-channel, 12 mm in length (200 µm in width, 10 µm depth), entrapping the ISE membrane, after its deposition into the chip. The channel possesses a junction structure (point D, in Figure 1a) (500 µm length, 20 µm in width, 10 µm depth) which allows the ISE membrane to contact the sample passing through the sample channel passage. As shown in the diagram, this U-channel can be filled completely with the organic membrane cocktail. Fabrication of the Sensor. The glass-glass ISE chips were fabricated using standard photolithography, etching, and thermalbonding techniques.18 Prior to thermal bonding with a glass plate, hole reservoirs of 1 mm in diameter were drilled (as represented by the circles in Figure 1a) in order to provide inlet and outlet openings into the chip. The fabrication process had been described elsewhere.19 To assemble the ISE chip, the organic cocktail must be trapped in the U-channel, while keeping the sample channel free from the organic material. The deposition technique for trapping the membrane material inside the U-channel will now be discussed (12) Manz, A.; Harrison, D. J.; Verpoorte, E.; Widmer, H. M. Adv. Chromatogr. 1993, 33, 1. (13) Bessoth, F. G.; deMello, A. J.; Manz, A. Anal. Commun. 1999, 36, 213215. (14) Harrison, D. J.; Manz, A.; Fan, Z.; Luidi, H.; Widmer, H. M. Anal. Chem. 1993, 65, 1481. (15) Ramsey, J. M.; Jacobson, S. C.; Ramsey, J. M. J. Microcoloumn Sep. 1998, 10(4), 313-319. (16) Crabtree, H. J.; Kopp, M. U.; Manz, A. Anal. Chem. 1999, 71 (11), 21302138. (17) Kappes, T.; Hauser, P. C. Anal. Commun. 1998, 35, 325-329. (18) Qin, D.; Xia, Y.; Rogers, J. A.; Jackman, R. J.; Zhao, X-M.; Whitesides, G. M. Top. Curr. Chem. 1998, 194, 1-20. (19) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897.
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Figure 1. Micromachined ISE chip. (a) Schematic drawing of a sensor chip design with channels and reservoirs: point A, inlet for U-channel; point B, outlet for U-channel; point C, inlet for sample channel; point D, junction structure, where membrane contacts sample solution; and point E, outlet for sample channel. The diagram illustrates the complete filling of the silanized U-channel with organic membrane cocktail. Circles denote drilled holes. The diagram also illustrates the measuring system. (b) Scaled diagram of the 12 mm × 6 mm chip.
in detail. The junction channel is so narrow in comparison to the U-channel that, when liquid is introduced into the U-channel, the junction channel gets automatically filled. The first step involved silanization of the U-channel with silane solution, to make this channel more hydrophobic. A microliter quantity of the silane solution was introduced into the reservoir (point A in Figure 1a) with the use of a syringe needle. Simultaneously, this solution was aspirated (at point B in Figure 1a) by the use of a vacuum pump, until the reservoir at point A was half empty. To trap the solution in the U-channel and prevent outflow into the sample channel, Ar gas was introduced into the sample channel (via point C, in Figure 1a). Gas flow into the channel was successfully controlled by the use of a needle valve. The trapped silane solution in the U-channel was left for approximately 2-3 min before it was aspirated out completely using vacuum (via point B, in Figure 1a). The silane solution was then reintroduced into the channel and aspirated again in the same manner, before achieving successful silanization. After successful silanization, the second step involves the introduction of a microliter amount of the organic cocktail into the U-channel (via point A, in Figure 1a). The organic cocktail should fill the silanized channel spontaneously. Problems such as bubble entrapment were not encountered during the filling process. Once trapped, the membrane was stored in a desiccator and allowed to air-dry overnight. To complete the assembly of ISE chip electrode, reservoir valves were mounted on the
appropriate openings (points A and E, in Figure 1a). Reservoir valves were made from sections of ∼4-mm micropipet tips glued (using epoxy resin) to the glass surface around the 1-mm drilled holes, providing a reservoir volume of ∼40 µL each. To complete the electrochemical cell, the ISE half-cell connector was constructed. This involves filling the reservoir valve (at point A, in Figure 1a) with 0.1 M of BaCl2 as the internal filling solution. Finally, Ag:AgCl wire was dipped in the BaCl2 solution and held there with silicone rubber. The electrode was stored for a few hours to stabilize before use. Reagents and Solutions. Poly(vinyl chloride) (PVC), 2-nitrophenyl octyl ether (o-NOPE), potassium tetrakis(4-chlorophenyl)borate (TPB salt), barium ionophore 1, V 163 (N,N,N′,N′tetracyclohexyloxybis(o-phenyleneoxy)diacetamide) (Vogtle) ionophore, and barium chloride were bought from Fluka. Repelcote silanizing solution was obtained from Merck. The chemicals used were of analytical reagent grade or better. BaCl2 (0.1 M) stock solution was made up fresh. Dilution to appropriate concentration is achieved just prior to conducting calibration measurements. All aqueous solutions were prepared with deionized water. Membrane. The solvent polymeric membranes were prepared in accordance to the guidelines specified in the Fluka catalog.20 The cocktail for the PVC-based sensor membrane used by Kappes and Hauser17 was modified in an attempt to improve membrane adhesion inside the chip. As a result, the following membrane formulation was used: o-NOPE (1 mL), PVC (70 mg), TPB salt (2 mg), and Vogtle ionophore (7 mg), all dissolved in THF (0.5 mL). The cationic exchanger membrane consists of the same membrane formulation, but in the absence of the Vogtle ionophore. Experimental Setup. In the flow-through manifold, the cell was equipped with an electrolyte management system consisting of two syringe pumps (Harvard Apparatus), a syringe switch (Biotech Instruments), and a peristaltic pump (Biotech Instruments), as shown in Figure 2. The two syringe pumps and the syringe switch allow the analyte to be injected in the sample stream, at various concentrations, without interruption of flow. The sample stream passes the ISE contact and eventually ends up in the outlet opening. The outlet (point E in Figure 1a) consists of an open valve reservoir, in which the reference electrode is attached. For stable and reproducible potentiometric measurements, a commercially available miniaturized Ag/AgCl electrode (with its own internal filling solution) was employed as the reference. This outlet valve reservoir is also connected to a peristaltic pump via PTFE tubing. This allows the system to pump waste out into the waste reservoir. The basic design of the measuring system is also shown in Figure 2. Owing to high resistivity of the membrane (which requires high-input impedance), the electrode signal is first brought to a buffer amplifier (World Precision Instruments). The amplifier converts high-input impedance into low-output impedance. The output signal is then measured using a PICO-analog digital converter (ADC, Pico Instruments) data acquisition system. To provide electrostatic shielding, the chip sensor and batteryoperated amplifier are enclosed inside a metal Faraday cage. (20) Fluka-Chemika Catalog. Selectophore: Ionophore, Membranes and MiniISE., Fluka Chemie AG, Buchs, Switzerland, 1996; pp 13-16.
Figure 2. Block diagram of the continuous flow-through design.
Electrode Testing and Calibration Procedure. Testing of individual electrodes shows that the most common form of instability is drift. Electrodes commonly show drift, shortly after fabrication and during the first part of the calibration procedure. Therefore, prior to conducting potentiometric measurements, there is a need to condition the electrode with 0.1 M BaCl2 for 1 h, in which the drift should become smaller. Emf measurements were made at room temperature (range 20-25 °C). The potential data recorded by the data acquisition system were collected under non-stopped-flow conditions (flow rates were set at 1 µL/min). Electrodes were evaluated by studying their behavior in various BaCl2 concentrations (ranging from 10-1 to 10-8 M). Screening of the electrodes was carried out by determining the slope of the calibration curve (emf versus log [c]). For conditioning overnight, the electrodes were stored in 0.1 M BaCl2 when no measurement was performed. RESULTS AND DISCUSSION Figure 3 shows the potential-time function for a typical electrode (made from a membrane of the Vogtle ionophore formulation). The signal of the sensor in the flow-through manifold was stable, capable of giving rapid responses in the range of 10-110-5 M. In particular, the response to changes to BaCl2 in the range of 10-4-10-1 M is always complete within seconds taken to flush the new solution through. While a static sensor may take several minutes to equilibrate, this flowing system allows rapid analysis due to an increase in diffusion flux of the analyte to the ISE membrane. The plot shows unstable potentials at low concentration range, i.e., below 10-5 M. To further analyze the performance characteristics, a calibration curve (Figure 4), from data points derived from Figure 3, was plotted. The error bars represent the amplitude of fluctuations in the measured emf signal. The calibration plot (Figure 4) is shown to give slope of 36 mV/decade (theoretical Nernstian response, 29 mV/decade at 25 °C). The slightly higher slope Analytical Chemistry, Vol. 72, No. 13, July 1, 2000
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Figure 4. Calibration graph for Ba2+ Vogtle ISE on chip. Ba2+
Figure 3. Potential-time function of Vogtle ISE on chip, at various BaCl2 (dissolved in water) concentrations, as indicated by the arrows.
obtained can be attributed to the fact that the experimental emf values have not been corrected for changes in the liquid junction potential by using the Henderson formula21 or temperature variation. From the plot, the detection limit is 10-6 M. According to IUPAC,22 the detection limit of a measurement made with ISE is defined as the intersection of the two extrapolated linear segments of the calibration graph. The ISE chip is a highimpedance system and therefore will be susceptible to noise problems. Measurements conducted at concentrations below 10-6 M result in unstable emf signals and therefore accurate measurements cannot be made. The characteristics of the micromachined ISE chip described can be favorably compared with those previously reported for a barium sensor.23 Many similarities appear when comparisons are made with ionophore-based electrodes. For example, in the study conducted by Bouklouze and co-workers,24 a new class of Ba2+ ionophore belonging to the naphthylpolyether family was synthesized and incorporated in an ethylene-vinyl acetate copolymer. In this study, electrodes were constructed by dipping the end of a micropipet tip into polymeric membrane solution and then evaluated for analysis of barium ion. The resulting potentiometric (21) Morf, W. E. The Principles of Ion-Selective Electrodes and of Membrane Transport; Scientific Publishing Co.: Amsterdam, Holland, 1981; Chapter 5. (22) CRC Handbook of Ion-Selective Electrodes; CRC Press: Boca Raton, FL, 1990. (23) Jaber, A. M. Y.; Moody, G. J.; Thomas, J. D. R. Analyst 1976, 101, 179186. (24) Bouklouze, A. A.; Vire, J.-C.; Cool, V. Anal. Chim. Acta 1993, 273, 153163. (25) Feng, Y. P.; Goodlet, G.; Islam, M. M.; Moody, G. J.; Thomas, J. D. R. Analyst 1991, 116, 469-472.
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response was linear in the range of 10-1-10-6 M with a slope of 30 mV/decade. For comparison purposes only, an ISE chip that employed a cation-exchange membrane (a recipe similar to the Vogtle membrane but in the absence of the ionophore) was studied. In summary, the results show that the presence of the ionophore has a big influence on performance in terms of speed of response, stability of the response at steady-state conditions, and effective linear calibration range. Feng and co-workers, who have employed di-2-nitrophenyl ether alone as the cation exchanger,25 observed similar results. CONCLUSIONS It has been shown that a novel ISE chip, in analogy to ISE microelectrodes, responds to free analyte ion activities. An attempt to develop an ISE detection for Ba2+ using a Vogtle membrane cocktail (trapped in a PVC matrix membrane) was successful, and the electrodes were able to monitor [Ba2+] levels in the range of 10-1-10-6 M. The micromachined ISE chip showed response characteristics comparable to those for previous barium ionselective electrodes of both the liquid membrane and PVC matrix membrane varieties. ACKNOWLEDGMENT This research was sponsored by Schlumberger. The clean room used for fabrication of microchips was financially supported by Smithkline Beecham and Zeneca, as well as for the BBSRC and JREI. The authors also thank Gareth Jenkins for assistance in chip fabrication. Received for review January 7, 2000. Accepted March 30, 2000. AC000036+
