Anal. Chem. 2005, 77, 5385-5388
Fabrication of a Poly(dimethylsiloxane)-Based Electrochemiluminescence Detection Cell for Capillary Electrophoresis Jilin Yan, Xiurong Yang,* and Erkang Wang*
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
An easy but effective technique is described here for quick fabrication of low-cost electrochemiluminescence detection cells for capillary electrophoresis. The technique is based on molding of poly(dimethylsiloxane) (PDMS) with a capillary column inserted into a pipet tip. Two access holes are left in the PDMS slab; they provide neat accommodations for the separation capillary and the working electrode made with the same type of tip. Since the access holes are well-aligned, the electrode and the capillary are automatically aligned; thus, end-column detection is easily obtained. Fabrication of the detection cell is straightforward; no micromechanical operation is included. Also the principle for the procedure makes it possible to efficiently batch production detection cells with good reproducibility. Because of the end-column scheme, the cell can be adopted for electrophoresis with electrochemical detection as well. During the past decade, electrochemiluminescence (ECL) of tris(2,2′-bipyridyl)ruthenium (Ru(bpy)32+) has been found a useful scheme for direct and sensitive detection of oxalate and a variety of amine-containing analytes;1-4 the method is widely adopted for flow injection and HPLC analysis.2-4 As a new development, its application in capillary electrophoresis (CE-ECL) also attracts much attention of researchers; various systems have been developed for this purpose.5-15 The detection cell design is one * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Gerardi, R. D.; Barnett, N. W.; Lewis, S. W. Anal. Chim. Acta 1999, 378, 1-41. (2) Richter, M. M. Chem. Rev. 2004, 104, 3003-3036. (3) Knight A. W. Trends Anal. Chem. 1999, 18, 47-62. (4) Fa¨hnrich, K. A.; Pravda, M.; Guilbault, G. G. Talanta 2001, 54, 531-559. (5) Huang, X. J., Fang, Z. L. Anal. Chim. Acta 2000, 414, 1-14. (6) Forbes, G. A.; Nieman, T. A.; Sweedler, J. V. Anal. Chim. Acta 1997, 347, 289-2936. (7) Liu, J. F.; Yan, J. L., Yang, X. R.; Wang, E. K. Anal. Chem. 2003, 73, 36373642. (8) Wang, X.; Bobbitt, D. R. Anal. Chim. Acta 1999, 383, 213-220. (9) Wang, X.; Bobbitt, D. R. Talanta 2000, 53, 337-345. (10) Hendrickson, H. P.; Anderson, P.; Wang, X.; Pittman, Z.; Bobbitt, D. R. Microchem. J. 2000, 65, 189-195. (11) Chiang, M. T.; Whang, C. W. J. Chromatogr., A 2001, 934, 59-66. (12) Huang, X. J.; Wang, S. L.; Fang, Z. L. Anal. Chim. Acta 2002, 456, 167175. (13) Liu J.; Cao W.; Qiu, H.; Sun, X.; Yang X.; Wang E. Clin. Chem. 2002, 48, 1049-1058. (14) Cao, W.; Liu, J.; Yang, X.; Wang, E. Electrophoresis 2002, 21, 36833691. 10.1021/ac050581g CCC: $30.25 Published on Web 07/16/2005
© 2005 American Chemical Society
of the kernel parts of the CE-ECL technique. Ru(bpy)32+ is electrochemically oxidized to its active state Ru(bpy)33+ at the vicinity of the outlet of the separation column and instantly reacts with the electrokinetically driven out analytes.4 The detection cell has some characteristics similar to those of the cell for conventional electrochemical detection for capillary electrophoresis (CE-EC), and most of the researchers set up their cells as derived from the previously reported ones for CE-EC.5-7 Both off-column detection and end-column detection have been reported.6-16 The previous one is popular for its elimination of the complex procedure of the preparation of decouplers. In this scheme, the electrode is placed some distance from the outlet of the separation column to initiate ECL reaction there. This setup remains a high demand for the electrode alignment. A mechanism for microadjustment is needed for this operation, and often micropositioners or 3-dimensional screws are included in the cell designs.5-11 The corresponding assembly and alignment procedures are complex and tedious. Poly(dimethylsiloxane) (PDMS) is a soft material recently accepted in the field of microfabrication.17-20 The material has been intensively reported for fabrication of microfluidic chips and other microdeveices.20 The main course of its fabrication includes preparation of the template and curing of the PDMS precursor on it. The method has advantages over the conventional one in a variety of aspects, such as ease of operation, low cost, high precision and reproducibility, etc. Structures down to the micrometer level can be easily obtained without any special facilities. Here we attempt to take this material into the construction of a simplified CE-ECL cell. Micromolding of the material provides an easy way to replicate cells with a high efficiency. Furthermore, the flexibility of the material greatly benefits the assembly operation; end-column detection can be obtained without any special effort. The cell can be successfully used for CE-ECL analysis. Also the cell is applicable for electrochemical detection when a normal size electrode is used. (15) Chiang, M. T.; Lu, M. C.; Whang, C. W. Electrophoresis 2004, 24, 30333039. (16) Huang, X.; Zare, R. N. Anal. Chem. 1991, 63, 189-192. (17) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550-575. (18) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H.; Schueller, O. J. A.; Whitesides, G. M. Electrophoresis 2000, 21, 27-40. (19) McDonald, J. C., Whitesides, G. M. Acc. Chem. Res. 2002, 35, 491-499. (20) Ng, J. M. K.; Gitlin, I.; Stroock, A. D.; Whitesides, G. M. Electrophoresis 2002, 23, 3461-3473.
Analytical Chemistry, Vol. 77, No. 16, August 15, 2005 5385
Figure 2. Scheme of the working electrode (A) (1, metal wire; 2, pipet tip; 3, epoxy sealing) and assembly of the CE-ECL detection cell (side view) (B) (4, working electrode; 5, separation capillary; 6, glass slide; 7, PDMS slab; 8, electrophoresis ground electrode; 9, reference electrode; 10, counter electrode. Magnification of the interface between the working electrode and the capillary outlet (C). Dimensions are not to scale.
Figure 1. Preparation of the PDMS slab for the cell. (A) Setup for the PDMS mold: (1) a pipet tip, (2) capillary column, (3, 4) two cut plastic vials, (5) glass slide. (B) Pouring in of PDMS: (6) PDMS mixture. (C) PDMS slab as the detection cell body.
EXPERIMENTAL SECTION Chemicals and Materials. Tris(2,2′-bypiridyl)dichlororuthenium(II) hexahydrate (Ru(bpy)32+), tripropylamine (TPA), lidocaine, carbohydrates, and sodium hydroxide were purchased from Aldrich (Milwaukee, WI), L-proline was purchased from Shanghai Biochemical Co. (Shanghai, China), a Syglard 184 PDMS kit was purchased from Dow Corning (Midland, MI), and all other reagents were of analytical grade and used as received. Solutions were frequently prepared with deionized (DI) water processed with a Milli-Q system (18.2 MΩ‚cm, Millipore, Bedford, MA). Capillary columns (25 µm i.d., 375 µm o.d.) were obtained from Reafine Chromatograph Device Ltd. (Yongnian, Hebei, China). A length of 60 cm was taken for electrophoresis separation. It was rinsed with 1 M NaOH solution overnight before the initial use, then washed with DI water, and conditioned with the running buffer for at least 15 min just before each series of electrophoresis. Preparation of the PDMS Cell for CE-ECL Detection. The procedure for making the PDMS cell body is shown in Figure 1. First the container as well as the template for the PDMS molding was provided by two well-cut plastic sample vials (with diameters of 8 and 30 mm). Appropriate access holes were drilled in the sidewall of the vials (3 mm from the bottom) for inserting a pipet tip (200 µL, Eppendorff, Germany) with a short capillary column (8 cm). The vials were sealed to a flat glass with instant adhesive. 5386 Analytical Chemistry, Vol. 77, No. 16, August 15, 2005
The adhesive was applied out of the ring area between the two vials to maintain a flat surface. After the capillary together with the tip was inserted into the holes, 6 g of a degassed mixture of PDMS monomer and curing agent (in a 10:1 ratio) was poured into the ring part. A subsequent 40 min baking at 100 °C was done to accelerate the mixture’s curing, and then the inserted pipet tip together with the capillary was pulled out. PDMS was peeled from the mold, and the cell body was obtained. Working Electrode and Assembly of the Cell. The same type of pipet tips as used above were adopted for the preparation of working electrodes (Figure 2). The top point of the tip was a hole of diameter ca. 400 µm. A slice of the top part of the tip should be cut off to give a neat accommodation for a 500 µm platinum wire. Epoxy (WSR6101, Xingchen New Chemical Material Co. Ltd., Wuxi, China) was applied to secure the wire in the tip. After the epoxy’s curing, the protruded part of the platinum wire was cut off with a blade. The left surface was ground with sandpaper and further well polished with alumina, to obtain a smooth disk surface. The electrode was thoroughly ultrasonically rinsed with DI water and ethanol before use. Similarly, a copper disk electrode was made with a wire of 380 µm diameter, except that no further cutting off of the top part of the tip was needed in this operation. The assembly procedure for the cell was quite simple. A flat glass slide was reversibly attached to the bottom of the PDMS slab, and the cell was formed. The working electrode and separation column were inserted into their corresponding preset access holes. They were automatically aligned without any further manual effort (Figure 2). The distance between the electrode and capillary could be controlled by adjusting the capillary column. The electrophoresis ground electrode, a platinum counter electrode, and a Ag/AgCl (with saturated KCl) reference electrode were added into the cell from the top. Apparatus. CE separation and ECL detection were performed with a model MPI-A capillary electrophoresis electrochemiluminescence system (Xi’an Remax Electronics Inc., Xi’an, China, and Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China). The system provided a high-voltage resource for electrophoresis, a luminescence detector, and an electrochemical potentiostat. Electrochemical detection was performed with a CHI 800 electrochemical analyzer (CHI Instrument,
Austin, TX). The detection cell was placed in a metal dark box, which diminished both the optical and electric disturbance of the surrounding environment. RESULT AND DISCUSSION As most of the CE-ECL cells reported in previous works, the one described here also takes a scheme that originates from CE-EC.10-15 An end-column scheme was adopted, and the electrode worked in a wall-jet configuration.13,14 The main concept of our cell design is the well-aligned access holes in the PDMS cell body. The principle of this cell is similar to a previous CE-EC one reported by Fermier et al.,21 but it is designed in a much simplified way. The pipet tip adopted in the experiment had a small hole of diameter ca. 400 µm at the top point; the size was just a little larger than the outer diameter (o.d.) of the capillary column (375 µm). This ensured that the inserted column located almost the very center of the hole. The working electrode was made of the same type of tip. Because of the diameter of the metal wire adopted, the so-made disk electrodes also lay at the center of the pipet tip’s top. When the electrode (in the tip) and capillary column were inserted into their corresponding preset holes in the PDMS cell body, they directly formed a straight line; no further manual effort was needed for the alignment operation. The distance between the capillary outlet and electrode can be adjusted by protrusion or withdrawal of the capillary column; the column remains aligned with the electrode within this operation for guiding of the access hole. The PDMS slab can seal to some flat surfaces either reversibly or irreversibly.19,22 Here reversible sealing was done for its convenience of operation. After a thorough rinse of the surfaces and conforming them together, a flat glass slide could be easily incorporated onto the bottom of the PDMS cell body. The sealing formed through van der Waals contact between the two was successful and robust.22 Interaction between the PDMS and the plastic surface of the tip or polyimide coating of the capillary may have a different principle,20 but these contacts also seemed tight. This was mainly attributed to the neat accommodation in the PDMS slab for the inserted objects and the elastic property of the material. All adherences between these surfaces were firm. Though no screws or glue were adopted in the assembly, the cell exhibited high robustness and was leakage free. PDMS is a soft material; a device made of complete PDMS may have a relatively large distortion under external pressure.19 This is crucial in the application as a material for the cell body, for even a little distortion will greatly affect the alignment and distance control between the electrode and capillary. In the application described here, this property was found to have little side effect on our cell. The glass slide on the cell bottom gave a stubborn support of the PDMS cell body; also no external pressure was applied on the cell in the CE-ECL detection procedure. The cell remained in its original configuration in the detection process. CE-ECL performance of the so-developed cell was characterized through separation and detection of mixed samples. Phosphate buffer (pH 8.2) was used for the experiment. A concentration of 10 mM was adopted as the running buffer; 0.3 mL of a solution (21) Fermier, A. M.; Gostkowski, M. L.; Colo`n L. A. Anal. Chem. 1996, 68, 16611664. (22) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984.
Figure 3. Electrophoresis separation and ECL detection of the mixtures of TPA (1; 2 µM), proline (2; 2 µM), and lidocaine (3; 20 µM) (A). The concentrations of the analytes in electropherograms B and C are 2 and 3 times that in electropherogram A. Electrophoresis conditions: a 60 cm fused-silica capillary column (25 µm i.d., 375 µm o.d.); runner buffer, 10 mM phosphate (pH 8.2); buffer in the detection cell, 50 mM phosphate and 5 mM Ru(bpy)32+ (pH 8.2); electrokinetic injection, 10 kV for 10 s; separation field strength, 250 V/cm; electrochemical initiation, 1.20 V (vs Ag/AgCl); PMT bias, 750 V.
of 50 mM phosphate and 5 mM Ru(bpy)32+ was placed in the detection cell. Samples were obtained by a series of dilutions of the stock solution with DI water. They were electrokinetically injected into the electrophoresis system by applying a 10 kV high voltage for 10 s. The distance between the electrode and the capillary outlet was optimized at 100 µm, which effectively eliminated the influence of the capillary high voltage with less hindrance on the separation efficiency. ECL reaction was electrochemically triggered under a potential of 1.20 V. The glass slide on the bottom also acted as the luminescence detection window here, and the signal was collected with a multiple-photon tube (PMT) beneath the cell. Analyses of TPA and proline were successfully performed; linear ranges of 20-0.05 and 10-0.05 µM were obtained for these two analytes, respectively, with correlation coefficients of 0.9989 and 0.9993. Detection limits were obtained at 5.2 × 10-9 and 1.3 × 10-8 M (S/N ) 3). Also the CE-ECL system exhibited a good reproducibility; a consecutive analysis of a 2 µM TPA sample (n ) 9) provided RSDs of 3.43% for the peak area and 0.89% for the migration time. Electrophoretic separation of three analytes, TPA, proline, and lidocaine, was well done under the same conditions (Figure 3). A stable baseline was presented in the electropherogram. The calculated plate numbers were 58000, 127000, and 28000 per meter for the three analytes, respectively. The performance of the cell is comparable to that of a previous chip type one developed by our group7 (LOD obtained at 1.4 × 10-9 mol/L for TPA and 1.6 × 10-7 mol/L for proline with calculated theoretical plate numbers of 52000 and 105000 per meter, respectively). The sensitivity of our CE-ECL system is better than that of the ITO-based cell by Chiang et al., with a lower column efficiency.15 Another merit of the fabrication technique described here is the convenience for replicating the cells. Most of the cells described earlier were produced individually; the reproducibility between cells remains a problem. In our method, after the mold is ready, only a little labor is required if more cells are needed. Analytical Chemistry, Vol. 77, No. 16, August 15, 2005
5387
Figure 4. Separation and ECL detection of a mixture of TPA and proline (1 µM each) with five (1-5) different PDMS-based cells. Electrophoresis conditions: a 60 cm fused-silica capillary column (25 µm i.d., 375 µm o.d.); runner buffer, 10 mM phosphate (pH 8.2); buffer in the detection cell, 50 mM phosphate and 5 mM Ru(bpy)32+ (pH 8.2); electrokinetic injection, 10 kV for 10 s; separation field strength, 250 V/cm; electrochemical initiation, 1.20 V (vs Ag/AgCl); PMT bias, 750 V.
They can be quickly replicated (less than 1 h for a PDMS unit) at a rather low cost. In a continuous operation, five detection cell bodies were obtained with the same mold in our laboratory. The same working electrode and separation column were used to assemble five different cells. These cells were tested in a continuous separation and detection of a TPA and proline mixture (1 µM each). The RSDs of the migration time were 0.49% and 1.13%, while those for peak area were obtained at 9.6% and 11.4% (Figure 4). The value of the reproducibility of the peak area was not completely satisfactory, but was still acceptable, and this was the first reproducibility study between detection cells for CE-ECL. The cell design is suitable for multiple-purpose applications. All electrodes made with the same type of pipet tips and capillaries of the same o.d. can be adopted on this type of platform. The old capillary or electrode could be easily replaced with a new one. After adjustment of the distance, the cell could go into another use within 2 or 3 min. The same type of cell was adopted for a CE-EC application with a 380 µm disk copper electrode. The system was characterized by separation and detection of carbohydrates. Freshly prepared 100 mM NaOH solution was used as the running buffer. The carbohydrates were negatively charged under this environment and could be separated according to their charge/mass ratio difference.23 Samples were diluted to the desired concentration with the running buffer and injected into the electrophoresis system at 10 kV for 5 s. Amperometric detection was performed under a working potential of 0.70 V. Sucrose, lactose, and glucose (23) Ye J. N., Baldwin R. P. Anal. Chem. 1993, 65, 3525-3527.
5388 Analytical Chemistry, Vol. 77, No. 16, August 15, 2005
Figure 5. Separation of a carbohydrate mixture (1, sucrose; 2, glucose; 3, fructose; 30 µM each) with electrochemical detection under various field strengths: 200 (A), 250 (B) and 300 (C) V/cm. Electrophoresis conditions: a 60 cm fused-silica capillary column (25 µm i.d., 375 µm o.d.); runner buffer, 100 mM NaOH; injection, 15 kV for 5 s; electrochemical detection, 0.70 V vs Ag/AgCl.
were well separated (Figure 5). Theoretical plate numbers of about 100000 per meter were obtained for the carbohydrates (98000 per meter for sucrose, 122000 per meter for lactose, and 98000 per meter for glucose) under a field strength of 200 V/cm. The limit of detection for glucose was obtained at 1.8 µM. The analytical performance of the cell for CE-EC application is similar to those of the previous CE-EC applications.21,22 CONCLUSION Here we have presented an easy technique for the buildup of detection cells for capillary electrophoresis with Ru(byp)32+ electrochmiluminescence. The cell can be effectively batch produced from the same mold at a rather low cost. Electrode alignment needed for end-column detection can be easily obtained with little manual effort. Some other operations on the cell, such as changing the electrode or capillary, are also greatly simplified. This design is applicable for electrochemiluminescence and electrochemical detection with a normal size electrode. Furthermore, the cells may also be adoptable in cases with microelectrodes (electrodes with a diameter of less than 100 µm) when proper tips or other guiding tubes (with holes of the same diameter as the electrode adopted) can be obtained. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant Nos. 20299030 and 20335040). Received for review April 6, 2005. Accepted June 7, 2005. AC050581G
