Electrophoresis 2005, 26, 3007–3012


Dong-Mung Tsai1 Kuan-Wen Lin1 Jyh-Myng Zen1 Hung-Yu Chen2 Ray-Hua Hong2

A new fabrication process for a microchip electrophoresis device integrated with a three-electrode electrochemical detector


We report here a novel and simple process for the fabrication of a poly(methyl methacrylate) (PMMA)-based microchip electrophoresis device, integrated with a screenprinted three-electrode electrochemical detector that does not require a replicate mold. In this approach, a photoresist layer constitutes both an adhesion layer and side walls of 50 mm wide and 50 mm tall microfluidic channels on a screen-printed threeelectrode PMMA substrate. Openings were drilled for buffer reservoirs on an additional piece of PMMA, then the final device was bonded in a PMMA/photoresist/PMMA sandwich configuration. This process is inexpensive, less time-consuming, and simpler compared with traditional fabrication methods. The combination of this PMMAbased microchip fabrication together with screen-printed electrode technology holds great promise for the mass production of a single-use micrototal analytical system. Successful determination of uric acid and L-ascorbic acid with the presented system validates its utility. In combination with a suitable electrochemical detector, this device holds much promise for the determination of other analytes in various biological samples for medical and clinical diagnosis.

Department of Chemistry Institute of Precision Engineering, National Chung Hsing University, Taichung, Taiwan


1 Introduction Microfluidic devices are being increasingly used in many analytical applications [1–3]. Both glass and plastic substrates have been used to fabricate microfluidic systems in the past [4–9]. In general, glass fabrication processes are time-consuming and relatively expensive. Replicationbased plastic microfluidic devices, formed from casting, embossing, or injection molding [4, 5, 7] to ensure device reproducibility, are relatively simple and inexpensive in mass production [1, 10]. Conventional replication methods require the use of a replicate mold typically formed by electroplating or etching on quartz [11, 12] or silicon [8, 13]. This is followed by molding with polymers, such as poly(methyl methacrylate) (PMMA), polycarbonate, polyethylene, or poly(dimethylsiloxane) (PDMS) [1]. We report here a novel and simple process to fabricate a microchip electrophoresis device, integrated with a screen-printed three-electrode electrochemical detector. In this approach, a photoresist layer was used to make a Correspondence: Dr. Jyh-Myng Zen, Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan E-mail: [email protected] Fax: 1886-4-2286-2547 Abbreviations: AA, L-ascorbic acid; PDMS, poly(dimethylsiloxane); PMMA, poly(methyl methacrylate); SEM, scanning electron microscopy; UA, uric acid

© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

50 6 50 mm2 microfluidic channel on a PMMA substrate containing a screen-printed three-electrode system. Screen-printing fabrication technology has been shown to offer the possibility of large-scale and inexpensive mass production of a CE detector [10]. For successful electrochemical detection, the presented microchip design alleviates the need to realign the working electrode with the separation channel with a separate extrinsic reference electrode or auxiliary electrode. After drilling holes for buffer reservoirs on another piece of PMMA, a final device having a PMMA/photoresist/PMMA sandwich configuration was completed by a bonding process. This is as effective as a UV-glue bonding process for microchip as reported by Landers and co-workers [14]. The coupling of low-cost PMMA-based microchips with massproduced screen-printed technology holds great promise for creating single-use disposable CE electrochemical microsystems for a wide range of applications. In this article, we demonstrate the attractive performance and characteristics of this low-cost disposable microsystem. The separation of uric acid (UA) and L-ascorbic acid (AA) in human urine is presented using this microchip device. UA, a major nitrogenous compound in urine, is the product of purine metabolism in human body and is related to many clinical disorders (urinary stone, gout, etc.) [15]. Thus, the screenings of UA in human physiological fluids are indispensable for the diagnosis of patients suffering


Keywords: Human urine / Microchip electrophoresis / Miniaturization / Screen-printed electrode DOI 10.1002/elps.200500107


D.-M. Tsai et al.

from a range of disorders associated with altered purine metabolism. UA and AA are present together in biological fluids such as blood and urine, and are oxidized at similar potentials [16]. Therefore, it is important to separate UA in the presence of AA for quantification by amperometry.

2 Materials and methods 2.1 Chemicals and reagents The negative photoresist (THB-120N) was purchased from JSR (Sunnyvale, CA, USA). UA and AA were obtained from Aldrich (Milwaukee, WI, USA). The stock pH 7.0 run buffer solution was prepared by mixing 0.01 M Na2HPO4 and 0.01 M H3PO4. The human urine sample, collected 2 h after

Electrophoresis 2005, 26, 3007–3012 the donor consumed 500 mg vitamin C, was diluted tenfold with the run buffer solution prior to determination.

2.2 Microchip fabrication and assembly Photolithographic masks (Fig. 1A) were designed using a standard computer software (AutoCad 2000) and transferred onto a film by a professional photo corporation (Gem Line Technology, Taichung, Taiwan) using a commercial high-resolution printer with 4000 dpi (,6 mm) resolution. The channel network was represented by a 50 mm wide black line, and the pattern border was a 1 cm wide black rectangle. The entire PMMA chip fabrication procedure is illustrated in Fig. 2. The PMMA plates were provided from a local technical corporation (Zensor R&D,

Figure 1. (A) Photolithographic mask design. Size of photomask is 50 6 100 mm2. (B) Schematic of whole microchip device including electrochemical detector, each section is explained below. (a) HV for separation, in which a1 is HV input and a2 is ground; (b) HV for injection, in which b1 is HV input and b2 is ground; (c) working electrode (carbon); (d) auxiliary electrode (carbon); (e) reference electrode (Ag/AgCl). Reservoir 1 is filled with sample, and reservoirs 2, 3, and 4 are filled with run buffer solution. (C) Scanning electron microscope (SEM) image of channels made from photoresist for a microchip CE device fabricated based on a PMMA chip base. This image shows a straight cross-link of the channel.

© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2005, 26, 3007–3012

New fabrication process for a microchip electrophoresis device

Figure 2. Flow sheet of the fabrication procedure. (1) Coating a 50 mm thick layer of photoresist on a PMMA chip base. (2) A transparency template then serves as the photomask for photolithography. When the photoresist is exposed to UV light, solidification occurs. (3) The device is then subjected to the developer solution (SD-W), thus exposing the microchannels. (4) The entire unit is bonded at a pressure about 10 psi and temperature about 907C. (5) Completed microchip made by this fabrication.

Taichung, Taiwan). The size (L 6 W) was 50 6 100 mm2 and 30 6 80 mm2 for the top and bottom plates, respectively, and the thickness for both was 3 mm. The PMMA (50 6 100 mm2) chip base was cleaned with ethanol before fabrication. Electrodes were fabricated by screenprinted techniques on the lower PMMA substrate. The pattern of electrode on the microchip was designed by a Corel Draw 9.0 computer software and transferred to a silk screen as a final 250 mesh silk screen halftone pattern having a thickness of 10 mm by Sammo Co. (Taipei, Taiwan). The silk screen was used to print the electrodes. Carbon and silver inks (Acheson, Ontario, CA, USA) were screen-printed on the lower PMMA plate followed by baking at 1007C for 30 min to form the three-electrode system [17, 18], as depicted in Fig. 1B. The Ag/AgCl reference electrode, made from silver ink, was conditioned with 50 mL of 15% H2O2 (SHOWA, Tokyo, Japan) for 15 min and then 50 mL of 3 M KCl (TEDIA, Fairfield, OH, USA) for 15 min. The high-voltage (HV) power supply, used for separation, has an adjustable voltage range of 0 to 130 kV (model CZE1000R, Spellman). Input was con-

© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


nected to a1 and b1 and ground was connected to b2 and a2. A laboratory-prepared switch alternates the power source between separation (a1) and injection (b1). The detection system includes an amperometric detector (model LC-4C, BAS) and a chromatographic data station (model ADV24, Scientific Information Service Corporation, Taiwan) coupled to the working (c), auxiliary (d), and reference (e) electrodes through sockets. Electrodes lie beneath the separation channel, thus permitting buffer to flow past the detector. Similar to the preparation of working electrodes on CE microchips, the ground electrode also serves as the decoupler. During electrophoretic separation, the high electric field is sufficient for the electrolysis of water, which may cause gas evolution at the ground electrode and interfere with the electrochemical signal. For the decoupler onto which hydrogen ion is reduced and adsorbed, the unique property is that hydrogen can diffuse relatively fast. Therefore, before gas bubbles can develop, hydrogen is removed from the decoupler by the solution due to EOF. Taking advantage of the decoupler, single or multiple working electrodes can be placed across the separation channel. The problem of gas bubble in the channel disrupting the process does not exist in this three-electrode system. A photolithography fabrication technique was next used to make the microfluidic channels on the PMMA chip base. Briefly, the PMMA substrate was spin coated with a 50 mm layer of negative photoresist, and soft baked at 907C for 7 min. This assembly was then exposed to UV light (500 W) in 10 s through a mask. The excess liquid photoresist, not exposed to UV light, was rinsed using a developer solution (JSR). The upper PMMA layer (30 6 80 mm2) has four openings that align with the ends of the microchannels on the lower PMMA layer that houses the screen-printed three-electrode system. The access holes on the upper PMMA plate (30 6 80 mm2) were drilled using a 2 mm diameter stainless-steel drill. Note that there is a special design for the part of access holes on photomask as shown in Fig. 1A. A funnel type channel design can effectively keep the connecting section (between the channel and access holes) flowing freely. Without this design, the connecting section is sometimes obstructed by collapsing photoresist during the bonding procedure. The final PMMA/photoresist/PMMA sandwich configuration was completed under mild bonding conditions at pressures and temperatures less than 10 psi and 907C in 30 min, respectively. The injection portion of the microchannel is 2 cm and the separation channel has a 5 cm effective separation length. The size of the entire microchip assembly is 5 6 10 cm2 with a calculated manufacturing cost of ,10 USD per piece.


D.-M. Tsai et al.

2.3 Electrophoresis procedure Before any CE experiment, the microchannels are rinsed with DI water (resistance .18 MO/cm) and a phosphate buffer solution (pH 7.0) sequentially for 30 min each. UA and AA standard solutions (10 mm) were prepared in DI water, and diluted by run buffer to the desired concentration. The urine sample was diluted tenfold by the run buffer prior to injection. Reservoirs 2, 3, and 4, as seen in Fig. 1B, were filled with the run buffer solution and reservoir 1 was filled with sample. The volume of sample was controlled by varying the applied potential during the injection mode for a fixed time. Injection voltage was applied at 100 V/cm for 20 s. Subsequently, separation began by switching to voltages of 50, 100, or 200 V/cm across the separation channel. The oxidation signal obtained at the negative side of the current-time graph was uniformly taken as a quantitative parameter for further CE analysis.

3 Results and discussion A scanning electron microscopic (SEM) image of the photoresist microfluidic channels is depicted in Fig. 1C. The channel dimension originally designed using a photomask width of 50 mm is in accordance with the final product. The observed sidewall images, depicting smooth surfaces and sharp edges, confirm the uniformity of this patterned photoresist material. Microchannel fabrication using photoresist provides an appreciable control of channel dimension [19]. Because the photoresist layer is part of the microfluidic channel, it is essential that the surface properties of photoresist after exposure to UV light may remain similar to PMMA. In this way the hydrodynamics of sample flow through the microchannel might be easily calculated [20, 21]. More importantly, the patterned photoresist microchannel shape in the PMMA/ photoresist/PMMA structure should remain unchanged after the bonding process. This latter concern is confirmed true through optical microscope. Conventional photolithographic techniques result in readily produced micrometer-sized conducting metal film EC electrodes directly on a wide range of substrate materials via sputtering or vapor deposition [22, 23]. But these procedures are tedious and the associated high mass production costs make a disposable microchip device impractical. The printed electrode has been used in microchip devices with the advantages of low-cost and rapid fabrication [10]. The rough substrate of printed electrode on glass, however, would result in an incomplete PDMS fabrication casting of the PDMS microchannel on the glass substrate. To employ these types of

© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2005, 26, 3007–3012 electrodes, it is necessary to include a complicated endcolumn detection cell [24, 25]. In our approach, we have successfully produced a microchip with in-column screen-printed electrode detection using PMMA fabrication in a much simpler configuration. The in-column configuration helps to eliminate some of the negative separation performance characteristics encountered with an end-channel configuration, especially with respect to the alignment of the working electrode at the end of the separation channel [26]. When compared to end-channel detection, in-channel amperometric detection exhibited a twofold decrease in plate height while lowering the peak skew by a factor of 10 [27]. Other bonding protocols are more demanding, requiring much higher temperatures and pressures or the use of plasmas for adhesion of the polymeric layers. As an application of this device, we demonstrate the detection of UA and AA in human urine using a screenprinted carbon working electrode. The effectiveness of the separation channel was confirmed by separating a mixture of UA and AA at 50, 100, and 200 V/cm. The resulting electropherograms are shown in Fig. 3. As can be seen, the higher the separation voltage the shorter the migration time. In these experiments, the signal of UA is more stable than that of AA. This is expected since AA can rapidly be oxidized in air and thus decayed at high temperature [28]. The details of detected data are shown in Table 1. Because of AA oxidizing easily compared with UA, the sensitivity, linearity, and RSD of AA is poorer than UA. Future improvements for the response of AA will include using a sol-gel or polymer modified screen-printed electrode [29, 30].

Figure 3. Electropherogram resulting from the separation of UA and AA at different separation voltages, the applied voltages are (a) 200 V/cm, (b) 100 V/cm, and (c) 50 V/cm. Concentration of UA and AA are both 1 mM. Injection voltage is 50 V/cm in 20 s. Detection voltage of working electrode is 10.7 V (vs. Ag/AgCl).

Electrophoresis 2005, 26, 3007–3012

New fabrication process for a microchip electrophoresis device

Table 1. Data of UA and AA detected by microchip CE system with an electrochemical detector at different concentrationsa) Analyte


Current (nA)



1 mM 100 mM 10 mM 1 mM 100 mM 10 mM

3750 349 29 3011 282 19

2.83 3.49 5.31 10.44 12.37 13.86


a) Separation voltage is 100 V/cm; the other conditions are the same as in Fig. 3. b) Ten replicates It is important to notice some tailing effect with AA signal in the CE graphs. This may be due to the difference in the zeta potential of two different flow profiles corresponding to the PMMA and photoresister materials, and/or with some adsorption characteristics. Further work is in progress to differentiate the effect by specific experiments. In a real sample microchip separation, a tenfold buffer diluted urine sample was sampled 2 h after the donor consumed 500 mg of vitamin C. UA and AA were spiked into the tenfold diluted urine sample at 1029 mol to confirm the composition of each peak. The resulting electrophoregram is shown in Fig. 4 and is similar to the reported results using a PDMS/glass microchip [31]. The rapid identification of UA in urine (,50 s) successfully demonstrates the PMMA microchip as high-throughput screening systems for clinical samples.

Figure 4. Electropherogram of a tenfold diluted urine sample collected 2 h after taking 500 mg of vitamin C. (A) Tenfold diluted urine sample, (B) 1029 mol of UA added in 1 mL tenfold diluted urine sample, (C) 1029 mol of AA added in 1 mL tenfold diluted urine sample. Separation voltage is 100 V/cm, and the other conditions are the same as Fig. 3. Peaks (1) and (2) are UA and AA, respectively.

© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


4 Concluding remarks We have developed a simpler procedure to fabricate microchips compared to traditional methods (such as glass etching and high-temperature bonding). The width and depth of microfluidic channels within this structure can be controlled easily using photoresist material, and the pattern is controlled through the use of an appropriate patterned photomask. Screen-printed electrodes, for electrochemical detection, can be easily fabricated and modified on a smooth PMMA substrate. Through the presented procedure, a microchip can be prepared by a more economical way than the metal deposition and glass etching methods. In the future, we will investigate the detection of other analytes in medically and clinically important biological samples using this microchip device. Received February 10, 2005

5 References [1] 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. [2] Holland, L. A., Leigh, A. M., Electrophoresis 2002, 23, 3649– 3658. [3] Backofen, U., Matysik, F.-M., Lunte, C. E., Anal. Chem. 2002, 74, 4054–4059. [4] Rodriguez, I., Zhang, Y., Lee, H. K., Li, S. F. Y., J. Chromatogr. A 1997, 781, 287–293. [5] Linder, V., Verpoorte, E., de Rooij, N. F., Sigrist, H., Thormann, W., Electrophoresis 2002, 23, 740–749. [6] Colyer, C. L., Mangru, S. D., Harrison, D. J., J. Chromatogr. A 1997, 781, 271–276. [7] Tan, W., Fan, Z. H., Qiu, C. X., Ricco, A. J., Gibbons, I., Electrophoresis 2002, 23, 3638–3645. [8] Hong, J. W., Fuji, T., Seki, M., Yamamoto, T., Endo, I., Electrophoresis 2001, 22, 328–333. [9] Mitnik, L., Carey, L., Burger, R., Desmarais, S., Koutny, L., Wernet, O., Matsudaira, P., Ehrlich, D., Electrophoresis 2002, 23, 719–726. [10] Wang, J., Pumera, M., Chatrathi, M. P., Escarpa, A., Konrad, R., Griebel, A., Dörner, W., Löwe, H., Electrophoresis 2002, 23, 596–601. [11] Dolnk, V., Liu, S., Jovanovich, S., Electrophoresis 2000, 21, 41–54. [12] Manica, D. P., Ewing, A. G., Electrophoresis 2002, 23, 3735– 3743. [13] Guijt, R. M., Baltussen, E., van den Steen, G., Schasfoort, R. B. M., Schlautmann, S., Billiet, H. A. H., Frank, J., van Dedem, G. W. K., van den Berg, A., Electrophoresis 2001, 22, 235–241. [14] Huang, Z., Sanders, J. C., Dunsmor, C., Ahmadzadeh, H., Landers, J. P., Electrophoresis 2001, 22, 3924–3929. [15] Horton, H. R., Moran, L. A., Ochs, R. S., Rawn, J. D., Scrimgeour, K. G., Principles of Biochemistry, 3rd ed., Prentice Hall, New Jersey 2000. [16] Ernst, H., Knoll, M., Anal. Chim. Acta 2001, 449, 129–134. [17] Shih, Y., Zen, J.-M., Electroanalysis 1999, 11, 229–233.


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[18] Zen, J.-M., Chung, H.-H., Kumar, A. S., Anal. Chim. Acta 2000, 421, 189–197. [19] Chen, Z., Gao, Y., Su, R., Li, C., Lin, J., Electrophoresis 2003, 24, 3246–3253. [20] Nicolas, P. C. S., Qiu, F., Gooch, K. A., Fisher, A. C., J. Phys. Chem. B 2000, 104, 7110–7114. [21] Fu, L.-M., Yang, R.-J., Lee, G.-B., Electrophoresis 2002, 23, 602–612. [22] Kenis, P. J. A., Ismagilov, R. F., Whitesides, G. M., Science 1999, 285, 83–85. [23] Baldwin, R. P., Roussel, T. J., Jr., Crain, M. M., Bathlagunta, V., Jackson, D. J., Gullapalli, J., Conklin, J. A., Pai, R., Naber, J. F., Walsh, K. M., Keynton, R. S., Anal. Chem. 2002, 74, 3690–3697. [24] Kappes, T., Schnierle, P., Hauser, P. C., Electrophoresis 2000, 21, 1390–1394.

© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2005, 26, 3007–3012 [25] Zeng, Y., Chen, H., Pang, D.-W., Wang, Z.-L., Cheng, J.-K., Anal. Chem. 2002, 74, 2441–2445. [26] Vandaveer IV, W. R., Pasas, S. A., Martin, R. S., Lunte, S. M., Electrophoresis 2002, 23, 3667–3677. [27] Martin, R. S., Ratzlaff, K. L., Huynh, B. H., Lunte, S. M., Anal. Chem. 2002, 74, 1136–1143. [28] Zen, J.-M., Hsu, C.-T., Hsu, Y.-L., Sue, J.-W., Conte, E. C., Anal. Chem. 2004, 76, 4251–4255. [29] Zen, J.-M., Tsai, D.-M., Kumar, A. S., Electroanalysis 2003, 15, 1171–1176. [30] Zen, J.-M., Tsai, D.-M., Yang, H.-H., Electroanalysis 2002, 14, 1597–1600. [31] Fanguy, J. C., Henry, C. S., Electrophoresis 2002, 23, 767– 773.

A new fabrication process for a microchip ... - Wiley Online Library

We report here a novel and simple process for the fabrication of a poly(methyl metha- crylate) (PMMA)-based microchip electrophoresis device, integrated with a screen- printed three-electrode electrochemical detector that does not require a replicate mold. In this approach, a photoresist layer constitutes both an adhesion ...

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