Fabrication and testing of high-performance detection ... - Springer Link

Biomed Microdevices (2008) 10:73–80 DOI 10.1007/s10544-007-9111-1

Fabrication and testing of high-performance detection sensor for capillary electrophoresis microchips Lung-Ming Fu & Chia-Yen Lee & Ming-Huei Liao & Che-Hsin Lin

Published online: 7 August 2007 # Springer Science + Business Media, LLC 2007

Abstract This study presents a new approach for highperformance detection sensors for MEMS-based capillary electrophoresis chips to substitute laser induced fluorescence (LIF) detection systems. The developed sensors easily integrate with well-known microfabrication techniques for glass-based microfluidic devices. Three-dimensional gold electrodes are structured in enveloping side channels by sputtering and patterned using a standard “lift-off” process. The variations in the capacitance between the electrodes in the side channels are measured as different samples and ions pass through the detection region of the capillary electrophoresis separation channel. Samples of beer, white wine and milk are each mixed in different buffer solutions, then successfully separated and detected using the developed device. The proposed high-performance detection sensors have microscale dimensions and provide a critical step towards the realization of the lab-on-a-chip concept. L.-M. Fu Department of Materials Engineering, National Pingtung University of Science and Technology, 912 Pingtung, Taiwan C.-Y. Lee (*) Department of Mechanical and Automation Engineering, Da-Yeh University, 515 Changhua, Taiwan e-mail: [email protected] M.-H. Liao Department of Veterinary Medicine, National Pingtung University of Science and Technology, 912 Pingtung, Taiwan C.-H. Lin (*) Department of Mechanical and Electro-Mechanical Engineering, National Sun Yat-sen University, 804 Kaohsiung, Taiwan e-mail: [email protected]

Keywords Detection sensors . Capillary electrophoresis chip . Lab-on-a-chip

1 Introduction Microfabrication analytical systems, known also as “lab-ona-chip” devices, can dramatically change the way that biochemical assays are performed (Erickson 2005; Yun and Yoon 2006; Gao et al. 2005; Li et al. 2005; Wei et al. 2006; Chang et al. 2006; Lee et al. 2006; Bown and Meinhart 2006). Particularly attractive are micromachined capillary electrophoresis (CE) microchips because of their fast and efficient separation capabilities (Du et al. 2005). The advantages of microchip separations over conventional analytical methods include extremely short analysis time, negligible consumption of reagents and samples, disposability of separation devices, and the possibility of being simply incorporated into portable devices. Traditional CE systems generally incorporate some form of optical detection system. Most reports on microchip CE devices rely on laser-induced fluorescence (LIF; Fu et al. 2003, 2005; Fu and Lin 2004; Huang et al. 2006; Tsai et al. 2006; Wu and Yang 2006; Lin et al. 2007) to detect the analytes at some points towards the end of the separation channel. LIF is fairly easy to implement with many biochemical analytes, though most of these require labeling with a fluorescent marker, adding an extra step in the analysis process. However, the system is relatively bulky and expensive. One miniaturized approach is to integrate optic fibers in the microchip device for LIF detection. (Lin et al. 2004a,b,c) Nevertheless, it is challenging to integrate the optic fibers with the on-chip CE system since a precise alignment for the integrated fibers is essential to obtain a good detection performance. Thus, end-channel (Heberta

74

and Brazill 2003), in-channel (Lagally et al. 2005) and offchannel (Vuorinen et al. 2003) detection approaches have been developed for enhancement of the electrochemical detection systems. Interest in electrochemical detection arrangements is growing. Among them, off-channel detection systems with two or more electrodes positioned at a small distance from the separation channels have attracted particular attention. Without the need for special transducers (e.g. photomultipliers), such systems directly permit electrical measurement to be carried out. For all manner of compounds, contactless electrical detectors are suitable universal detectors (Vuorinen et al. 2003). Recently, optical detection methods were developed in Ref. (Fu et al. 2004; Lin et al. 2004a). Optical waveguides like Su-8/spin-onglass (SOG; Lin et al. 2004a) and optical fibers (Fu et al. 2004) were buried beside the separation channels. The detection light propagated through the waveguide structure by a process of total internal reflection. As samples or cells flow through the detection region, the passage of the detection light is interrupted and the corresponding changes in the light intensity are detected by a second waveguide buried on the opposite side of the channel. The results indicated a successful detection of samples in separation channels with the coupled waveguides. In the past decade, many different arrangements for integrating contactless electrodes at a minimum distance from the capillary were developed to improve the detection sensitivity. A contactless conductometric detector was presented with an easily exchangeable capillary (Tùma et al. 2001). A mixture of inorganic cations were successfully separated and detected with electrodes spaced at 1 mm distance from a commercial capillary. Pumera et al. (2002) placed two planar aluminum film electrodes outside a poly(methyl methacrylate; PMMA) microchip as a contactless conductivity microchip detector and successfully measured the impedance of the solution in the separation channel. The planar electrodes were placed 700 μm away from each other and showed a feasible integration with microfabricated planar CE chips. On-chip capacitively coupled with four-electrode conductivity detectors for CE devices were proposed to experimentally verify that the measurement characteristics of the four-electrode configuration are better than those of the classical two-electrode detection setup (Laugere et al. 2003). Vuorinen et al. (2003) integrated a contactless conductivity detection (CCD) system and presented a highly promising detection method due to the universality of the system. Abad-Villar et al. (2004) demonstrated contactless conductivity measurements for both the conventional capillary and the microchip CE chip. Human immunoglobulin M (IgM) was used as a model analyte, and good detection performance was obtained by using the conventional capillary and the CE chip. Wang et al. (2003) introduced a movable contactless conductivity

Biomed Microdevices (2008) 10:73–80

detector with positioning the detector at different positions along the separation channel by “sliding” the electrode holder. By using the moving contactless conductivity detector, analytical performance can be enhanced and optimal separation at shorter analyzing time can be achieved. Wang and Pumera (2004) also proposed a dual conductivity/amperometric detection system for microchip capillary electrophoresis. The coupling of conductivity and amperometric detection modes in a single separation channel enhanced the sample characterization. The present study designs and fabricates a highperformance detection sensor for a capillary electrophoresis microchip. Using simple fabrication processes, capacitor electrodes are deposited in buried side channels located on either side of the separation channel. The experimental results show that the device is capable of separating and detecting various samples with high detection sensitivity. The proposed high-performance detection sensor provides a valuable contribution to the realization of the “Lab-on-aChip” concept.

2 Experimental section 2.1 Design The present study used “buried” shielding electrodes to detect the capacitance variation in a sample flow [Fig. 1(b)]. Since the measured capacitance response is proportional to the effective area of the sensing electrode, a simple method was used in the current study to sculpt 3-D shielding electrodes to increase the effective area of the

Fig. 1 Schematic of: (a) cross-shaped microchannel with two short shielding electrodes designed to detect the capacitance variation of the sample flow, and (b) close-up perspective view of the electrodes

Biomed Microdevices (2008) 10:73–80

75

Fig. 2 Overview of fabrication process used to form contactless capacitive detector for microchip capillary electrophoresis. (a) spinning on PR, (b) exposure, (c) developing, (d) BOE etching and PR stripping, (e) spinning on PR, (f) exposure, (g) developing, (h) sputtering Cr/Au, (i) patterning electrodes by “Liftoff” process, (j) cover glass drilling, and (k) alignment and vacuum fusion bonding

sensing electrodes. In compared with commonly-used planar electrodes deposited on one side of the channel surface, buried shielding electrodes provide a larger effective sensing area while sensing the analytes in the separation channel and result in a better detection performance. It is also possible to deposit thicker planar electrodes to increase the effective sensing area. However, current leakage usually caused by an incomplete bonding of the device can be eliminated using this approach. In the present study, individual electrodes were deposited near the microchannels of cross-shaped microfluidic devices [Fig. 1(a)]. Electrodes were located on both sides of the separation

channel for detecting the separated samples with higher sensitivity [Fig. 1(b)]. The spacing from the separation channel to the buried electrodes is 50 μm such that contactless detection scheme was achieved and the current leakage during detection can be avoided. The electrodes were used to measure the capacitance changes induced by the passing analytes inside the separation channel. Sensing performance can be significantly improved due to the use of 3-D buried shielding electrodes. We designed two electrode pairs was simply to improve the yield of the fabricated chip. It is nothing to do with neither the time resolve detection nor the differential measurement. As described in the

76

manuscript, the detection method is a contactless capacitive detection which measures the capacitance change in the microchannel using a high frequency signal. 2.2 Microfabrication The present high-performance detection sensor was fabricated on glass substrates (Assistant Inc., Germany). Prior to fabrication, the substrates were annealed at 400°C for 4 h to relieve any internal residual stress. Figure 2 presents a schematic illustration of the current fabrication process (Lin et al. 2004b; Lin et al. 2005; Tai et al. 2006; Tsai et al. 2007). After being annealed, the glass substrates were cleaned in a boiling Piranha solution (H2SO4 (%):H2O2 (%)=3:1) at 120°C for 10 min. Traditionally, a timeconsuming vacuum deposition process is used to fabricate the mask for glass etching in a HF-based etchant. However, this study used a 3-μm thick AZ4620 (Clariant Corp., USA) photoresist layer. The photoresist was spun coated on the glass substrate and then etched using a wet chemical etching process [Fig. 2(a)]. A standard UV lithography process [Fig. 2(b)] was then used to generate the required configuration of microchannels. Note that the microchannels were designed with a width of 30 μm. The PR layer was then developed by immersing the exposed substrate in a developer solution (AZ400k: DI water=1:3) for 70 s [Fig. 2(c)]. The patterned substrates were then etched in a 6:1 BOE (buffered oxide etchant, J. T. Baker, USA) bath aided by ultrasonic agitation for 30 min to form microfluidic trenches of depth 25 μm following the stripping of the PR layer [Fig. 2(d)]. The microchannels were formed using a modified glass etching technique with an etching rate of 0.9 μm/min (Lin et al. 2001). Importantly, the average surface roughness (Ra) of the etched surface was controlled such that it was less than 45 Å to ensure a high quality microchannel surface. Therefore, it was not necessary to protect the backside of the glass substrates during the etching procedure. The fabrication steps shown in Fig. 2(a–c) were then repeated, as shown in Fig. 2(e–g), to form the offchannel detector electrodes adjacent to the downstream region of the separation channel. After the photoresist stripping process, a thin layer of Cr (0.05 μm) was sputtered into the shielding electrode channels to form an adhesion layer for the subsequent deposition of a 0.4-μm Au layer [Fig. 2(h)]. A “lift-off” method was then used to pattern the Au layer to form the two electrical electrodes [Fig. 2(i)]. Meanwhile, via holes with a diameter of 1.5 mm were drilled in a second glass plate to form sample inlets and outlets [Fig. 2(j)]. The bottom and the cover plates were then fusionbonded in a vacuum furnace at 580°C for 20 min to form the completed microfluidic device [Fig. 2(k)]. The shielding electrode channels let these detection electrodes be “buried” inside the microchip so that it could

Biomed Microdevices (2008) 10:73–80

measure the capacitance values between the two electrodes beside the channels [Fig. 3(a)]. Thus measured capacitance values varied as separated ions passing through the detection area of the microchannels. Figure 3(b) presents a photo image of the proposed microfluidic chip after fabrication. The photographs of planar and high-performance Au electrodes are presented in Fig. 4. The microchannel width was 80 μm, and the diameter of the reservoirs was 1.5 mm for both figures. A planar Au electrode can also be observed in Fig. 4(b). The Au electrode was deposited on the cover plate, which just formed a planar electrode. In Fig. 4(c), the Au electrodes were deposited both on the cover plate and within the channel of the bottom plate. An overall high-performance Au electrode can be successfully fabricated as bonding the two plates.

3 Results and discussion Similar to the layout of the two-pairs-of-electrodes detector design, the read-out electronics had to be designed carefully owing to the low intensity signals to be measured. Figure 5 presents a schematic illustration of the current experimental setup used to test the performance of the developed capillary electrophoresis (CE) chip with high-performance electrodes. As shown, a high voltage programmable power

Fig. 3 Photographs of fabricated microchip device: (a) detecting electrodes and (b) completed chip assembly

Biomed Microdevices (2008) 10:73–80

77

Fig. 4 Photographs of 2-D planar and high-performance Au electrodes in the bonded chips. (a) Planar view, (b) cross-sectional view of the channel with a planar electrode, and (c) cross-sectional view of the channel with a high performance Au electrode

supply (MP-3500, Major Science, Taiwan) was used to generate the electrokinetic force required to inject the sample and drive it through the microchip. The injection step was driven by an electrical field of 200 V/cm applied over a 15-s loading time, while the separation step was performed using an electrical field of 300 V/cm applied over a 45-s separation time. Three different test samples were considered in this study, namely: (1) white wine [50 ppm (Na+), 1,100 ppm (K+), 110 ppm (Ca2+), and 52 ppm (Mg2+), Robert Mondavi Moobridge White Zinfandel (Robert Mondavi Co., USA)]; (2) beer (40.3 ppm (Li+), 7.3 ppm (Na+), 560 ppm (K+), 46.4 ppm (Ca2+), and 121 ppm (Mg2+), Taiwan Beer (Taiwan Tobacco and Liquor Co., Taiwan)]; and (3) milk [525 ppm (Na+), 1723 ppm (K+), 1426 ppm (Ca2+) and 192 ppm (Mg2+), NPUST milk (National

Fig. 5 Schematic diagram of experimental setup for contactless capacitive-type detector

Pingtung University of Science and Technology, Taiwan)] in a buffer fluid of 10 mM 2-(N-morpholino)ethanesulfonic acid (MES)/10 mM histidine(His; pH 6.1), respectively. The reported cation concentrations of the samples were listed in Table 1. Note that these values were obtained from the ingredient table listed on the product packages. A LCR meter (LCR-821, Good Will, Taiwan) was used to detect the instantaneous capacitance or impedance variations as the separated samples were driven through the detection region of the separation channel. The capacitance values measured during the course of the separation experiments were taken to represent the sample concentration by its velocities induced by the electrokinetic force (EOF) of the different cations, respectively. In order to prevent the sample leakage effect from degrading the detection perfor-

78

Biomed Microdevices (2008) 10:73–80

Table 1 Ion concentrations for different commercial drinks

White wine Beer Milk

Sodium

Calcium

Potassium

Magnesium

Ammonium

50 7.3 525

110 46.4 1,426

1,100 560 1,723

52 121 192

None 40.3 None

Note that the unit is ppm (mg/l).

tion electrodes. It is observed that each sample separation in the proposed CCD has four capacitance peaks, corresponding to the four fragments of the K+, Ca2+, Na+, and Mg2+cations [Fig. 8(b)]. The measured numbers of theoretical plates per meter for K+ are 25,000 and 80,000 plates/m in Fig. 8(a) and (b), respectively. Figure 9 represents two sets of electropherograms for a more realistic sample (a beer sample) which was also analyzed for inorganic cations by the two-pairs-of-electrodes CCDs device. In the present experiment, the beer sample measured of six fragments of the NH4+, K+, Ca2+, Na+, Mg2+, and Li+ inorganic cations. The peak intensities of the six samples are very clearly distinguishable in Fig. 9(a) and (b). The results indicate that the two-pairs-of-electrodes CCDs device successfully separate and detect sample fluids and therefore confirms its ability of the proposed high-performance detector. Figure 10 represents two sets of electropherograms for a milk sample which was also analyzed for inorganic cations by the two-pairs-of-electrodes CCDs device. Note that the

1.2 First pair CCD Second pair CCD

1.0

Relative peak response

mance of the microfluidic device, this study employed the double-L injection method (Fu and Lin 2003; Lin et al. 2004a,b,c; Tsai et al. 2005, 2006) shown in Fig. 6. In order to find the optimized operating conditions in terms of excitation frequency and waveform on the highperformance detection electrodes for the two-pairs-ofelectrodes CCDs, a sinusoidal excitation signal waveform was used and the output voltage was kept at 5 V peak-topeak. Sample plugs were transported through the highperformance detector using a field of 300 V/cm. Experiments were carried out at an operating frequency between 1 and 35 kHz. The heights of the sample peaks were measured on the electropherograms and plotted as a function of frequency. Figure 7 shows the effect of applied frequency on the high-performance detection electrodes at 5 V peakto-peak. At a frequency of 17 kHz, the two-pairs-ofelectrodes detectors response to the sample plug show a maximum peak. Note that both the capacitance and the impedance detection modes can get a similar but inversed results using the proposed microchip device. However, capacitance measurement provided a larger signal response such that only capacitance measurement results were given in the following discussion. Figure 8 shows the peak capacitance values observed at different times after the initial injection of the white wine sample with a separation electrical field of 300 V/cm in the detectors with planar and high-performance Au electrodes. Four cations were separated due to their different electrophoretic mobilities. In Fig. 8(a), the capacitance intensity of the planar-structured detector is weak as compared to the measured signals of the proposed high-performance detec-

0.8 0.6 0.4 0.2 0.0

Fig. 6 Double-L injection method in microfluidic chip: (a) injection step and (b) separation step

0

5

10 15 20 25 Frequency (kHz)

30

35

Fig. 7 Response effect of the applied frequency on the two pair detectors. At a frequency of 17 kHz, the response of the two-pairs-ofelectrodes detector to the sample plug is at a maximum peak of 5 V peak-to-peak

Biomed Microdevices (2008) 10:73–80 Fig. 8 Determination of inorganic cations in white wine sample using (a) planar contactless electrodes and (b) proposed high-performance detection system (K+, Ca2+, Na+ and Mg2+)

79 14

(a)

Capacitance (nF)

12

(b)

10 8

8 +

K

6

6

4

2+

2

0

10

20

30

milk samples were first denatured by addition of glacial acetic acid (Merck, final concentration of acetic acid in the milk samples, 1.5% v/v) and subsequently centrifuged at 5,000 rpm for 20 min. Four fragments of the K+, Ca2+, Na+, Mg2+ cations were separated and detected with high performance for the milk sample in Fig. 10(a) and (b).

4 Conclusions This study has successfully demonstrated an innovative detection sensor with high performance for capillary

Mg

2+

Ca

+

Na

40

4

2+

Mg

2+

Ca

50

60 0

10

20

Time (s)

30

Na+

2

40

50

0

60

Time (s)

electrophoresis microchips. A new fabrication process has been developed in which the detection sensor comprises microchannels for sample injection and separation, and shielding channels for two buried detection electrodes. The proposed detection sensor demonstrates that the electrokinetically driven sample can be separated in the CE channel and the separated cations are detected as they pass through the detection region for three different samples. The developed device provides a simultaneous sample separation and detection capability, and therefore provides a viable basis for the future development in micro-TAS devices.

35

(a)

+

K

2+

(a)

30

Ca +

6

K

25 2+

Mg

4

Capacitance Intensity (nF)

Capacitance Intensity (nF)

12

10

0

8

14

+

K

2+

Ca

+ NH4

2

+

Li

Na

(b)

8

+

+

K

6 2+

Mg

4

Na+

20 15 10

2+

Mg

5 2+

(b)

30

Ca +

K

25

Na+

20 15

2+

+

NH4

2

Ca

Na 0

0

2+

10 +

Li

+

5 10 15 20 25 30 35 40 45 50 55 60

Time (s) Fig. 9 Determination of inorganic cations in beer sample using proposed high-performance detection system. a, b: electropherogram obtained using the 1st and 2nd electrode pair, respectively. (NH4+, K+, Ca2+, Na+, Mg2+and Li+)

Mg

5 0

0

5 10 15 20 25 30 35 40 45 50 55 60

Time (s) Fig. 10 Determination of inorganic cations in milk sample using proposed high-performance detection system. a, b: electropherogram obtained using the 1st and the 2nd electrode pair, respectively. (K+, Ca2+, Na+ and Mg2+)

80 Acknowledgments The current authors gratefully acknowledge the financial support provided to this project by the National Science Council of Taiwan under Grant Number NSC-95-2314-B-020-001MY2 and NSC-95-2221-E-212-058.

References E.M.Abad-Villar, J. Tanyanyiwa, M.T. Femández-Abedul, A. Costa-Garcia, P.C. Hauser, Anal. Chem. 76, 1282 (2004) M.R. Bown, C.D. Meinhart, Microfluid. Nanofluid. 2, 513 (2006) Y.H. Chang, G.B. Lee, F.C. Huang, Y.Y. Chen, J.L. Lin, Biomed. Microdev. 8, 215 (2006) W.B. Du, Q.H. Fang, Q.H. He, Z.L. Fang, Anal. Chem. 77, 1330 (2005) D. Erickson, Microfluid. Nanofluid. 12, 310 (2005) L.M. Fu, C.H. Lin, Anal. Chem. 75, 5790 (2003) L.M. Fu, C.H. Lin, Electrophoresis 25, 3652 (2004) L.M. Fu, R.J. Yang, G.B. Lee, Y.J. Pan, Electrophoresis 24, 3026 (2003) L.M. Fu, R.J. Yang, C.H. Lin, G.B. Lee, Y.J. Pan, Anal. Chim. Acta 507, 163 (2004) L.M. Fu, R.J. Yang, C.H. Lin, Y.S. Chien, Electrophoresis 26, 1814 (2005) Y. Gao, G. Hu, F.Y.H Lin, P.M. Sherman, D. Li, Biomed. Microdev. 7, 301 (2005) N.E. Heberta, S.A. Brazill, Lab Chip 3, 241 (2003) M.Z. Huang, R.J. Yang, C.H. Tai, C.H. Tsai, L.M. Fu, Biomed. Microdev. 8, 309 (2006) E.T. Lagally, S. Lee, H.T. Soh, Lab Chip 5, 1053 (2005) F. Laugere, R.M. Guijt, J. Bastemeijer, G. van der Steen, A. Berthold, E. Baltussen, P. Sarro, G.W.K. van Dedem, M. Vellekoop, A. Bossche, Anal. Chem. 75, 306 (2003)

Biomed Microdevices (2008) 10:73–80 C.Y. Lee, C.M. Chen, G.L. Chang, C.H. Lin, L.M. Fu, Electrophoresis 27, 5043 (2006) F. Li, D.D. Wang, X.P. Yan, J.M. Lin, R.G. Su, Electrophoresis 26, 2261 (2005) C.H. Lin, G.B. Lee, Y.H. Lin, G.L. Chang, J. Micromech. Microeng. 11, 726 (2001) C.H. Lin, L.M. Fu, Y.S. Chien, Anal. Chem. 76, 5265 (2004a) C.H. Lin, G.B. Lee, L.M. Fu, S.H. Chen, Biosens. Bioelectron. 20, 83 (2004b) C.H. Lin, R.J. Yang, C.H. Tai, C.Y. Lee, L.M. Fu, J. Micromech. Microeng. 14, 639 (2004c). C.H. Lin, C.H. Tsai, L.M. Fu, J. Micromech. Microeng. 15, 935 (2005). C.H. Lin, C.H. Tsai, C.W. Pan, L.M. Fu, Biomed. Microdev. 9, 43 (2007) M. Pumera, J. Wang, F. Opekar, I. Jelínek, J. Feldman, H. Löwe, S. Hardt, Anal. Chem. 74, 1968 (2002) C.H. Tai, R.J. Yang, M.Z. Huang, C.W. Liu, C.H. Tsai, L.M. Fu, Electrophoresis 27, 4982 (2006) C.H. Tsai, R.J. Yang, C.H. Tai, L.M. Fu, Electrophoresis 26, 674 (2005) C.H. Tsai, Y.N. Wang, C.F. Lin, R.J. Yang, L.M. Fu, Electrophoresis 27, 4991 (2006) C.H. Tsai, H.T. Chen, Y.N. Wang, C.H. Lin, L.M. Fu, Macrofluid. Nanofluid. 3, 13 (2007) P. Tùma, F. Opeka, I. Jelinek, Electroanalysis 13, 989 (2001) J. Wang, M. Pumera, Anal. Chem. 74, 5919 (2004). J. Wang, G. Chen, A. Muck, Anal. Chem. 75, 4475 (2003). C.W. Wei, J.Y. Cheng, T.H. Young, Biomed. Microdev. 8, 65 (2006). C.H. Wu, R.J. Yang, Biomed. Microdev. 8, 119 (2006) K.S. Yun, E. Yoon, Biomed. Microdev. 7, 35 (2006) P.S. Vuorinen, M. Jussila, H. Sirén, S. Palonen, M. Riekkola, J. Chromatogr. A 990, 45 (2003)