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Metalless electrodes for capacitively coupled contactless conductivity detection on electrophoresis microchips Gerson Francisco Duarte Junior1, José Alberto Fracassi da Silva2,5, Kelliton José Mendonça Francisco3, Claudimir Lucio do Lago3, Emanuel Carrilho4,5 and Wendell K. T. Coltro1,5*
1
Instituto de Química, Universidade Federal de Goiás, Goiânia, Goiás, Brasil.
2
Instituto de Química, Universidade Estadual de Campinas, Campinas São Paulo, Brasil.
3
Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brasil.
4
Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos São Paulo, Brasil.
5
Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Campinas, São Paulo Brasil.
*
Corresponding Author:
Professor Wendell K. T. Coltro Instituto de Química, Universidade Federal de Goiás Campus Samambaia, 74001-970, P.O. Box 131 Goiânia, GO, Brasil Fax: +55 62 3521 1127 E-mail: [email protected]
Abbreviations: His, L-histidine; IPA, isopropanol; PANI, Polyaniline
Keywords: analytical instrumentation, electrochemical detection, ionic liquids, microfluidic devices, sensing electrodes.
Words: ca. 4,100
Received: 23-Jan-2015; Revised: 05-Mar-2015; Accepted: 13-Mar-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/elps.201500033. This article is protected by copyright. All rights reserved.
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Abstract This paper describes the use of ionic solutions as sensing electrodes for capacitively coupled contactless conductivity detection (C4D) on electrophoresis microchips. Initially, two channels were engraved in a PMMA holder by using a CO2 laser system and sealed with a thin adhesive membrane. PDMS electrophoresis chips were fabricated by soft lithography and reversibly sealed against the polymer membrane. Different ionic solutions were investigated as metalless electrodes. The electrode channels were filled with KCl solutions prepared in conductivity values from ca. 10 to 40 S/m. The best analytical response was achieved using the KCl solution with 21.9 S/m conductivity (2 mol/L). Besides KCl, we also tested NaCl and LiCl solutions for actuating as detection electrodes. Taking into account the same electrolyte concentration (2 mol/L), the best response was recorded with KCl solution due to its higher ionic conductivity. The optimum operating frequency (400 kHz) and the best sensing electrode (2 mol/L KCl) were used to monitor electrophoretic separations of a mixture containing K+, Na+ and Li+. The use of liquid solutions as sensing electrodes for C4D measurements has revealed great performance to monitor separations on chip-based devices, avoiding complicated fabrication schemes to include metal deposition and encapsulation of electrodes. The LOD values were estimated to be 28, 40 and 58 µmol/L for K+, Na+ and Li+, respectively, what is comparable to that of conventional metal electrodes. When compared to the use metal electrodes, the proposed approach offer advantages regarded to the easiness of fabrication, simplicity, and lower cost per device.
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1. Introduction Capacitively coupled contactless conductivity detection (C4D) has become a powerful system to monitor electrophoretic separations on chip-based devices [1-8]. In general, metallic electrodes can be embedded in the microchip, placed either underneath the bottom or on top of the device, or can also be external to the microchip electrophoresis (ME) devices [9-20]. In all examples, the detection electrodes are electrically isolated from the contact with the fluid inside the channel by a physical barrier with typical thickness from 10 to 150 µm. Due to some advantages as compatibility with microfabrication techniques and also absence of electrical interferences related to the coupling of the electric field applied for electrophoresis, C4D has become one of the most popular detection modes for ME devices. The electrodes for C4D on ME devices may be fabricated using different metal sources like platinum [21], gold [18, 22, 23], copper [14, 15, 19, 20, 24], and aluminum [9, 10, 25-27]. However, the conventional fabrication methods involve photolithographic and sputtering techniques, which are not always available to most of the researchers. For this reason, the development of alternative techniques for manufacturing detection electrodes has received growing attention. In this scenario, different authors have reported the use of silver ink [28, 29], metallic adhesive tapes [12, 13], printed circuitry boards [19, 20, 30], polymer [31], gallium [32], and alloys [33] as electrodes for C4D measurements. Metallic adhesive tapes offer simplicity, low cost and easiness of fabrication to obtain planar electrodes [7]. Despite the low cost, the use of conductive adhesive tapes limits the investigation of more complex geometries. In 2012, Henderson and colleagues proposed the use of polyaniline (PANI) electrodes to apply the AC voltages required for C 4D measurements [31]. The polymer electrodes were integrated into a laser-patterned ME device. The performance achieved on such devices was comparable to devices with metal electrodes and the cost was ca. 20 times lower [31]. In 2013, Thredgold and co-workers described a new
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approach for the fabrication of electrodes by concept of “injected electrodes” [32]. The microchip and electrodes were made in one-step by molding of PDMS. Molten gallium was then injected into electrodes channels and became solidified by addition of a seed crystal [32]. More recently, Gaudry et al. developed in-plane alloy electrodes for C4D on ME devices [33]. In this study, small pellets of Woods metal alloy were placed at the electrode contact and heated at temperature above its melting point (~70 oC). The heating allowed the melting of the alloy and ensured the filling of electrode channel. The use of injected electrodes, like gallium and alloy, are advantageous when compared to metal-deposition techniques. Basically, fluidic channels are filled with molten materials, which promote suitable conductivity for electrical measurements after solidified. In addition to the alternative metallic electrodes, Blaszczyk and co-workers reported a few years ago the use of KCl electrodes for C4D measurements [34, 35]. The authors used these pseudo-electrodes to monitor conductivity changes on flow injection devices prepared in PDMS. In comparison with CuSO4 and NaNO3 electrodes, they reported that the KCl pseudoelectrodes provided the higher changes in the baseline magnitude. When compared to the conventional fabrication technologies often adopted to prepare metallic electrodes, the use of electrolytic solutions as sensing electrodes for C4D measurements offers some advantages like low instrumental requirements and ability to allow easily the design of new geometries in short processing time. In the pioneering reports, the authors demonstrated the concept of liquid electrodes for C4D measurements monitoring conductivity changes during the hydrodynamic pumping of KCl solutions (as analyte) prepared at concentration range between 0.01 and 0.5 mol/L [34, 35]. This current study reports the first use of ionic solutions for actuating as sensing electrodes for C4D on ME devices. For this purpose, microfluidic channels were engraved in a PMMA plate and later filled with electrolytes for C4D measurements. The PMMA plate containing the electrode microchannels were sealed with an adhesive membrane and reversibly integrated with electrophoresis microchips fabricated in PDMS. The proof-of-concept has been successfully investigated using different ionic solutions as sensing electrodes to monitor electrophoretic separations of a model mixture of inorganic cations.
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2. Materials and methods 2.1. Chemicals and materials All reagents were of analytical grade. 2-(N-morpholine)ethanesulfonic acid (MES), Lhistidine (His), potassium chloride, sodium chloride, lithium chloride, calcium chloride, sodium hydroxide, and sodium sulphate were obtained from Sigma Aldrich Co. (St. Louis, MO). Running buffer and standard solutions were prepared by dissolving the reagents or diluting stock solutions in ultrapure water with resistivity 18.2 MΩ cm from Millipore (Kansas City, MO). The sample solutions for experiments were prepared diluting stock solutions in water. Sylgard 184 and curing agent were purchased from Dow Corning (Elizabethtown, KY). The DurexTM (45 mm wide and 40 µm thick) adhesive membrane was received from 3M (Manaus, Brazil). Poly(methyl methacrylate) (PMMA) plates (50 mm width × 90 mm long × 4 mm thick) were purchased from Vick (Campinas, Brazil).
2.2. Fabrication of channels in PMMA to be filled with ionic solutions Two microfluidic channels were engraved in a PMMA piece to be used as liquid electrodes (see labels e1 and e2 in Figure 1). Channels were engraved using a 30 W CO 2 Laser Engraver LS100 (Gravograph, La Chapelle St Luc, France) system under vector mode applying 20% power, 10% speed, 1000 × 1000 dpi resolution and 1 mm out of the focal plane. The electrode channel dimensions were 1-mm wide and 1-mm deep. Both electrode channels were arranged in a parallel orientation with total length of 36 mm and gap of 1 mm (see Fig. 1). In order to minimize the stray capacitance [1], the length of electrode channels in parallel was 2 mm. Details about the geometry and dimensions of the electrode channels can be seen in Figure S1, available in supporting information (SI). The PMMA piece with engraved channels was sealed with a 40-µm-thick adhesive membrane, as schematized in Figure 1. To avoid the evaporation of electrode solutions, reservoirs were made with cylindrical tubes (o.d. = 7 mm; i.d. = 5 mm; 5 mm height) and glued at the channel ends with a bicomponent epoxy resin (Araldite, São Bernardo do Campo, Brazil).
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2.3. Fabrication of PDMS electrophoresis devices and assembly PDMS electrophoresis microchips were fabricated by soft lithography using a high-relief master previously defined in silicon wafer as described elsewhere [36]. Briefly, PDMS monomer and the curing agent were mixed at a ratio of 10:1 (m/m), poured then on the master, and kept at 70 ºC during 30 min. Afterwards, PDMS replica was peeled off the master and reversibly sealed against the PMMA-engraved piece, previously coated with an adhesive membrane to provide the physical insulation between the electrode and fluidic channels. PDMS channels were produced in a simple cross-format with 40-µm-wide and 20-µm-deep. The electrophoresis devices consisted of a 4 cm long separation channel (from the intersection to the end of the separation channel) and 0.7 cm long side arms. The holes for reservoirs were produced using 3 mm biopsy puncher. Details about the device layout can be seen in Figure S-2, available in SI. Insert Figure 1
2.4. Electrophoresis procedure and Instrumentation Microchannels were firstly filled with isopropanol (IPA) by capillary action to avoid the formation of bubbles. Subsequently, IPA was replaced by running buffer solution composed of an equimolar mixture of MES and His (20 mmol/L each). Prior to injection, microfluidic channels were electrokinetically conditioned during 10 min. Sample injection was performed using gated mode. For this purpose, the potentials applied to sample and buffer reservoirs were, respectively, +0.8 and +1.0 kV keeping sample and buffer waste reservoirs grounded. Sample injection was then performed by floating the voltage at the buffer reservoir for 2 s. Injection and separation were accomplished by a bipolar two-channel high voltage sequencer model ER230 from Edaq (Denistone East, Australia). A C4D system was built according to the electronic circuit developed by Francisco and do Lago [37]. An external function generator (model GV-2002) acquired from ICEL (Manaus, Brazil) was used to apply a sinusoidal signal of 2 V (peak-to-peak) to the excitation electrode. The data acquisition was recorded in software written in LabVIEWTM. Platinum wires were used to make electrical contact with the ionic solution electrodes (see labels e1 and e2 in Fig. 1).
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3. Results and discussion The development of alternative and simple approaches to create and incorporate electrodes for C4D measurements on ME devices has been one of the current trends in the microscale separation community. One of the main motivations is related to the limited access to photolithographic and sputtering techniques. The replacement of metallic conductors by ionic solutions as sensing electrodes is quite advantageous once they can fill the microfluidic channels by capillarity at room temperature and remain with good stability over time. However, it is necessary to avoid the evaporation of the ionic solutions, once this phenomenon promotes an increase of conductivity and consequently affect the C4D response. It has been observed that for the evaporation rate of 50%, the solution conductivity raises from 26 to ca. 40 S/m. Besides the detector response, the solution evaporation may also causes the crystallization and consequently the blockage of inside channels. This effect would generate bubbles and interrupt the electrical conductivity of the liquid sensing electrodes. To minimize this problem, reservoirs were fixed at the extremities of the electrode channels and the electrolytic solutions were periodically replaced. Also, the reservoirs were useful to support platinum electrodes used for electrical connections. When compared to conventional technologies often explored to prepare electrodes like sputtering, evaporation or screen printing approaches, the use of liquid electrodes offers easiness of fabrication, simplicity and lower cost.
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3.1. Operating frequency and electrode concentration optimization We have systematically investigated the use of liquid electrodes as sensing electrodes for monitoring electrophoretic separations in microfluidic devices. In order to demonstrate our hypothesis, the operating parameters as well as the ionic solution conductivity were optimized. Typical electropherograms were successfully recorded with ionic electrodes composed of a 2 mol/L KCl solution at a frequency range between 200 and 500 kHz. The detector response performance has demonstrated dependence on the operating frequency, as expected. The best signal-to-noise ratio (SNR) was achieved for a 400-kHz frequency, as it can be seen in Figure S-3, available in SI. It is important to highlight the operational parameters should be optimized for different running buffers. The original C4D operated at 1.1 MHz, [37] which is a frequency appropriate for fused silica capillaries. However, the optimal frequency may be different for different geometries, sizes, and materials. In addition, two 2.2 nF 400 V capacitors were inserted between the electrodes and the electronics. In case of dielectric failure of the polymeric membrane, these capacitors represent an additional barrier to the DC high voltage and protect the electronic. The capacitive reactance at 400 kHz imposed by each capacitor is only 181 Ω, which is two order of magnitude lower than the impedance imposed by the liquid electrode. Therefore, they do not represent a significant contribution to the total impedance of the detection cell. In addition, it has been noticed that the ionic solution concentration affects the detector response, as shown in Figure 2A. Basically, different electropherograms were recorded for the detection of K+ (500 mol/L) using sensing electrodes prepared with KCl in concentration levels from 1 to 4 mol/L. For these solutions, the conductivity values ranged from ca. 10 to 40 S/m. According to the data shown in Figure 2A, the best SNR for the K+ peak was achieved using a 2 mol/L (21.9 S/m) KCl solution. However, the use of solutions prepared in the mol/L range also promotes the increase in the solution viscosity. For this
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reason, the drop in the SNR values for concentrations greater than 2 mol/L may be attributed to the increase in the cell impedance – induced by the solution viscosity. Besides the use of KCl solution, other electrolytic solutions were tested as sensing electrodes. First, we performed the comparison of the detector response using KCl, NaCl and LiCl solutions (2 mol/L each) as liquid electrodes. According to the results presented in Figure 2B, the best response was provided with 2 mol/L KCl solution due to its higher ionic conductivity. The data recorded with NaCl and LiCl solutions were not statistically different from each other. Additionally, the analytical response recorded using sensing electrodes prepared with ionic solutions composed of NaOH, CaCl2 and Na2SO4 was also investigated. Based on the electropherograms recorded (data not shown), the peak intensities recorded with CaCl2 and Na2SO4 (2 mol/L each) electrodes were slightly lower than those recorded with 2 mol/L KCl electrodes. On the other hand, the profile of the electropherogram recorded using NaOH electrodes was quite similar to the data achieved with KCl electrodes (data not shown).
Insert Figure 2 3.2. Analytical performance The best ionic solution as well as the best operating parameters were fixed and used for a preliminary investigation of the analytical performance of liquid electrodes for C4D measurements on ME devices. For this purpose, an equimolar mixture of K+, Na+ and Li+ (300 µmol/L each) was chosen as model mixture to demonstrate the proof-of-concept. As it can be seen in Figure 3, a series of four sequential electropherograms was recorded using the proposed electrodes. The RSD values for the migration times and peak areas related to all cationic species ranged from 2 to 4% and from 6 to 16%, respectively.
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Insert Figure 3 The LOD values were estimated (SNR = 3) to be 28, 40, and 58 µmol/L for K+, Na+ and Li+, respectively. The values achieved herein are higher than those typically reported with metal electrodes [26, 29, 30, 38], but they are lower than the LOD’s recently reported with inplane alloy electrodes (85326 µmol/L) [33]. Besides the detectability levels, the separation efficiencies for K+, Na+, and Li+ were 4,700 ± 800, 9,800 ± 500, and 34,000 ± 4,000 plates/m, respectively. Taking into account the short effective length (3.5 cm), the separation monitored with liquid electrodes exhibited an acceptable performance with baseline resolution (see electropherogram highlighted in Fig. 3). The fronting behavior observed in the peaks recorded in the electropherogram shown may be attributed to the electrodispersion phenomenon [39]. Furthermore, it is important to highlight that sensitivity achieved with ionic electrodes can be improved by optimizing of the detection cell geometry [7] as well as investigating other electrode shapes as recen[39]tly described by Lima and co-workers [27]. The results obtained for the C4D with liquid electrodes agree with the simplified electrical model for the C4D system. The model proposed herein includes an additional resistor element (Re), which was introduced in series with the detection cell (see Fig. S-4, available in SI). Since the impedance of Re is much smaller than the cell impedance, the frequency response of the detector is basically dictated by the cell, but better results in terms of peak height are obtained for ionic electrodes with higher conductivity (lower values of R e).
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4. Concluding remarks In summary, we have described the use of ionic solutions as sensing electrodes for contactless conductivity detection on ME devices. The main achievement is regarded to the possibility of employing liquid solutions instead metal electrodes. In comparison with metal or injected electrodes, the use of ionic solutions as detection electrodes offers simplicity and it allows an easy integration of electrodes for C4D on ME chips. In addition, other advantages of the proposed approach are related to the fabrication time and the possibility to create electrodes and electrophoresis channels in single-step technologies, like soft lithography [40, 41], direct printing [12, 26] methods, or 3D direct microfabrication [42]. Regarding the analytical feasibility, the replacement of metal by liquid electrodes did not compromise the analytical performance of the ME-C4D system. The detectability levels found (2858 µmol/L) are lower than the values recently reported with in-plane alloy electrodes[33] and comparable to those achieved with planar electrodes [43]. However, it is important to note that the detection cell geometry as well as the electrode shape can be optimized to enhance the sensitivity of the proposed approach, and opens new venues for exploration of electrolytes that could enhance sensitivity.
This project was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grants No. 478911/2012-2 and No. 448089/2014-9, Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Instituto Nacional de Ciência e Tecnologia de Bioanalítica (INCTBio). CNPq is also acknowledged for researcher fellowships granted to J.A.F.S. (grant No. 309778/2011-5), C.L.L. (grant No. 304239/2010-0), E.C. (grant No. 311323/2011-1) and W.K.T.C. (grant No. 311744/2013-3). The authors have declared no conflict of interest.
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5. References [1] Brito-Neto, J. G. A., da Silva, J. A. F., Blanes, L., do Lago, C. L., Electroanalysis 2005, 17, 1198-1206. [2] Brito-Neto, J. G. A., da Silva, J. A. F., Blanes, L., do Lago, C. L., Electroanalysis 2005, 17, 1207-1214. [3] Pumera, M., Talanta 2007, 74, 358-364. [4] Matysik, F.-M., Microchim. Acta 2008, 160, 1-14. [5] Kubáň, P., Hauser, P. C., Anal. Chim. Acta 2008, 607, 15-29. [6] Kubáň, P., Hauser, P. C., Electrophoresis 2011, 32, 30-42. [7] Coltro, W. K. T., Lima, R. S., Segato, T. P., Carrilho, E., de Jesus, D. P., do Lago, C. L., da Silva, J. A. F., Anal. Methods 2012, 4, 25-33. [8] Kubáň, P., Hauser, P. C., Electrophoresis 2015, 36, 195-211. [9] 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, 2537-2541. [10] Pumera, M., Wang, J., Opekar, F., Jelinek, I., Feldman, J., Lowe, H., Hardt, S., Anal. Chem. 2002, 74, 1968-1971. [11] Wang, J., Pumera, M., Collins, G. E., Mulchandani, A., Anal. Chem. 2002, 74, 61216125. [12] do Lago, C. L., da Silva, H. D. T., Neves, C. A., Brito-Neto, J. G. A., da Silva, J. A. F., Anal. Chem. 2003, 75, 3853-3858. [13] Tanyanyiwa, J., Abad-Villar, E. M., Fernandez-Abedul, M. T., Costa-Garcia, A., Hoffmann, W., Guber, A. E., Herrmann, D., Gerlach, A., Gottschlich, N., Hauser, P. C., Analyst 2003, 128, 1019-1022. [14] Kubáň, P., Hauser, P. C., Electrophoresis 2005, 26, 3169-3178. [15] Chen, Z., Li, Q., Li, O., Zhou, X., Lan, Y., Wei, Y., Mo, J., Talanta 2007, 71, 19441950. [16] Ding, Y., Garcia, C. D., Rogers, K. R., Anal. Lett. 2008, 41, 335-350. [17] Mahabadi, K. A., Rodriguez, I., Lim, C. Y., Maurya, D. K., Hauser, P. C., de Rooij, N. F., Electrophoresis 2010, 31, 1063-1070. [18] Segato, T. P., Coltro, W. K. T., de Jesus Almeida, A. L., de Oliveira Piazetta, M. H., Gobbi, A. L., Mazo, L. H., Carrilho, E., Electrophoresis 2010, 31, 2526-2533. [19] Coltro, W. K. T., da Silva, J. A. F., Carrilho, E., Anal. Methods 2011, 3, 168-172.
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Figure 1. Assembling of an integrated ME-C4D device based on the use of ionic solutions as sensing electrodes. The labels e1 and e2 mean the excitation and receiver electrodes, respectively.
Figure 1.
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Figure 2. Effect of the electrode solution conductivity on the signal-to-noise ratio (SNR) for the K+ peak (500 mol/L) on ME separation (n=3). In (A), the SNR was calculated using KCl solutions prepared in a concentration range from 1 (ca. 11 S/m) to 4 mol/L (ca. 40 S/m), as sensing electrodes. In (B), the SNR values were calculated using KCl, NaCl, or LiCl (2 mol/L each), as sensing electrodes. Operating frequency was 400 kHz.
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Figure 3. Typical electropherograms recorded sequentially (n=4) for the separation of K+, Na+, and Li+ (300 µmol/L each) using 2 mol/L KCl as sensing electrodes. Running buffer: 20
C4D Signal
mmol/L MES/20 mmol/L His (pH 6.1). Other conditions: see Fig. 2.
1000 AU 0
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Metalless electrodes for capacitively coupled contactless conductivity detection on electrophoresis microchips Gerson Francisco Duarte Junior1, José Alberto Fracassi da Silva2,5, Kelliton José Mendonça Francisco3, Claudimir Lucio do Lago3, Emanuel Carrilho4,5 and Wendell K. T. Coltro1,5*
1
Instituto de Química, Universidade Federal de Goiás, Goiânia, Goiás, Brasil.
2
Instituto de Química, Universidade Estadual de Campinas, Campinas São Paulo, Brasil.
3
Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brasil.
4
Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos São Paulo, Brasil.
5
Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Campinas, São Paulo Brasil.
*
Corresponding Author:
Professor Wendell K. T. Coltro Instituto de Química, Universidade Federal de Goiás Campus Samambaia, 74001-970, P.O. Box 131 Goiânia, GO, Brasil Fax: +55 62 3521 1127 E-mail: [email protected]
Abbreviations: His, L-histidine; IPA, isopropanol; PANI, Polyaniline
Keywords: analytical instrumentation, electrochemical detection, ionic liquids, microfluidic devices, sensing electrodes.
Words: ca. 4,100
Received: 23-Jan-2015; Revised: 05-Mar-2015; Accepted: 13-Mar-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/elps.201500033. This article is protected by copyright. All rights reserved.
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Abstract This paper describes the use of ionic solutions as sensing electrodes for capacitively coupled contactless conductivity detection (C4D) on electrophoresis microchips. Initially, two channels were engraved in a PMMA holder by using a CO2 laser system and sealed with a thin adhesive membrane. PDMS electrophoresis chips were fabricated by soft lithography and reversibly sealed against the polymer membrane. Different ionic solutions were investigated as metalless electrodes. The electrode channels were filled with KCl solutions prepared in conductivity values from ca. 10 to 40 S/m. The best analytical response was achieved using the KCl solution with 21.9 S/m conductivity (2 mol/L). Besides KCl, we also tested NaCl and LiCl solutions for actuating as detection electrodes. Taking into account the same electrolyte concentration (2 mol/L), the best response was recorded with KCl solution due to its higher ionic conductivity. The optimum operating frequency (400 kHz) and the best sensing electrode (2 mol/L KCl) were used to monitor electrophoretic separations of a mixture containing K+, Na+ and Li+. The use of liquid solutions as sensing electrodes for C4D measurements has revealed great performance to monitor separations on chip-based devices, avoiding complicated fabrication schemes to include metal deposition and encapsulation of electrodes. The LOD values were estimated to be 28, 40 and 58 µmol/L for K+, Na+ and Li+, respectively, what is comparable to that of conventional metal electrodes. When compared to the use metal electrodes, the proposed approach offer advantages regarded to the easiness of fabrication, simplicity, and lower cost per device.
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1. Introduction Capacitively coupled contactless conductivity detection (C4D) has become a powerful system to monitor electrophoretic separations on chip-based devices [1-8]. In general, metallic electrodes can be embedded in the microchip, placed either underneath the bottom or on top of the device, or can also be external to the microchip electrophoresis (ME) devices [9-20]. In all examples, the detection electrodes are electrically isolated from the contact with the fluid inside the channel by a physical barrier with typical thickness from 10 to 150 µm. Due to some advantages as compatibility with microfabrication techniques and also absence of electrical interferences related to the coupling of the electric field applied for electrophoresis, C4D has become one of the most popular detection modes for ME devices. The electrodes for C4D on ME devices may be fabricated using different metal sources like platinum [21], gold [18, 22, 23], copper [14, 15, 19, 20, 24], and aluminum [9, 10, 25-27]. However, the conventional fabrication methods involve photolithographic and sputtering techniques, which are not always available to most of the researchers. For this reason, the development of alternative techniques for manufacturing detection electrodes has received growing attention. In this scenario, different authors have reported the use of silver ink [28, 29], metallic adhesive tapes [12, 13], printed circuitry boards [19, 20, 30], polymer [31], gallium [32], and alloys [33] as electrodes for C4D measurements. Metallic adhesive tapes offer simplicity, low cost and easiness of fabrication to obtain planar electrodes [7]. Despite the low cost, the use of conductive adhesive tapes limits the investigation of more complex geometries. In 2012, Henderson and colleagues proposed the use of polyaniline (PANI) electrodes to apply the AC voltages required for C 4D measurements [31]. The polymer electrodes were integrated into a laser-patterned ME device. The performance achieved on such devices was comparable to devices with metal electrodes and the cost was ca. 20 times lower [31]. In 2013, Thredgold and co-workers described a new
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approach for the fabrication of electrodes by concept of “injected electrodes” [32]. The microchip and electrodes were made in one-step by molding of PDMS. Molten gallium was then injected into electrodes channels and became solidified by addition of a seed crystal [32]. More recently, Gaudry et al. developed in-plane alloy electrodes for C4D on ME devices [33]. In this study, small pellets of Woods metal alloy were placed at the electrode contact and heated at temperature above its melting point (~70 oC). The heating allowed the melting of the alloy and ensured the filling of electrode channel. The use of injected electrodes, like gallium and alloy, are advantageous when compared to metal-deposition techniques. Basically, fluidic channels are filled with molten materials, which promote suitable conductivity for electrical measurements after solidified. In addition to the alternative metallic electrodes, Blaszczyk and co-workers reported a few years ago the use of KCl electrodes for C4D measurements [34, 35]. The authors used these pseudo-electrodes to monitor conductivity changes on flow injection devices prepared in PDMS. In comparison with CuSO4 and NaNO3 electrodes, they reported that the KCl pseudoelectrodes provided the higher changes in the baseline magnitude. When compared to the conventional fabrication technologies often adopted to prepare metallic electrodes, the use of electrolytic solutions as sensing electrodes for C4D measurements offers some advantages like low instrumental requirements and ability to allow easily the design of new geometries in short processing time. In the pioneering reports, the authors demonstrated the concept of liquid electrodes for C4D measurements monitoring conductivity changes during the hydrodynamic pumping of KCl solutions (as analyte) prepared at concentration range between 0.01 and 0.5 mol/L [34, 35]. This current study reports the first use of ionic solutions for actuating as sensing electrodes for C4D on ME devices. For this purpose, microfluidic channels were engraved in a PMMA plate and later filled with electrolytes for C4D measurements. The PMMA plate containing the electrode microchannels were sealed with an adhesive membrane and reversibly integrated with electrophoresis microchips fabricated in PDMS. The proof-of-concept has been successfully investigated using different ionic solutions as sensing electrodes to monitor electrophoretic separations of a model mixture of inorganic cations.
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2. Materials and methods 2.1. Chemicals and materials All reagents were of analytical grade. 2-(N-morpholine)ethanesulfonic acid (MES), Lhistidine (His), potassium chloride, sodium chloride, lithium chloride, calcium chloride, sodium hydroxide, and sodium sulphate were obtained from Sigma Aldrich Co. (St. Louis, MO). Running buffer and standard solutions were prepared by dissolving the reagents or diluting stock solutions in ultrapure water with resistivity 18.2 MΩ cm from Millipore (Kansas City, MO). The sample solutions for experiments were prepared diluting stock solutions in water. Sylgard 184 and curing agent were purchased from Dow Corning (Elizabethtown, KY). The DurexTM (45 mm wide and 40 µm thick) adhesive membrane was received from 3M (Manaus, Brazil). Poly(methyl methacrylate) (PMMA) plates (50 mm width × 90 mm long × 4 mm thick) were purchased from Vick (Campinas, Brazil).
2.2. Fabrication of channels in PMMA to be filled with ionic solutions Two microfluidic channels were engraved in a PMMA piece to be used as liquid electrodes (see labels e1 and e2 in Figure 1). Channels were engraved using a 30 W CO 2 Laser Engraver LS100 (Gravograph, La Chapelle St Luc, France) system under vector mode applying 20% power, 10% speed, 1000 × 1000 dpi resolution and 1 mm out of the focal plane. The electrode channel dimensions were 1-mm wide and 1-mm deep. Both electrode channels were arranged in a parallel orientation with total length of 36 mm and gap of 1 mm (see Fig. 1). In order to minimize the stray capacitance [1], the length of electrode channels in parallel was 2 mm. Details about the geometry and dimensions of the electrode channels can be seen in Figure S1, available in supporting information (SI). The PMMA piece with engraved channels was sealed with a 40-µm-thick adhesive membrane, as schematized in Figure 1. To avoid the evaporation of electrode solutions, reservoirs were made with cylindrical tubes (o.d. = 7 mm; i.d. = 5 mm; 5 mm height) and glued at the channel ends with a bicomponent epoxy resin (Araldite, São Bernardo do Campo, Brazil).
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2.3. Fabrication of PDMS electrophoresis devices and assembly PDMS electrophoresis microchips were fabricated by soft lithography using a high-relief master previously defined in silicon wafer as described elsewhere [36]. Briefly, PDMS monomer and the curing agent were mixed at a ratio of 10:1 (m/m), poured then on the master, and kept at 70 ºC during 30 min. Afterwards, PDMS replica was peeled off the master and reversibly sealed against the PMMA-engraved piece, previously coated with an adhesive membrane to provide the physical insulation between the electrode and fluidic channels. PDMS channels were produced in a simple cross-format with 40-µm-wide and 20-µm-deep. The electrophoresis devices consisted of a 4 cm long separation channel (from the intersection to the end of the separation channel) and 0.7 cm long side arms. The holes for reservoirs were produced using 3 mm biopsy puncher. Details about the device layout can be seen in Figure S-2, available in SI. Insert Figure 1
2.4. Electrophoresis procedure and Instrumentation Microchannels were firstly filled with isopropanol (IPA) by capillary action to avoid the formation of bubbles. Subsequently, IPA was replaced by running buffer solution composed of an equimolar mixture of MES and His (20 mmol/L each). Prior to injection, microfluidic channels were electrokinetically conditioned during 10 min. Sample injection was performed using gated mode. For this purpose, the potentials applied to sample and buffer reservoirs were, respectively, +0.8 and +1.0 kV keeping sample and buffer waste reservoirs grounded. Sample injection was then performed by floating the voltage at the buffer reservoir for 2 s. Injection and separation were accomplished by a bipolar two-channel high voltage sequencer model ER230 from Edaq (Denistone East, Australia). A C4D system was built according to the electronic circuit developed by Francisco and do Lago [37]. An external function generator (model GV-2002) acquired from ICEL (Manaus, Brazil) was used to apply a sinusoidal signal of 2 V (peak-to-peak) to the excitation electrode. The data acquisition was recorded in software written in LabVIEWTM. Platinum wires were used to make electrical contact with the ionic solution electrodes (see labels e1 and e2 in Fig. 1).
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3. Results and discussion The development of alternative and simple approaches to create and incorporate electrodes for C4D measurements on ME devices has been one of the current trends in the microscale separation community. One of the main motivations is related to the limited access to photolithographic and sputtering techniques. The replacement of metallic conductors by ionic solutions as sensing electrodes is quite advantageous once they can fill the microfluidic channels by capillarity at room temperature and remain with good stability over time. However, it is necessary to avoid the evaporation of the ionic solutions, once this phenomenon promotes an increase of conductivity and consequently affect the C4D response. It has been observed that for the evaporation rate of 50%, the solution conductivity raises from 26 to ca. 40 S/m. Besides the detector response, the solution evaporation may also causes the crystallization and consequently the blockage of inside channels. This effect would generate bubbles and interrupt the electrical conductivity of the liquid sensing electrodes. To minimize this problem, reservoirs were fixed at the extremities of the electrode channels and the electrolytic solutions were periodically replaced. Also, the reservoirs were useful to support platinum electrodes used for electrical connections. When compared to conventional technologies often explored to prepare electrodes like sputtering, evaporation or screen printing approaches, the use of liquid electrodes offers easiness of fabrication, simplicity and lower cost.
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3.1. Operating frequency and electrode concentration optimization We have systematically investigated the use of liquid electrodes as sensing electrodes for monitoring electrophoretic separations in microfluidic devices. In order to demonstrate our hypothesis, the operating parameters as well as the ionic solution conductivity were optimized. Typical electropherograms were successfully recorded with ionic electrodes composed of a 2 mol/L KCl solution at a frequency range between 200 and 500 kHz. The detector response performance has demonstrated dependence on the operating frequency, as expected. The best signal-to-noise ratio (SNR) was achieved for a 400-kHz frequency, as it can be seen in Figure S-3, available in SI. It is important to highlight the operational parameters should be optimized for different running buffers. The original C4D operated at 1.1 MHz, [37] which is a frequency appropriate for fused silica capillaries. However, the optimal frequency may be different for different geometries, sizes, and materials. In addition, two 2.2 nF 400 V capacitors were inserted between the electrodes and the electronics. In case of dielectric failure of the polymeric membrane, these capacitors represent an additional barrier to the DC high voltage and protect the electronic. The capacitive reactance at 400 kHz imposed by each capacitor is only 181 Ω, which is two order of magnitude lower than the impedance imposed by the liquid electrode. Therefore, they do not represent a significant contribution to the total impedance of the detection cell. In addition, it has been noticed that the ionic solution concentration affects the detector response, as shown in Figure 2A. Basically, different electropherograms were recorded for the detection of K+ (500 mol/L) using sensing electrodes prepared with KCl in concentration levels from 1 to 4 mol/L. For these solutions, the conductivity values ranged from ca. 10 to 40 S/m. According to the data shown in Figure 2A, the best SNR for the K+ peak was achieved using a 2 mol/L (21.9 S/m) KCl solution. However, the use of solutions prepared in the mol/L range also promotes the increase in the solution viscosity. For this
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reason, the drop in the SNR values for concentrations greater than 2 mol/L may be attributed to the increase in the cell impedance – induced by the solution viscosity. Besides the use of KCl solution, other electrolytic solutions were tested as sensing electrodes. First, we performed the comparison of the detector response using KCl, NaCl and LiCl solutions (2 mol/L each) as liquid electrodes. According to the results presented in Figure 2B, the best response was provided with 2 mol/L KCl solution due to its higher ionic conductivity. The data recorded with NaCl and LiCl solutions were not statistically different from each other. Additionally, the analytical response recorded using sensing electrodes prepared with ionic solutions composed of NaOH, CaCl2 and Na2SO4 was also investigated. Based on the electropherograms recorded (data not shown), the peak intensities recorded with CaCl2 and Na2SO4 (2 mol/L each) electrodes were slightly lower than those recorded with 2 mol/L KCl electrodes. On the other hand, the profile of the electropherogram recorded using NaOH electrodes was quite similar to the data achieved with KCl electrodes (data not shown).
Insert Figure 2 3.2. Analytical performance The best ionic solution as well as the best operating parameters were fixed and used for a preliminary investigation of the analytical performance of liquid electrodes for C4D measurements on ME devices. For this purpose, an equimolar mixture of K+, Na+ and Li+ (300 µmol/L each) was chosen as model mixture to demonstrate the proof-of-concept. As it can be seen in Figure 3, a series of four sequential electropherograms was recorded using the proposed electrodes. The RSD values for the migration times and peak areas related to all cationic species ranged from 2 to 4% and from 6 to 16%, respectively.
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Insert Figure 3 The LOD values were estimated (SNR = 3) to be 28, 40, and 58 µmol/L for K+, Na+ and Li+, respectively. The values achieved herein are higher than those typically reported with metal electrodes [26, 29, 30, 38], but they are lower than the LOD’s recently reported with inplane alloy electrodes (85326 µmol/L) [33]. Besides the detectability levels, the separation efficiencies for K+, Na+, and Li+ were 4,700 ± 800, 9,800 ± 500, and 34,000 ± 4,000 plates/m, respectively. Taking into account the short effective length (3.5 cm), the separation monitored with liquid electrodes exhibited an acceptable performance with baseline resolution (see electropherogram highlighted in Fig. 3). The fronting behavior observed in the peaks recorded in the electropherogram shown may be attributed to the electrodispersion phenomenon [39]. Furthermore, it is important to highlight that sensitivity achieved with ionic electrodes can be improved by optimizing of the detection cell geometry [7] as well as investigating other electrode shapes as recen[39]tly described by Lima and co-workers [27]. The results obtained for the C4D with liquid electrodes agree with the simplified electrical model for the C4D system. The model proposed herein includes an additional resistor element (Re), which was introduced in series with the detection cell (see Fig. S-4, available in SI). Since the impedance of Re is much smaller than the cell impedance, the frequency response of the detector is basically dictated by the cell, but better results in terms of peak height are obtained for ionic electrodes with higher conductivity (lower values of R e).
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4. Concluding remarks In summary, we have described the use of ionic solutions as sensing electrodes for contactless conductivity detection on ME devices. The main achievement is regarded to the possibility of employing liquid solutions instead metal electrodes. In comparison with metal or injected electrodes, the use of ionic solutions as detection electrodes offers simplicity and it allows an easy integration of electrodes for C4D on ME chips. In addition, other advantages of the proposed approach are related to the fabrication time and the possibility to create electrodes and electrophoresis channels in single-step technologies, like soft lithography [40, 41], direct printing [12, 26] methods, or 3D direct microfabrication [42]. Regarding the analytical feasibility, the replacement of metal by liquid electrodes did not compromise the analytical performance of the ME-C4D system. The detectability levels found (2858 µmol/L) are lower than the values recently reported with in-plane alloy electrodes[33] and comparable to those achieved with planar electrodes [43]. However, it is important to note that the detection cell geometry as well as the electrode shape can be optimized to enhance the sensitivity of the proposed approach, and opens new venues for exploration of electrolytes that could enhance sensitivity.
This project was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grants No. 478911/2012-2 and No. 448089/2014-9, Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Instituto Nacional de Ciência e Tecnologia de Bioanalítica (INCTBio). CNPq is also acknowledged for researcher fellowships granted to J.A.F.S. (grant No. 309778/2011-5), C.L.L. (grant No. 304239/2010-0), E.C. (grant No. 311323/2011-1) and W.K.T.C. (grant No. 311744/2013-3). The authors have declared no conflict of interest.
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5. References [1] Brito-Neto, J. G. A., da Silva, J. A. F., Blanes, L., do Lago, C. L., Electroanalysis 2005, 17, 1198-1206. [2] Brito-Neto, J. G. A., da Silva, J. A. F., Blanes, L., do Lago, C. L., Electroanalysis 2005, 17, 1207-1214. [3] Pumera, M., Talanta 2007, 74, 358-364. [4] Matysik, F.-M., Microchim. Acta 2008, 160, 1-14. [5] Kubáň, P., Hauser, P. C., Anal. Chim. Acta 2008, 607, 15-29. [6] Kubáň, P., Hauser, P. C., Electrophoresis 2011, 32, 30-42. [7] Coltro, W. K. T., Lima, R. S., Segato, T. P., Carrilho, E., de Jesus, D. P., do Lago, C. L., da Silva, J. A. F., Anal. Methods 2012, 4, 25-33. [8] Kubáň, P., Hauser, P. C., Electrophoresis 2015, 36, 195-211. [9] 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, 2537-2541. [10] Pumera, M., Wang, J., Opekar, F., Jelinek, I., Feldman, J., Lowe, H., Hardt, S., Anal. Chem. 2002, 74, 1968-1971. [11] Wang, J., Pumera, M., Collins, G. E., Mulchandani, A., Anal. Chem. 2002, 74, 61216125. [12] do Lago, C. L., da Silva, H. D. T., Neves, C. A., Brito-Neto, J. G. A., da Silva, J. A. F., Anal. Chem. 2003, 75, 3853-3858. [13] Tanyanyiwa, J., Abad-Villar, E. M., Fernandez-Abedul, M. T., Costa-Garcia, A., Hoffmann, W., Guber, A. E., Herrmann, D., Gerlach, A., Gottschlich, N., Hauser, P. C., Analyst 2003, 128, 1019-1022. [14] Kubáň, P., Hauser, P. C., Electrophoresis 2005, 26, 3169-3178. [15] Chen, Z., Li, Q., Li, O., Zhou, X., Lan, Y., Wei, Y., Mo, J., Talanta 2007, 71, 19441950. [16] Ding, Y., Garcia, C. D., Rogers, K. R., Anal. Lett. 2008, 41, 335-350. [17] Mahabadi, K. A., Rodriguez, I., Lim, C. Y., Maurya, D. K., Hauser, P. C., de Rooij, N. F., Electrophoresis 2010, 31, 1063-1070. [18] Segato, T. P., Coltro, W. K. T., de Jesus Almeida, A. L., de Oliveira Piazetta, M. H., Gobbi, A. L., Mazo, L. H., Carrilho, E., Electrophoresis 2010, 31, 2526-2533. [19] Coltro, W. K. T., da Silva, J. A. F., Carrilho, E., Anal. Methods 2011, 3, 168-172.
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Figure 1. Assembling of an integrated ME-C4D device based on the use of ionic solutions as sensing electrodes. The labels e1 and e2 mean the excitation and receiver electrodes, respectively.
Figure 1.
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Figure 2. Effect of the electrode solution conductivity on the signal-to-noise ratio (SNR) for the K+ peak (500 mol/L) on ME separation (n=3). In (A), the SNR was calculated using KCl solutions prepared in a concentration range from 1 (ca. 11 S/m) to 4 mol/L (ca. 40 S/m), as sensing electrodes. In (B), the SNR values were calculated using KCl, NaCl, or LiCl (2 mol/L each), as sensing electrodes. Operating frequency was 400 kHz.
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Figure 3. Typical electropherograms recorded sequentially (n=4) for the separation of K+, Na+, and Li+ (300 µmol/L each) using 2 mol/L KCl as sensing electrodes. Running buffer: 20
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