Critical points in the fabrication of microfluidic devices on glass substrates

Available online at www.sciencedirect.com

Sensors and Actuators B 130 (2008) 436–448

Critical points in the fabrication of microfluidic devices on glass substrates a , Diego F. Pozo Ayuso b , Miguel Garc´ıa Granda b , ´ Mario Casta˜no-Alvarez M. Teresa Fern´andez-Abedul a , Jose Rodr´ıguez Garc´ıa b , Agust´ın Costa-Garc´ıa a,∗ a

Departamento de Qu´ımica F´ısica y Anal´ıtica, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain b Departamento de F´ısica, Universidad de Oviedo, 33007 Oviedo, Asturias, Spain Available online 19 September 2007

Abstract Miniaturized total analysis systems are becoming a powerful tool for analytical and bioanalytical applications. In this work, microfluidic channels have been fabricated on glass substrates using photolithography and wet etching. Although these techniques are well-known and established, the influence of different parameters on the fabrication process of microchannels is of great importance. Thus, practical aspects and critical points of the procedure were considered and evaluated. Resulting glass chips with microfluidic channels were sealed with a cover plate to enclose the channels. Different low and high temperature bonding procedures have been evaluated. Microfluidic chips have been used in combination with a metal-wire end-channel amperometric detector for capillary electrophoresis (CE). The microfluidic channels and the amperometric detector have been tested using p-aminophenol as model analyte demonstrating that these devices are useful for analytical applications. © 2007 Elsevier B.V. All rights reserved. Keywords: Glass microfluidic; Electrophoresis microchip; Photolithography; Wet etching; Thermal bonding; Electrochemical detection

1. Introduction Micro-total analysis systems (␮TAS) have gained a great interest, especially since capillary electrophoresis (CE) microchips were proposed by Manz et al. [1,2]. These devices have several characteristic features such as speed, versatility, high performance, negligible consumption of reagent/sample and waste generation as well as the possibility of integration of various analytical steps, including sample preparation, mixing, reaction, separation and detection. Microfluidic fabrication clearly depends on the material employed. These devices have been mainly made of silicon and glass substrates using standard photolithographic and etching techniques [3–5]. In the last years, polymer materials are of increasing interest because their potentially low manufacturing costs may allow them to be disposable. Several polymers, including poly(methylmethacrylate) (PMMA) [6,7], poly(dimethylsiloxane) (PDMS) [8,9], polycarbonate (PC) [10], polyester [11], polystyrene (PS) [12] and poly(ethyleneterephthalate) (PET) [13], have been employed. Recently, cyclic olefin polymers and copolymers

∗

Corresponding author. Tel.: +34 9 85103488; fax: +34 9 85103125. E-mail address: [email protected] (A. Costa-Garc´ıa).

0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.09.043

such as Topas [14] or Zeonor [15,16] have also received attention. Fabrication processes on glass substrates are time-consuming and clean-room facilities are required. Therefore, the resulting microchips are usually expensive for use as disposable devices. However, characteristics such as good optical properties, efficiency in dissipating heat, well-understood surface characteristics as well as high resistance to mechanical stress and chemicals make them the first option. Moreover, their behavior is quite similar to traditional fused silica electrophoresis capillaries [17]. Ideal geometries of resulting microfluidic channels involve a high aspect ratio channel, which means deep channels with parallel sides. Non-parallel walls occur in glass with wetetching procedures because this process occurs on the exposed glass surface; hence, as the channel etches deeper, the walls are also etched. The result is channels wider at the top than at the base. An alternative to produce very deep channels with parallel sides is the use of dry etching techniques, such as powder blasting [18,19], plasma or deep reactive ion etching (DRIE) [20] and laser ablation [21]. However, these techniques require high cost instrumentation set-up and maintenance. After channels microfabrication, the system has to be assembled enclosing the channel networks or microstructures to allow fluids to flow through the device. In most cases, a cover plate made of the same material as the base is sealed to the device.

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The procedure for glass-to-glass bonding is not always straightforward as is shown in bibliography. Thus, various bonding methods such as anodic bonding, silicon fusion bonding and thermal bonding have been developed [4,5,22–25]. The thermal bonding process is used often in quartz and glass microchannels, although channel deformations are possible due to the high temperature required. Alternatively, low temperature process based on intermediate layers [26–32] has been used for glass-to-glass bonding. Glass microfluidic devices can be employed for capillary electrophoresis (CE). These capillary electrophoresis microchips require a sensitive and miniaturized detection system. Electrochemical detection (EC), especially amperometric detection, has been successfully employed in CE microchips due to advantages such as inherent miniaturization, sensitivity, low cost, portability and compatibility with microfabrication technology [33]. Electrochemical detection requires an appropriate design of the detector in order to ensure electrical isolation from the high separation voltage. Three approaches have been reported for coupling EC detection to CE: end-channel, inchannel and off-channel [34]. Although in-channel [35,36] and off-channel [37] formats are supposed to be more sensitive, the end-channel configuration has been more widely employed due to the simplicity and variety of detector designs [9,11,14,38–40]. In this work, a fast, low cost and reliable process for the fabrication of microfluidic devices on different glass substrates has been developed. The requirement for clean-room conditions was less rigorous than those reported in the bibliography. A rigorous study on the critical points affecting the fabrication procedure has been performed. Considerable attention has been directed toward simplifying and improving the reliability of the different manufacturing steps. Finally, a simple procedure for glass-to-glass bonding has been also described. The developed microchip has been applied to capillary electrophoresis demonstrating its analytical application. 2. Experimental 2.1. Reagents and materials p-Aminophenol (pAP), tris(hydroxymethyl) aminomethane (Tris), glycine (Gly) and sodium silicate solution (SiO2 ·Na2 O, 35%) were purchased from Sigma–Aldrich (St. Louis, MO 63103, USA). Potassium chloride, sodium hydroxide, ammonium hydroxide (25%), hydrogen peroxide (30%), acetone, isopropyl alcohol, nitric (65%), acetic (99%), sulfuric (95–97%) and hydrochloric (37%) acid were obtained from Merck (D-6100 Darmstadt, Germany). Hydrofluoric acid (48%), ammonium fluoride solution (40%) were supplied by Fluka (9471 Buchs, Switzerland). p-Aminophenol solution was prepared daily in the running buffer and protected from light. All solutions were filtered through nylon syringe filters (Cameo 30N, 0.1 ␮m, 30 mm) obtained from Osmonics (Minnetonka, MN 55343, USA).

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Water was purified employing a Milli-Q plus 185 equip from Millipore (Bedford, MA 01730). All other reagents were of analytical reagent grade. Micropipettes, 0.250 and 1.0 mL tips, and 1.5 mL tubes obtained from Eppendorf (22331 Hamburg, Germany) were also employed. The rest of volumetric material (flasks, pipettes, vessels. . .) was of analytical reagent grade. 2.2. Microchip fabrication Microfluidic channels have been fabricated in different glass substrates using photolithography and wet-etching technique. Fabrication process has been performed in a clean room and involved various steps (Fig. 1): 2.2.1. Substrate preparation Different glass substrates have been evaluated: commercially available soda-lime microscope glass slides (76 mm × 25 mm × 1 mm, Menzel-Gl¨asser, Germany), polished borofloat, and soda-lime glass (75 mm × 25 mm × 1 mm, Vitrotec S.A., Spain). Pyrex wafers (10 cm diameter, Vitrotec S.A., Spain), which were cut in small pieces, have been used too. Glass surfaces have to be adequately cleaned before photoresist deposition. Different washing procedures have been evaluated. The first one (C1) is described as follows: (1) Substrates were left soaking in deionized water with commercial detergent overnight. (2) They were sonicated in deionized water with commercial detergent at 40 ◦ C for 30 min. (3) Substrates were rinsed several times with deionized water in an ultrasonic bath. (4) They were sonicated in isopropyl alcohol for 30 min in order to eliminate the water. (5) Finally, they were blown dry with nitrogen gas. The second washing (C2) consisted of: (1) Substrates were left soaking in deionized water with commercial detergent overnight. (2) Substrates were rinsed with deionized water and acetone in order to eliminate organic material. (3) They were cleaned in piranha solution (H2 SO4 :H2 O2 , 3:1) for 15 min in an ultrasonic bath. (4) They were rinsed with deionized water in an ultrasonic bath. (5) They were sonicated in isopropyl alcohol for 30 min in order to eliminate the water. (6) Finally, they were blown dry with nitrogen gas. The last washing tested (C3) was based on: (1) Substrates were left soaking in deionized water with commercial detergent overnight.

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(4) They were rinsed with deionized water in an ultrasonic bath. (5) They were sonicated in isopropyl alcohol for 30 min in order to eliminate the water. (6) Finally, they were blown dry with nitrogen gas. After washing, substrates were dried in an oven at 90 ◦ C for 15 min. 2.2.2. Photolithography Glass substrates, depending of photoresist to be used, were spin-coated with Microposit Primer (Shipley, UK) in order to improve the photoresist adherence. The primer-treated or un-treated substrates were coated with a positive photoresist. Two different photoresist, S1813 (Shipley, UK) and ma-P1275 (Microresist Technology, Germany), were used in this study. Conditions of the spin-coating step (speed, acceleration and time) were optimized. After photoresist deposition, the substrates were soft-baked in an oven at 90 ◦ C for 30 min. The UV lithography was processed using chromium photomasks. These masks have been fabricated using a low cost laser photomask system developed in our laboratory [41]. This makes possible an easy modification of the designed pattern, as shown in Fig. 2, obtaining high-resolution mask (<5 ␮m). The glass substrates, coated with photoresist, were exposed to UV radiation (λ = 365 nm), through the fabricated mask, placed by simple contact on top of the photoresist. Thus, a mask aligner is not necessary to use. The exposure dose was calculated considering the power of the lamp, which has been previously characterized. The density of energy used was 150 and 252 mJ cm−2 for S1813 and ma-P1275, respectively. Development of the photoresist was accomplished in 90 s by immersing the exposed substrate into the developer, MF-311 (Shipley, UK) for S1813 and ma-D331 (Microresist Technology, Germany) for ma-P1275. After rinsing with deionized water, the temperature and time of the photoresist hard baking were studied for each photoresist. 2.2.3. Wet chemical etching Finally, glass substrates were immersed in the etching solution. Different HF-based solutions and etching times as well as several types of stirring have been evaluated. After etching, substrates were rinsed with deionized water and the photoresist was removed by dipping in acetone.

Fig. 1. Schematic representation of microfabrication process on glass substrates.

(2) Substrates were rinsed with deionized water and acetone in order to eliminate organic material. (3) Substrates were cleaned with NaOH 0.1 M for 15 min in an ultrasonic bath.

2.2.4. Glass bonding After microchannel fabrication, the system has to be assembled, enclosing the channel networks or microstructures to allow fluids to flow through the device. The device assembly has been made using different bonding processes. Before bonding, it is necessary to make connection holes to the flow channel (for supplying buffer and sample solutions). Those were manufactured by drilling the microslide with diamond drills (2 mm diameter) at the end of the microfluidic channels. Since glass bonding depends on the chemical surface state of the wafers to be sealed, the glass has to be adequately clean and flat. Different techniques based on intermediate layer as well

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Fig. 2. Photomask pattern with (I) twin T and (II) Π designs.

as thermal bonding have been evaluated. The micromachined substrate and cover plate were treated as follows: (1) Cleaning: sonicate in deionized water for 30 min and furtherly in acetone for 15 min. (2) Hydrophilic treatment: the substrates to be bonded were treated with NH4 OH:H2 O2 (3:1) solution for 30 min to give them hydrophilic properties. Next, the substrates were rinsed with deionized water, isopropyl alcohol and blown dry with nitrogen gas. (3) Intermediate layer: the cover plate was treated with several intermediate layers. A two-component epoxy glue (Araldit), a thin adhesive tape and a solution of sodium silicate (SiO2 ·Na2 O) were chosen for sealing the glass plates. Different procedures for deposition of the intermediate layers have been evaluated. (4) Bonding: the treated cover plate surface was immediately brought in contact with the glass substrate. Different bonding temperatures have been evaluated for annealing the device. 2.3. Electrochemical detector The amperometric detector was situated in the waste reservoir (B) with a three-electrode configuration. The reference and counter electrodes were coupled in a 250 ␮L micropipette tip. The reference electrode consisted of a 1 mm diameter silver wire anodized in saturated KCl, introduced in a tip through a syringe rubber piston. The tip is filled with saturated KCl solution and contains a low resistance liquid junction. A platinum

wire (0.3 mm diameter) that acted as auxiliary electrode was externally fixed with insulating tape. The working electrode was manually aligned at the outlet of the separation channel (as previously reported [14]) and it consisted of a 250 ␮m diameter gold wire (Alfa Aesar, Germany). The wire was introduced in the reservoir with the aid of a micropipette tip, then, it was adhered to the microchip with Araldit (Vantico AG, Basel, Switzerland). Finally, 1 cm long piece of a 250 ␮L micropipette tip is adhered to the chip hole fixing the wire. 2.4. Apparatus In order to fabricate the photomasks, a chromium layer must be deposited on top of a glass substrate. This layer was deposited by evaporation using an Emitech K950X (UK). The substrates were placed at the top of a high vacuum chamber. This chamber is pumped by a Varian DS-102 rotary pump assisted by a Turbomolecular pump (Boc Edwards, UK) reaching a pressure of less than 10−5 bar. In the bottom of the chamber, the chromium is situated inside a tungsten basket. After high vacuum was reached, a current of 10 A was applied through the basket, increasing its temperature and thus evaporating the metal, which is spread all over the chamber walls and substrates. A thin layer of the metal is obtained on the glass substrates and the layer thickness (100 nm) is controlled by a K150X Film Thickness Monitor (Emitech, UK). Photoresist deposition was made by spin-coating using a single wafer spin processor model WS-400A-6NPP from Laurell Technologies Corporation (North Wales, USA). The process

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involves depositing a small puddle of photoresist onto the centre of the glass substrate and then spinning the substrate at high speed fixed previously. Centripetal acceleration will cause the photoresist to spread to, and eventually off, the edge of the substrate leaving a thin film of photoresist on the surface [42]. A 473 nm Nd:YAG laser (CrystaLaser, USA) was employed for the direct photoresist exposure and the fabrication of photomasks. The glass substrates with the photoresist were moved during the laser beam exposure using two high-accuracy translation stages M-605.2DD (Physik Instrumente, Germany) of 50 mm (Y-axis) and 25 mm (X-axis). A shutter SH05 (ThorLabs, USA) was also employed for controlling the laser emission. Translation stages and shutter were interfaced to Pentium 3, 500 MHz, 128 MB RAM computer system and controlled by custom software based on LabView. The final exposure of the samples has been made employing a 365 nm UV lamp (LTF Labortechnik, Germany). Thermal bonding was made in a muffle furnace from Nabertherm (Germany). An ultrasonic bath, a magnetic stirrer Agimatic-E and a digitally controlled oven from JP Selecta (Spain) were also employed. Characterization of microfluidic channels were performed using a confocal microscope Leica TCS SP2/AOBS (Leica Microsystems, Germany). The images were processed employing Leica confocal software. Amperometric detection was performed with an Autolab PGSTAT 10 (ECO Chemie, Netherlands) potentiostat interfaced to a Pentium Celeron, 333 MHz, 64 MB RAM computer system and controlled by Autolab GPES software version 4.9 for Windows 98. 2.5. Electrophoresis procedure The separations in CE microchips were electrokinetically driven using two high-voltage power supplies (HVPS, MJ series) with a maximum voltage of +5000 V from Glassman High Voltage (High Bridge, NJ 08829-0317, USA). They were interfaced to a Pentium Celeron, 333 MHz, 64 MB RAM computer system and monitored by a DT300 Series Board, DT Measure Foundry 4.0.6 software for Windows 98. Positive connections were located in A and C reservoirs and grounds where situated in B and D reservoirs (Fig. 2). In this way, a high voltage between A and B (separation channel) and between C and D (injection channel) can be applied. High-voltage electrodes consisted of 0.3 mm diameter, 1 cm long platinum wires (Goodfellow) were inserted into each of the reservoirs and connected by means of crocodile clips to the HVPS. Prior to electrophoresis, microchips were initially rinsed with Milli-Q water for 15 min and then with the running buffer for 10 min. Washing was made with the aid of a simple vacuum system and reservoirs were filled with the running buffer solution. The microchip was fixed in its holder and a Faraday cage was used for housing it in order to minimize electrical interferences. After baseline stabilization, reservoir C is filled with the sample solution and injections were performed by applying the desired voltage between sample (C) and sample waste (D) reser-

voir. Since no voltage was applied to the other two reservoirs, an “unpinched” injection was performed. Separation was carried out by applying the corresponding voltage to the running buffer reservoir (A) with the detection reservoir (B) grounded. Then, the detection potential is applied and the electropherogram is recorded. All experiments were performed at room temperature. 2.5.1. Safety considerations High-voltage power supplies should be handled with extreme care to avoid electrical shock. 3. Results and discussion 3.1. Fabrication parameters Glass is an isotropic material that is wet etched with HF-based solutions in a non-directional manner, resulting in hemispherical channel structures. Ideal geometries involve high aspect ratio channels, that means, deep channels with parallel sides. Different parameters that affect the aspect ratio of the channel have been evaluated and optimized. Pretreatment of the glass substrates has been the first parameter studied. Substrates surface has to be adequately cleaned in order to get a good adherence of the photoresist (PR). Thus, neutral (C1, deionized water), acid (C2, piranha) and alkaline (C3, NaOH) solutions have been tested. Similar results in the final microchannel have been shown with the different washings. However, C2 washing was employed in the next part of the work due to this washing allowed improving the initial conditions of the substrate for the final bonding procedure. Next, the substrates were dried in an oven since this process can reduce the thermal stress between the glass substrate and the photoresist, subsequently added, resulting in a longer survival time of the photoresist during the wet etching. After washing, the photoresist has to be deposited on the glass substrates by spin-coating technique. The surface of glass materials oxidize very easily. The surface oxide forms long range hydrogen bonds with water adsorbed from the air. When the PR is spun onto surface, it adheres to the water vapour rather than to the surface, and poor adhesion results. Water adsorbed can be eliminated by soft-baking in an oven. Other two procedures, based on the modification of the interface surface, have been evaluated for improving the adherence of the PR (S1813). First, 100 nm thickness chromium layer was evaporated onto the glass. The Cr layer did not improve the PR adherence and the survival time of the PR in the wet etching decreased. In the second procedure, a hexamethyldisilizane (HMDS) based primer solution was deposited on the glass substrate during 20 s and then, it was spin-coated at 4000 rpm for 25 s. The primed surface improved the PR adherence; thus, peeling of the PR was not observed for a long time. Therefore, in all next studies that involved the use of the S1813 PR the primer solution was employed. The performance of the S1813 PR was compared with the maP1275 PR. Both of them have a very different viscosity, which affects the thickness of the coating layer. Final film thickness and other properties will depend, therefore, on the nature of the resin

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Fig. 3. Profile comparative obtained by confocal microscopy for (A) S1813 and (B) ma-P1275 photoresist with an etching time of 30 and 40 min, respectively.

(viscosity, drying rate, percent solids, surface tension, etc.) and the parameters chosen for the spin process. Factors such as final rotational speed and acceleration contribute to the properties of coated films. The S1813 PR was spun onto the glass substrate using speeds ranging from 3000 to 6000 rpm and times between 20 and 40 s. In all cases, a thickness layer around 1 ␮m was obtained. A speed of 5500 rpm and a time of 30 s have been used in the next steps of the work, due to the higher homogeneity of the PR layer deposited throughout the entire glass surface. PR ma-P1275 was deposited using two speed ramps due to its high viscosity. Thus, the PR has to be initially extended on the substrate by spin coating with a speed of 250 rpm for 10 s. Finally, it was spun on at 3000 rpm for 20 s. In these conditions, a layer around 7 ␮m thickness was obtained. An increase in the thickness layer allowed a long survival time during the wet chemical etching, reaching deeper channels. Moreover, this PR has shown a better adherence than S1813 PR, even without using the primer solution. An example of this is shown in Fig. 3, where two samples, made with these two different photoresists are compared. These images were taken using a confocal microscope, which gives a three-dimensional image of the sample (Fig. 3, right), as well as its depth profile in a selected region (Fig. 3, left). In some depth profiles, an unreal noise was observed in the base of channels, which was due to residual reflection during the acquisition of the confocal images. Using these depth profiles, the geometric factor (channel width divided by channel depth) of each sample can be calculated, reaching the conclusion that for the same fabrication parameters, ma-P1275 PR allows to get

a better value of this parameter. The best geometric factor should present a value proximal to 1. After PR deposition, the channel pattern has to be transferred to the resist. Thus, the PR was exposed by two different irradiation methods: direct writing and masked exposure. For direct writing method, a laser writing system has been designed and implemented. The basic principle of this system is the focalization of a low cost Nd:YAG laser light (λ = 473 nm) on the photoresist, creating a minimum light spot, which moves along the surfaces describing the desired motive (Fig. 2). By controlling the power of light and the velocity of the movements, it is possible to create on the photoresist a pattern of irradiated lines with the desires width. This method resulted very time-consuming and was not able to easily reproduce the same pattern many times. However, it was successfully used to make photomasks, which have been employed in the second method. In masked exposure method, a UV lamp was used to transfer the pattern from the photomask to the PR layer on substrates as can be seen in Section 2. Thus, the use of a mask-aligner is not required. The UV light modified the chemical structure of the PR, making it soluble on the developer solution. The development time was optimized to 90 s. With smaller times, the complete development of the PR was not obtained. Higher times could raise part of the non-exposed PR. Once PR has been developed, a post-develop bake or hardbake was used to improve resist’s wet-etching resistance by hardening it. This makes the PR more difficult to remove in aggressive etches with HF-based solutions. Thus, temperatures

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Table 1 Etching rates for different glass substrates and HF-based solutions HF-based solutions

Soda-lime microscope glass slides

Soda-lime glass

Borofloat glass

Pyrex

– – 0.8 ± 0.1 0.6 ± 0.1 0.9 ± 0.2

– – 0.4 ± 0.1 0.15 ± 0.05 0.10 ± 0.02

– 0.6 ± 0.1 – 0.10 ± 0.02 –

(␮m/min)a

Etching rate 5% (HF:NH4 F 1:7):9.25% HCl HF:H2 O 1:10 HF:NH4 F:H2 O 1:5:5 (BOE) HF:NH4 F 1:6 (BHF) HF:HNO3 :AcOH:H2 O 1:2:1:4 (HNA) a

0.25 0.5 1.3 0.7 1.3

± ± ± ± ±

0.07 0.1 0.2 0.1 0.3

Etching rate expressed as mean ± S.D., where “S.D.”: standard deviation of at least three samples.

comprised between 100 and 145 ◦ C as well as times ranging from 30 to 45 min have been tested. A temperature of 130 ◦ C for 30 min (S1813 PR) and 110 ◦ C for 30 min (ma-P1275 PR) was chosen taking into account the PR layer survival during the wet etching. Last step, and the most critical in the microfabrication process, was the wet etching of the glass substrates. Glass is a complex mixture based mainly on SiO2 and other compounds such as Na2 O, CaO, MgO, etc. HF is used to dissolve the glass and other chemical compounds, which are difficult to dissolve in water. Some authors [4,5] suggested, for glass substrates, a wet etching using HF-based solution with HCl, which is supposed to dissolve the formed precipitates. Moreover, we demonstrated experimentally that different stirring forms affect directly the good performance of wet etching. Thus, ultrasonic-vigorously and magnetic stirring were tested. The best results were obtained with magnetic stirring (3000 rpm), while the other ones caused the peeling and rising of the PR. In this work different etching solutions, based on hydrofluoric acid, with and without HCl, have been tested. In Table 1 the different etching rates obtained for each solution, acting on different kinds of glass substrates are shown. The higher etching rate (for soda-lime microscope glass slides) was obtained using the buffered oxide etch (BOE-HF:NH4 F:H2 O 1:5:5). As can be seen, the NH4 F adjust the etching performance increasing the etching rates when the BOE and the buffered HF (BHFHF:NH4 F 1:6) solutions are compared with HF:H2 O (1:10) solution. Adding nitric and acetic acid to the etching solution (HNA-HF:HNO3 :AcOH:H2 O 1:2:1:4) also improved the etching ratio, nevertheless, this solution was very aggressive decreasing the survival time of the photoresist (<15 min). When the soda-lime microscope glass substrates are compared with the other soda-lime glass substrate a decrease in the etching rate is observed. This difference can be attributed to the fabrication process of the glass, so that, the two glasses can present different SiO2 composition. Similar results are shown between the common glass (soda-lime) and borosilicate glass (borofloat and Pyrex). The borofloat and Pyrex glass present a higher proportion of SiO2 than soda-lime and then, they are more difficult to etch, decreasing the etching rate. The only advantage of using borosilicate glass was that no white precipitates were found on the surface while precipitates whether were observed with common glass. In Table 2 the geometric factor (channel width divided by channel depth) is shown for soda-lime microscope glass-slides, etched with each solution. Fig. 4 presents a comparative between

the aspects of the channels obtained in each case. In this case, a lower geometric factor meant a better aspect ratio (narrow and deep channels). Thus, from these results, it can be seen that the best geometric factor (for soda-lime substrates) corresponds to BHF solution. Due to its better geometric factor, good etching rate and longer survival time of the photoresist (approximately 60 min), the BHF solution was used in the next studies. As the literature proposes the use of HCl in order to dissolve the formed precipitates and increase the etching rate, this compound was added to the BHF solution in different concentrations. Thus, when HCl was added to the BHF solution between 0 and 15%, the geometric factor was increased from 5 to 12 for 0 and 15% HCl, respectively. Therefore, the performance of wet-etching with HCl was worse resulting channels with a bad aspect ratio, it means, very wide and less deep channels. Thus, although the HCl can dissolve the formed precipitates, it can also raise the photoresist making wider channels. In order to compare the shape and quality of each kind of etching process, Fig. 5 depicts microscope images and depth profiles of the obtained microchannels. As can be seen on the depth profiles (Fig. 5, left), when the HCl concentration was increased, the channel wide was also increased from 100 ␮m (0% HCl) to 300 ␮m (15% HCl). However, the depth was not increased with the same ratio, it even decreased. Therefore, the best etching solutions for soda-lime glass have resulted to be the BOE solution and especially the BHF solution without using HCl. The optimized wet-etching process makes unnecessary the use of more expensive methods such as RIE, DRIE, powder blasting or laser ablation [18–21]. 3.2. Bonding process The system has to be assembled in order to close the microchannels. Thus, different bonding processes have been Table 2 Geometric factors for different HF-based etching solutions HF-based solutions

Geometric factor

Soda-lime microscope glass slides 5% (HF:NH4 F 1:7):9.25% HCl HF:H2 O 1:10 HF:NH4 F:H2 O 1:5:5 (BOE) HF:NH4 F 1:6 (BHF) HF:HNO3 :AcOH:H2 O 1:2:1:4 (HNA)

63 72 7 5 9

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Fig. 4. Profile comparative obtained by confocal microscopy for different HF-based etching solutions (A) HF:NH4 F 1:6 (BHF), (B) HF:NH4 F:H2 O 1:5:5 (BOE), (C) HF:HNO3 :AcOH:H2 O 1:2:1:4 (HNA), (D) HF:H2 O 1:10 and (E) 5% (HF:NH4 F 1:7):9.25% HCl.

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Fig. 5. Profile comparative for wet etching with BHF solution with increasing levels of HCl, (A) 0%, (B) 1%, (C) 5% and (D) 15%.

tested. Although currently the procedure used to seal the microchannels is the thermal bonding, low temperature procedures based on intermediate layers have also been investigated. Commercial adhesive (Araldit) and adhesive tape were rejected. The first procedure was discarded because of a nonhomogeneous glue layer was obtained. Therefore, the cover

plate was not adequately sealed along the channel and thus, the fluid cannot flow through the microchannel. Similar results were obtained with the adhesive tape; moreover, the film had not resistance enough, which caused an ease rupture. The next procedure evaluated was based on an intermediate layer of SiO2 ·Na2 O (water glass). This procedure had been pre-

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Table 3 Optimal conditions for microfluidic fabrication on soda-lime glass substrates Step

Optimal conditions

Pre-treatment

Commercial detergent, deionized water, acetone, piranha solution (H2 SO4 :H2 O2 , 3:1), isopropyl alcohol and blown dry with N2 (approximately 1 h)

Lithography Spin-coating Soft-baked UV exposure Developed Hard-baked Wet etching Thermal bonding

Primer (HMDS) 4000 rpm, 25 s Photoresist S1813 5500 rpm, 30 s 90 ◦ C, 30 min 115 s (150 mJ cm−2 ) MF-311 developer → 90 s 130 ◦ C, 30 min HF:NH4 F 1:6, 30 min 650–675 ◦ C, 5 h

viously tested with good results [30,43]. Thus, different solution concentrations (1–35%), spin-coating speeds (3000–8000 rpm) and times (5–30 s) as well as several temperatures (20–700 ◦ C) have been evaluated. When high concentrations of silicate sodium and low spincoating speed are used, the intermediate layer is too thick and therefore a water layer is formed avoiding the good sealing of the glass. On the other hand, if low concentrations and high spincoating speed are employed, a thin layer is obtained causing short hardened time and unsuccessful adhesion of the cover plate occurs. The optimal conditions were obtained using a solution concentration of 3%, with a spin speed of 5000 rpm during 30 s. Initially, low temperatures (RT, 100 ◦ C) were used to seal the system, but a reversible bonding was obtained and the solutions did not flow properly through the channels. Then, the substrate was heated in a muffle furnace at 650–675 ◦ C for 5 h obtaining a successfully sealing of the system. Usually, high temperature procedures do not require an intermediate layer; hence, the thermal process was successfully made without using the intermediate layer. When the glass temperature was proximal to the melting point a homogenous external mechanical pressure throughout the glass was necessary for the accomplishment of the thermal bonding. This pressure was applied by a homemade refractory steel device, compound by an external chassis in which two sample holders can be inserted. The glass cover plate and the micromachined substrate were placed on these sample holders, and then they are covered by two steel plates and compressed by two screws. Fig. 6 shows the scheme of the device employed for thermal bonding. This thermal bonding method avoids the use of much more expensive equipments such as those used in anodic bonding [44]. In Table 3 the optimal conditions of each step to fabricate a microfluidic device on soda-lime glass substrates with channels of 50 ␮m width and 20 ␮m depth approximately are shown. 3.3. Microchip performance The resulting microfluidic system with a 4.5 cm long separation channel (between running buffer reservoir, A, and waste/detection reservoir, B) and 1.5 cm long injection chan-

Fig. 6. Schematic representation and picture of the thermal bonding device.

nel (between sample reservoir, C, and sample waste reservoir, D) was employed for capillary electrophoresis (CE) with amperometric detection. The performance of CE-microchip and the electrochemical response of the gold wire working electrode were studied using p-aminophenol (pAP) as model analyte. A 50 mM Tris–Gly pH 9.0 was employed as running buffer. At this pH, pAP (pKa = 10.46) is a neutral mark, so that, it can be used for characterizing the electroosmotic flow (EOF). Measuring the migration time of pAP and knowing the effective length of the channel (distance from injection to detection point: Leff = 4 cm), it is possible to calculate the electroosmotic velocity (cm s−1 ). The electroosmotic mobility (cm2 V−1 s−1 ) can be obtained dividing the electroosmotic velocity by the electric field applied (V cm−1 ) in the channel. A 100 ␮M solution of pAP was employed in the evaluation of the glass microchip using an injection voltage of +1500 V applied for 10 s. Since pAP presents an oxidation process, a detection potential of +0.75 V was used [14,39]. Parameters such as peak current (ip ), migration time (tm ) and half-peak width (w1/2 ) are shown in Table 4 as well as the theoretical plate number (N), electroosmotic velocity (veo ) and mobility (μeo ) for pAP using different electric fields (E). When the separation voltage was varied from +1000 to +1500 V, an increase in the peak current and a decrease in the

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Table 4 Analytical characteristics for pAP (100 ␮M) using different separation voltages in the glass-microchip with an end-channel gold wire detector Voltage (V)

E (V/cm)

ip (nA)

tm (s)

w1/2 (s)

N (m−1 )

EOF (veo , cm/s)

μeo (×10−4 cm2 /V s)

+1000 +1500 +2000

222 333 444

6.6 18 11

77.2 39.6 23.3

22.2 10.2 7.4

1700 2100 1400

0.05 0.10 0.17

2.3 3.0 3.9

Conditions, injection: 10 s at +1500 V; Ed = +0.75 V (vs. Ag/AgCl), running buffer: 50 mM Tris–Gly pH 9.0.

Fig. 7. Electropherogram for 100 ␮M pAP using glass-microchip with an end-channel gold wire detector. Conditions: Vsep = +1500 V; injection: 10 s at +1500 V; Ed = +0.75 V (vs. Ag/AgCl), running buffer: 50 mM Tris–Gly pH 9.0.

half-peak width and migration time was observed. If the separation voltage was increased to +2000 V a decrease in the half-peak width and migration time was also observed, but a lower peak current was measured. Moreover, in this case, a higher baseline noise level as well as bubbles formation due to excessive Joule heating happened. The best performance of the glass CE-microchip in combination with the end-channel gold wire detector was obtained using a separation voltage of +1500 V. Fig. 7 depicts an electropherogram for a 100 ␮M pAP solution using a separation voltage of +1500 V. Analytical characteristics of the new glass microchip with the amperometric detector were quite similar to those shown previously on commercial microchips [14,39] demonstrating, thus, its possible analytical and bioanalytical applications. Furthermore, the glass microdevices have demonstrated a longer life time than polymer commercial microchips. 4. Conclusions Microfluidic channels have been successfully fabricated in different glass substrates using photolithography and wetetching technique. Practical aspects and critical points that affect to all of the different parameters on the fabrication process of glass microfluidic devices have been rigorously evaluated and optimized. An accurate, fast and low cost methodology has been developed, permitting the fabrication of a butch of complete microchips in less than 8 h. The importance of the substrate pretreatment and the effect of photoresist thickness as well as the different methodologies for transferring the pattern design to the PR have been discussed.

The most critical step of microfabrication was the wet etching of the glass substrates. Different etching solutions, based on hydrofluoric acid, with and without HCl, as well as several types of stirring, were evaluated. The best microfluidic channels, with better geometric factor, were attained using BHF (HF:NH4 F 1:6) solution with a magnetic stirred. The resulting channels were worse when HCl was added to the etching solution since it increased the geometric factor. The device assembly has been made using different bonding processes. Different techniques based on intermediate layer were initially tested and discarded. The best sealing of channels was obtained by thermal bonding at 650–675 ◦ C for 5 h. Resulting microfluidic devices have been successfully used for capillary electrophoresis with electrochemical detection. An end-channel gold wire detector has been easily integrated on the glass microchip for amperometric detection. Thus, the electroosmotic flow (EOF) of the new glass microchip has been characterized using pAP as neutral mark. This advances the possible analytical and bioanalytical applications of the new glass microfluidic device. Acknowledgements This work has been supported by the FICYT under projects ´ IB05-151C1 and IB05-151C2. Mario Casta˜no Alvarez thanks FICYT-Principado de Asturias for the award of a Ph.D. grant. References [1] A. Manz, N. Graber, H.M. Widmer, Miniaturized total chemical analysis systems: A novel concept for chemical sensing, Sens. Actuators B: Chem. 1 (1990) 244–248. [2] A. Manz, D. Harrison, E. Verpoorte, J. Fettinger, A. Paulus, H. Ludi, H. Widmer, Planar chips technology for miniaturization and integration of separation techniques into monitoring systems: capillary electrophoresis on a chip, J. Chromatogr. A 593 (1992) 253–258. [3] T. McCreedy, Fabrication techniques and materials commonly used for the production of microreactors and micro total analytical systems, Trend Anal. Chem. 19 (2000) 396–401. [4] M. Stjernstr¨on, J. Roeraade, Method for fabrication of microfluidic systems in glass, J. Micromech. Microeng. 8 (1998) 33–38. [5] C.-H. Lin, G. -B- Lee, Y. -H- Lin, G.-L. Chang, A fast prototyping process for fabrication of microfluidic systems on soda-lime glass, J. Micromech. Microeng. 11 (2001) 726–732. [6] A. Muck Jr., J. Wang, M. Jacobs, G. Chen, M.P. Chatrathi, V. Jurka, Z. V´yborn´y, S.D. Spillman, G. Sridharan, M.J. Sch¨oning, Fabrication of poly(methylmethacrylate) microfluidic chips by atmospheric molding, Anal. Chem. 76 (2004) 2290–2297. [7] R.-H. Horng, P. Han, H.-Y. Chen, K.-W. Lin, T.-M. Tsai, J.-M. Zen, PMMA-based capillary electrophoresis electrochemical detection microchip fabrication, J. Micromech. Microeng. 15 (2005) 6–10.

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Biographies ´ ˜ Alvarez Mario Castano obtained his BSc degree in chemistry, focus in analytical chemistry, in 2002 (University of Oviedo) and his MSc degree in analytical chemistry in June 2005 (title: Amperometric detectors for capillary electrophoresis microchips). He is currently a PhD candidate at University of Oviedo and his work is focused on fabrication and characterization of microfluidic devices with electrochemical detection. Diego Francisco Pozo Ayuso received the degree in physics from the University of Oviedo, Spain. Since 2003, he is working in optical waveguide fabrication and characterization in the Physics Department of the University of Oviedo. In 2005, he received a grant to doctoral courses from this University. Moreover, this year he received the pedagogic aptitude for teachers of Secondary Education, obtained a master in management of the environment from Asturias Business School and worked in an environmental control company. He is currently working as researcher for a coordinated project between the inmunoelectroanalisis group and the integrated optic group of the University of Oviedo. His research interests are fabrication and characterization of optical waveguides, capillary electrophoresis microchips (MCE) and biosensors microdevices based on Mach–Zehnder interferometer. Miguel Garc´ıa Granda received his degree in physics in 2003 from the University of Oviedo (Spain). During 2003 he joined the Hanh-Meitner Institut

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in Berlin (Germany) where he worked in chalcopyrite solar cells fabrication techniques and structural haracterization. In 2004, he received a doctoral grant from the Spanish Ministry of Education and Science. Currently, he is pursuing his PhD which is directed towards the study of optical waveguides design, fabrication and characterization, as well as Mach–Zehnder optical modulators, within a cooperation framework between the Universities of Oviedo (Spain) and Paderborn (Germany). Mar´ıa Teresa Fern´andez Abedul obtained her BSc degree in chemistry, focus in analytical chemistry, in 1989 (University of Oviedo) and the PhD in chemistry in 1995 (University of Oviedo). Since September 1994, she is senior lecture in analytical chemistry (University of Oviedo). She works in the Immunoelectroanalysis Research Group of the University of Oviedo and has been supervisor of several research projects developed at the Electrochemistry laboratories of the Department of Physical and Analytical Chemistry of the University of Oviedo. Nowadays his research is focused on the development of electrochemical immunosensors and genosensors employing both enzymatic and non-enzymatic labels as well as fabrication and characterization of microfluidic devices with electrochemical detection. Jos´e Rodr´ıguez Garc´ıa is a professor at the University of Oviedo (Spain). He received the teaching degree from the University of Oviedo, and the BSc and

Extraordinary PhD degrees in physics from the University of Santander (Spain). From 1982 to 1988, he worked in electromagnetic field analysis on dielectric waveguides in the Electronics Department of the University of Santander. As member of the COST-216 European Project, he has worked at the ETH (Zurich) as well as for the CTNE and NESTLE Companies. Since 1988, he has been a professor at the University of Oviedo and since 1993, supervisor and coordinator of the Secondary School. He is a member of the Electromagnetism Academy (USA) and member of the SPIE society. He was selected as Man of the Year 1997 by the ABI and he was included in: Who’s Who in electromagnetics, Who’s Who in the world and Who’s Who in contemporary achievements. He conducts research in the following areas: electromagnetic field theory, modelling, characterization and evaluation of integrated optical waveguides and devices. Agust´ın Costa Garc´ıa obtained his BSc degree in chemistry, focus in analytical chemistry, in 1974 (University of Oviedo) and the PhD in chemistry in 1977 (University of Oviedo). Since February 2000, he is professor in analytical chemistry (University of Oviedo). He leads the Immunoelectrochemical Research Group of the University of Oviedo and has been supervisor of several research projects developed at the Electrochemistry laboratories of the Department of Physical and Analytical Chemistry of the University of Oviedo. Nowadays his research is focused on the development of electrochemical immunosensors and genosensors employing both enzymatic and non-enzymatic labels.