Article pubs.acs.org/ac
Fabrication of Biofunctionalized Microfluidic Structures by LowTemperature Wax Bonding María Díaz-González* and Antoni Baldi Instituto de Microelectrónica de Barcelona (IMB-CNM, CSIC), Spain S Supporting Information *
ABSTRACT: In this work, a new fabrication technology for microfluidics based on the use of wax is described. Microfluidic structures are assembled using wax as both a thermoplastic adhesive layer between two glass substrates and a spacer layer defining the microchannels. Wax patterns with dimensions down to 25 μm are easily produced on glass substrates using specially developed decal-transfer microlithography. A complete microfluidic system is created by bonding the wax patterned layer with an additional glass substrate. On the basis of the special melting behavior of waxes, an effective glass-wax bonding is achieved at 40 °C by applying a soft pressure and without the requirement of any glass pretreatment. Wax bonding provides an effective sealing of the fluidic networks even on nonflat glass substrates (i.e., containing metal electrodes). The mild conditions required for the bonding process enables the fabrication of lab-on-a-chip devices incorporating biomolecules, as is demonstrated with the implementation of a simple heterogeneous immunoassay in a microfluidic device with amperometric detection.
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for bonding glass/glass14,15 or poly(dimethylsiloxane) (PDMS) to plastics, glass, or gold.16−21 However, they involve the use of plasma, UV radiation, or silane chemistry which may affect the functionality of biomolecules. The solution most often adopted in the fabrication of microfluidic systems for heterogeneous immunoassays consists in immobilizing the biomolecules in the microchannels by injection of different reagents through fluidic inlets after the bonding step.22,23 This solution ensures the integrity of the biomolecules but limits the capacity for multiplex detection and is not suitable for mass production. Therefore, there is a need for the development of lowtemperature bonding processes compatible with the presence of biofunctionalized surfaces. In the present work, an alternative fabrication technique that meets this requirement is proposed. A patterned wax layer, generated by specially developed decaltransfer microlithography, is used both as an adhesive layer between two glass substrates and as the spacer layer defining the microchannels. This approach shares similarities with the rapid prototyping techniques, using a patterned double side pressure-sensitive adhesive tape,24−26 but enables much smaller feature size and provides higher potential for mass production. In the field of microfluidics, wax has already been used to create microfluidic channels on the development of low-cost lateral-flow paper-based microfluidic devices.27 Wax has also been used for substrate bonding in microfluidic chip assembly.28,29 However, the fabrication procedures used in
icrofluidic devices offer a great variety of exciting applications in several areas including biomedical research, clinical diagnostics, or environmental monitoring.1,2 Heterogeneous immunoassays, which require the immobilization of one of the immunoreactants onto a solid support, are one of the most intensively studied applications of microfluidic systems.3 Regardless of the application and the detection mode, microfluidic devices are often composed of two substrates which are fabricated separately and finally assembled together using a bonding process.4 The challenge of bonding a structured substrate with a cover plate to form enclosed microfluidic networks without deforming the small features or clogging the channels has engendered the development of a wide variety of techniques.4,5 Bonding techniques can be classified into two major categories: direct bonding and bonding with an intermediate layer (adhesive bonding). Direct bonding techniques provide high bonding strength and hermetic sealing, but they require harsh conditions, extremely clean and flat surfaces, and the access to sophisticated equipment.6 On the contrary, adhesive bonding7 is a costeffective, low-temperature, and less demanding surface quality alternative that can be used in nonflat substrates. Adhesive leakage into the microchannels, limited temperature stability, and nonuniform bonding of substrates due to the formation of voids are some of the main disadvantages of these techniques. Many different approaches have been investigated in order to overcome the above-mentioned drawbacks.8−21 Some of them require heating the substrates above room temperature,8−13 which may be disastrous if temperature sensitive biomolecules are first immobilized in the microfluidic areas. Different room temperature alternatives have been described in the literature © 2012 American Chemical Society
Received: June 3, 2012 Accepted: August 20, 2012 Published: August 20, 2012 7838
dx.doi.org/10.1021/ac301512f | Anal. Chem. 2012, 84, 7838−7844
Analytical Chemistry
Article
Figure 1. Schematic illustration of wax patterning process. (a) Wax was melted on the flat PDMS substrate. (b) Nonadherent PDMS mold was pressed onto flat substrate for molding wax. (c) After lowering the temperature, the PDMS mold was peeled-off. (d) The PDMS stamp was brought into conformal contact with a glass substrate. (d) The PDMS stamp was peeled away from the glass surface.
Figure 2. Schematic illustration of (a) glass coverslip functionalization and (b) wax-glass bonding to assemble a microfluidic system.
and was left to solidify by cooling. EVA is a rubbery plastic like material used to impart hardness and strength to paraffin wax. To obtain an EVA/paraffin mixture with a melting point similar to beeswax (mp 62−65 °C), a paraffin wax with melting point of 53−57 °C was selected. Wax Patterning. Figure 1 illustrates the procedure for wax patterning based on decal-transfer microlithography (DTM) methodology.30 This type of soft-lithography relies on controlled adhesion properties to transfer molded thin film materials from stamps onto compatible substrates. Here, a molded wax decal was transferred to a glass substrate using a PDMS stamp. Nonadherent PDMS molds were prepared as described in the Supporting Information. Wax was melted (80 °C for beeswax and 90 °C for paraffin−EVA mixtures) on top of a flat PDMS substrate and molded with the nonadherent PDMS mold by applying a pressure of 130 g/cm2 for 30 s (Figure 1a,b). These PDMS molds can be reused more than 10 times without the requirement of any additional treatment before each use. To increase the adhesion of wax to the PDMS stamp surface and prevent detachment of the features during demolding, the stamp can be activated by O2 plasma (1−3 min, 500 W) before its use. After lowering the temperature to ca. 0 °C, the PDMS mold was peeled-off (Figure 1c). Subsequently, the PDMS stamp having wax patterns was brought into contact with the receiving glass substrate at 40 °C (Figure 1d,e) and
these previous works were not compatible with the presence of temperature sensitive biocomponents.
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EXPERIMENTAL SECTION Materials. Poly(dimethylsiloxane) (PDMS Slygard 184 kit, which includes the Sylgard 184 silicone elastomer base and the Sylgard 184 silicone elastomer curing agent) was purchased from Dow Corning (Midland, MI, USA). Natural beeswax (mp 62−65 °C) was obtained from a local candle making supply shop. Paraffin wax (mp 53−57 °C), EVA (poly(ethylene-covinyl acetate)), 25 wt % vinyl acetate, mouse immunoglobulin G (mIgG), rabbit immunoglobulin G (rIgG), antimouse IgG peroxidase conjugate (anti-mIgG-HRP), bovine serum albumin (BSA), Tween 20, and 2′-azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS) were obtained from Sigma-Aldrich Co. (St Louis, MO 63103, USA). 1H,1H,2H,2H-Perfluorooctyltriethoxysilane was purchased from ABCR GmbH & Co (Germany). All other chemicals were of analytical grade. Glass slides (26 × 76 mm, 1.0 mm thick) and coverslips (20 × 20/22 × 40 mm, 0.13−0.16 mm thick) were obtained from MenzelGläser GmbH (Germany). Araldite epoxy adhesive and silver conductive paint were obtained from RS Amidata (Spain). Paraffin/EVA Blends. EVA (10% wt) and paraffin were mixed by fusion at 100 °C and mechanically stirred for 2 h. The hot solution was sucked into a 50 mL polystyrene container 7839
dx.doi.org/10.1021/ac301512f | Anal. Chem. 2012, 84, 7838−7844
Analytical Chemistry
Article
Figure 3. Optical microscope of wax (positive and negative) patterns on glass: (a) Beeswax, 25 μm height, and (b) EVA/paraffin, 100 μm height. SEM images of wax (25 μm height) positive patterns on glass: (c) Beeswax 50 μm squares molded with nonoxidized PDMS; (d) Beeswax 25 μm squares molded with plasma oxidized PDMS; (e) EVA/paraffin lines (50 μm wide, 1000 μm long) molded with nonoxidized PDMS; (f) EVA/ paraffin lines (25 μm wide, 500 μm long) molded with plasma oxidized PDMS. Scale bars = 50 μm.
°C and completely melting at about 64 °C.31 This special melting behavior makes waxes well-suited for low-temperature bonding applications. It is interesting to note that beeswax is a natural and renewable material. However, since beeswax chemical compositions differ to some degree depending on the species of bee, its geographical origin, etc.,32 a paraffin/EVA mixture has also been studied here as alternative wax material. The present approach to the construction of microfluidic systems has involved the development of two different processes: wax patterning and wax-glass bonding. Results and discussion of these two processes are presented next, followed by the characterization results of the analytical microfluidic device. Wax Patterning. Positive (unconnected) and negative (openings) wax features of diverse size (25 to 1000 μm) and density were successfully produced on glass surface by DTM (Figure 3). The overall printing and transfer method is mainly governed by the adhesion between wax and the different substrates involved during the process. The strength of the adhesion is influenced by many factors, e.g., polymer viscoelasticity, surface roughness, reactivity and hydrophobycity of surfaces, or temperature.33,34 To create wax patterns onto flat PDMS, the strength of the adhesion between this substrate and wax must be larger than that between wax and PDMS molds. To achieve this, PDMS molds were treated with 1H,1H,2H,2H-perfluorooctyltriethoxysilane to render the surface antisticking for an easy mold release. This treatment was enough for patterning large wax features on flat PDMS but failed with the smaller features tested in this work (≤50 μm), which remained adhered to the molds. Plasma treatment of the flat PDMS receiving substrate was found to notably improve this step. A 3 min (500W) treatment was required for beeswax transfer while 1 min was enough when working with paraffin/EVA blends. A cleaning step of PDMS35 was included before any treatment to obtain a more stable surface modification.36 It is well reported that cleaning PDMS in a series of solvents removes low molecular weight oligomers, improving the surface for subsequent PDMS functionalization.
then slowly peeled away. Wax patterns of two different thicknesses (100 and 25 μm) were prepared with this procedure. Bond Strength. Test beeswax patterns deposited on a glass slide were covered with a glass coverslip at 40 °C by applying different pressures at a fixed time (10 min). The patterns were 100 μm thick and 10 × 10 mm in size. Bond strength was determined by performing a tensile load test at three different temperatures between 25 and 35 °C. To apply the load to the samples, a wire was glued to the coverslips with epoxy resin. Microfluidic Chips. A simple analytical microfluidic device was designed to test the compatibility of the fabrication process with temperature sensitive biocomponents and with the presence of metal electrodes. Wax microfluidic patterns comprising an inlet channel, an outlet channel, and a microreaction chamber were transferred to a glass substrate containing a three gold electrode system (0.16 mm2 working electrode, 0.36 mm2 counter electrode, and 0.08 mm2 reference electrode; all 150 nm-thick). The electrodes were fabricated at the CNM-IMB clean room according to standard photolithographic techniques. The structure was bonded to a glass substrate (a 160 μm-thick coverslip with inlet and outlet orifices) that had been functionalized in order to incorporate mouse antibodies (Figure 2a). After the bonding of the microfluidic system at 40 °C and a pressure of 120 g/cm2 (Figure 2b), a chronoamperometric enzymatic immunoassay was carried out. Details of the fabrication of the gold microelectrodes, the antibody functionalization, and the chronoamperometric immunoassay procedure are included in the Supporting Information.
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RESULTS AND DISCUSSION Wax (beeswax) is the oldest thermoplastic material known to man. Like hot melt adhesives, waxes are hot melted on substrates to be bonded and develop adhesion after cooling. However, unlike other homogeneous chemical compounds, beeswax does not melt immediately on heating but passes through several intermediate states (solid−plastic−semiplastic−semiliquid−liquid), starting at a temperature of about 40 7840
dx.doi.org/10.1021/ac301512f | Anal. Chem. 2012, 84, 7838−7844
Analytical Chemistry
Article
the double-peak profile becomes more significant as the line width increases. The same trend was observed for 100 μm thickness PDMS molds. At 130 g/cm2 pressure, the doublepeak profile is still observable for the wider lines but vanishes at widths
