3772 Swomitra K. Mohanty Dongshin Kim David J. Beebe Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA Received April 17, 2006 Revised June 22, 2006 Accepted June 23, 2006 Electrophoresis 2006, 27, 3772–3778 Research Article Do-it-yourself microelectrophoresis chips with integrated sample recovery We present a microelectrophoresis chip that is simple to fabricate using the mfluidic tectonics (mFT) platform (Beebe, D. J. et al., Proc. Natl. Acad. Sci. USA 2000, 97, 13488–13493; Agarwal, A. K. et al., J. Micromech. Microeng. 2006, 16, 332–340). The device contains a removable capillary insert (RCI) for easy sample collection after separation (Atencia, J. et al., Lab Chip 2006, DOI: 10. 1039/b514068d). Device construction is accomplished in less than 20 min without specialized equipment traditionally associated with microelectrophoresis chip construction. mFT was used to build a PAGE device utilizing two orthogonal microchannels. One channel performs standard separations, while the second channel serves as an access point to remove bands of interest from the chip via the RCI. The RCI contains an integrated electrode that facilitates the removal of bands using electrokinetic techniques. The device was characterized using prestained proteins (Pierce BlueRanger and TriChromRanger). Samples were loaded into the microelectrophoresis device via a standard micropipette. An electrical field of 40 V/cm was used to separate and collect the proteins. The microPAGE device is simple to fabricate, benefits from microscale analysis, and includes an on-chip collection scheme that interfaces the macroworld with the microworld. Keywords: Microelectrophoresis / mfluidic tectonics / Removable capillary insert DOI 10.1002/elps.200600238 1 Introduction Miniaturization of electrophoresis analysis via microelectrophoresis chips has been an area of active research in recent years [1–3]. Advantages of miniaturization include reduced material usage, compact analysis space, and high speed analysis. However obstacles to microelectrophoresis chips are cost and fabrication techniques. These chips require expensive cleanroom facilities or specialized equipment not available to most biological researchers. In addition, most chips do not contain a convenient method for sample collection, which may further discourage researchers from using the technology, as sample analysis after separation is often needed. Historically, electrophoresis is one of the most common methods to separate charged molecules such as DNA and proteins [4]. These molecules have unique physical Correspondence: Mr. Swomitra Mohanty, Department of Biomedical Engineering, Room 2139 ECB, 1550 Engineering Drive, Madison, WI 53706, USA E-mail: [email protected] Fax: 1608-265-9239 Abbreviations: ìFT, mfluidic tectonics; RCI, removable capillary insert; PIBA, poly iso-bornyl acrylate © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim characteristics based on size, charge, and shape that allow researchers to separate them in the presence of a sieving matrix and electric field. Typical separations are done in either slab gels or capillaries. Slab gel electrophoresis is a well-established technique that has been used for decades. It has proven to be economical and yield reliable analytical results. However, separations done using gel electrophoresis are very time consuming (often taking several hours to complete). In the 1990s CE was introduced, which had several desirable attributes including reduced separation time, reduced sample/ reagent use, and automated analysis. However, CE requires expensive complex equipment and is not always appropriate for all separations. As microelectrophoresis chips are not readily available, one alternative that some researchers have employed to improve upon traditional methods is inexpensive microslab gel electrophoresis systems [5]. The microslab gels made it possible to achieve higher electric field strength with lower voltages, decreasing analysis time. However, fabrication of microslab gels is cumbersome and creating small loading wells is difficult. Recently a simple method for making microslab gels was reported [6]. This method casts gels (25 6 30 6 0.6 mm) between a www.electrophoresis-journal.com Miniaturization Electrophoresis 2006, 27, 3772–3778 microscope glass slide (24 6 30 mm) and transparency film. By cutting injection holes into the film, defined injection wells in the gel were created by taking advantage of polymerization inhibition properties of oxygen. The gel is polymerized at the film interface where oxygen is not present. However, gel at the injection holes were exposed to oxygen which inhibits polymerization. One draw back to this method is that band collection after separation is difficult. The size of the gel and space between the bands is very small making traditional band cutting challenging. Researchers continue to focus on improving electrophoresis through continued miniaturization [7–13]. As mentioned above, miniaturization improves electrophoretic analysis by reducing material usage, analysis space, and analysis time. Due to high surface to volume ratios at the microscale, Joule heating and temperature gradients are less problematic allowing for higher electric fields strengths (compared to microslab gels) in shorter distances. In addition, the reduced space used for microelectrophoresis allows for development of high-throughput systems that can be interfaced with commercial automation devices. One caveat to increased miniaturization is that sample collection after on-chip analysis becomes increasingly difficult. The details for off chip sample manipulations must be addressed for microelectrophoresis chips to be fully useful. In cases where separation of an unknown DNA sample is conducted, a specific band of interest is present that needs to be extracted for further analysis (for example Southern blot, PCR, DNA sequencing). The trend often observed in microfluidics is that the more miniaturized analysis devices become, the more inclined these devices are to use automated systems/custom instrumentation for sample loading and recovery. However, these automated liquid handling systems add to the cost of microelectrophoresis chips providing the same economical obsta- 3773 cle to researchers that CE does. Another issue in the cost of microelectrophoresis chips is in the materials used and fabrication methods. Using alternative materials and fabrication techniques can make microelectrophoresis chips more economical and accessible to life science labs. Various materials have been used to fabricate microelectrophoresis chips including polydimethylsiloxane (PDMS), glass, and silicon [9, 14]. Each type of material provides advantages and disadvantages with regard to ease of fabrication, cost, and repeatable results (Table 1). PDMS is a class of elastomers that is made up of an inorganic siloxane backbone and organic methyl groups [15]. When polymerized, PDMS is optically clear and generally considered non-toxic, making it a useful construction material for microfluidic devices involving biological material. Using a process known as soft lithography [16], it is possible to make micro cell culture constructs [17], micro in vitro fertilization devices [18], and electrophoresis devices [14]. Although PDMS is easy to handle, it is not optimal for electrophoretic separations without surface modification [19]. For example the surface of PDMS attracts biomolecules causing them to stick to the channel instead of separating out during electrophoresis. In addition, molding PDMS requires using relief master molds. The master mold fabrication process can take several hours depending on the thickness and feature size. Although a master can be used many times to make a device, the materials and equipment needed to make masters can be costly. Glass and silicon are materials that work well for microelectrophoresis devices utilizing acrylamide gel or capillary fluid because of their surface properties and precise fabrication processes. Glass is the material used in traditional devices (PAGE electrophoresis systems), thus its choice for microscale devices is logical. Acrylamide gel polymerizes within glass, which helps create a uniform gel Table 1. Comparison of microelectrophoresis fabrication methods Fabrication technique Advantages Disadvantages Silicon/glass microelectromechanical system (MEMS) Sub-micrometer resolution machining, well-established techniques for repeatable devices Requires cleanroom and expensive specialized equipment, time- and labor-intensive, complicated device assembly PDMS 10-mm resolution , simple fabrication method, easy device assembly, selfbonding material Additional surface modification needed for electrophoresis separations, requires fabrication of a relief master mold that adds to material costs and construction time mfluidic tectonics Fast/simple fabrication method, no masters needed, easy device assembly, no bonding step required Highest resolution 20–50 mm, limited to photo-definable materials © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com 3774 S. K. Mohanty et al. matrix for separation. For capillary separations, the glass surface allows charged layers to develop facilitating EOF. The charged layers are important for many types of separations involving positive and negative charged molecules. Silicon is a material traditionally used in integrated circuit chip fabrication. As a result, micromachining techniques have already been developed for silicon, making microelectrophoresis chip fabrication achievable with little process adjustment. Fabrication in silicon also allows for integration of electronic components such as detectors, heaters, and electrodes, making it very attractive for complete lab-on-a-chip applications. Since silicon fabrication involves batch processing, lab-on-a-chip devices can be made simultaneously on a massive scale reducing the cost per chip. However, such mass produced chips are not readily available to life science researchers. Successful devices have been made using glass and silicon [20, 21]. However, they are not as simple to build as PDMS devices. Traditional micromachining techniques are time consuming and require expensive cleanroom facilities not available to most researchers. A typical glass electrophoresis device takes several hours to complete. Assembly of devices is also an issue generally requiring anodic bonding to create a completely sealed channel. Anodic bonding requires extremely clean surfaces, relatively high temperatures, and high voltages to achieve. It is more labor intensive than PDMS bonding. While silicon and glass make excellent microelectrophoresis chips, the particular fabrication methodology that is traditionally employed makes it impractical for most life sciences research laboratories. We have addressed the issues of high cost microelectrophoresis chips and sample collection using alternative materials and fabrication methods than those previously described. We present a microelectrophoresis chip containing a removable sample collection component fabricated using the mfluidic tectonics (mFT) platform [22, 23]. The device is simple to fabricate with minimal costs and equipment requirements. The mFT platform uses poly isobornyl acrylate (PIBA) and liquid-phase polymerization to pattern microstructures such as microfluidic channels, mixers and valves [24]. PIBA is a high glass transition temperature (Tg 887C) polymer which is solid at room temperature. It has excellent adhesion properties to various substrates, such as glass, plastic, and metal. PIBA can be polymerized by liquid-phase photopolymerization, and shows minimal change (5–10%) in volume after polymerization. Photopolymerization of PIBA is done quickly with typical polymerization times ranging from 5 to 40 s. After polymerization, PIBA is stiff and optically clear. Devices are fabricated in a matter of minutes allowing for quick design improvements if needed. This contrasts with traditional glass and PDMS fabrication where both methods take hours to complete a device. While PDMS de© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2006, 27, 3772–3778 vices require a master (inverse of the desired shape) for fabrication, mFT creates the channels via direct photopolymerization of the final device material eliminating the need for masters. One other attractive aspect of mFT is no bonding is required for creating microchannels since they are automatically sealed during the channel formation. 2 Materials and methods 2.1 Device overview A scheme for the microelectrophoresis device and removable capillary insert (RCI) [25] is shown in Fig. 1. The device contains two orthogonal channels with RCI. Each RCI contains integrated electrodes (platinum wire) for electric field application and sample recovery. When a band of interest is identified, it is directed down the attached orthogonal channel by activating the electrodes at the end of the channel. This method of on-chip sample extraction has been previously reported by Burns et al. [26], giving complete details modeling electric fields at the intersection and design criteria. The sample recovery methods described here expand on this work by adding the RCI for easy off-chip removal. Figure 1. Scheme of microelectrophoresis device. Two orthogonal channels are shown with an RCI at each end. Each RCI has an integrated electrode to work as an anode or cathode during electrophoretic separation and collection. Proteins are initially sent down a separation channel. When a band of interest is identified and reaches the intersection, it is directed down the access channel by activating the electrodes in that channel and shutting off electric field in the separation channel. 2.2 Device fabrication Microelectrophoresis chips were fabricated with three layers of materials: glass-PIBA-glass (Fig. 2). Two adhesive spacers of 0.5 cm width, 5.5 cm length, and 125 mm www.electrophoresis-journal.com Electrophoresis 2006, 27, 3772–3778 Miniaturization 3775 Figure 2. Fabrication process for microelectrophoresis chip. Microelectrophoresis chips are fabricated of three layers of materials, glass-PIBA-glass. (a) Two adhesive spacers are placed on the edges of the bottom glass. The top slide (No. 1 cover slip) is drilled with 1-mm holes to provide access to the patterned channels and to create reservoirs for sample loading/collection. (b) The cover glass is placed on the adhesive forming a chamber. (c) PIBA is introduced using capillary action and a (d) photo-mask is visually aligned over the substrate. The substrate is exposed to UV light. (e) Any un-polymerized PIBA is vacuumremoved, leaving an empty channel ready for acrylamide gel. (f) The RCI is placed at the output of the separation channel and ends of the access channel. Acrylamide gel solution is placed into the channel through the RCI. This allows the gel to not only fill the channel but to remain in the RCI itself for polymerization. Once filled, the device is exposed to UV light again. This results in microchannels and RCI filled with acrylamide gel. (g) Platinum wire is placed in the RCI directly in the gel and acts as an electrode for the separation process. The input reservoir of the channel also has a platinum electrode in electrical contact to serve as the cathode for the separation. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com 3776 S. K. Mohanty et al. thickness (Grace Bio-Labs Secure-Seal Adhesive SA-S1L) were placed on the edges of the bottom glass (Corning 50 mm 6 75 mm plain microslide). The top slide (Fisher No. 1 premium cover glass 35650 mm) was drilled with 1-mm holes using a diamond tip drill bit (Eurotool Diamond Bur fine/med grit 1 mm, 1500 rpm) to provide access holes for the patterned channels and to create reservoirs for sample loading/collection. Since the cover glass was so thin, drilling took less than 10 s per hole with minimal breakage (typically one in eight slides cracked). A photo-definable PIBA solution was made as described by Beebe et al. [22]. The cover glass was placed on the adhesive forming a chamber. PIBA was introduced using capillary action and a photomask printed on an acetate sheet with a clear field pattern was visually aligned over the substrate. Features patterned for this work ranged from 500 mm to 1000 mm in width by 3 cm in length; however, features sizes down to 20–50 mm can be created. The substrate was exposed to UV light (Acticure EFOS A4000) at 6.7 mW/cm2 for 17 s. PIBA was cured by UV light, therefore areas of the mask that blocked light form the channel shape. The ends of the channel were aligned with the holes on the cover glass. A vacuum pump was placed over the holes using a vacuum cup interface (Integrated Dispensing Solutions part no. 8880264) to remove any un-polymerized PIBA leaving an empty sealed channel ready for acrylamide gel. Channels were cleaned out using deionized (DI) water to remove any PIBA residue on the surface of the glass. 2.3 Device interface and RCI construction Electrophoresis 2006, 27, 3772–3778 wich served as the cathode for the separation. The electrode was kept 2 cm away from the loading zone to minimize effects on the protein markers (bubbles generated from electrolysis). A drop of 0.56 Tris-borate-EDTA buffer (TBE) was placed over the reservoir once the separation process started to insure electric field continuity. 2.4 Device operation Characterization of the device was done performing separations on prestained protein markers (Pierce BlueRanger and TriChromRanger). TriChromRanger/BlueRanger consists of a stabilized and lyophilized formulation of seven proteins, ranging from 16.5 K to 210 K and is easily visualized in normal light. The marker solution (1 mL) was loaded into the channel reservoir. An electric field strength of 40 V/cm was applied (Thermo EC 6000P electrophoresis power supply) for 15 min. Typically in other microfluidic systems electrokinetic injection is used to inject samples into a channel [27]. This setup is complex and requires extra work and equipment (extra power supplies, controllers, programing) to manipulate electric fields. To simplify sample loading and make it more accessible to life science labs, we used standard methods via a micropipette for the experiments described here. Experiments were visualized on a standard stereoscope (Olympus SZX12) using Leica DFC300 FX camera for imaging. 3 Results and discussion 3.1 Band separation/collection Connectors to interface with the device were fabricated out of PDMS via a coring process (no masters were used). PDMS was poured over an adhesive (Grace Bio-Labs Secure-Seal Adhesive SA-S-1L) and cured. Small 1 cm 6 1 cm squares were cut and holes were bored through creating an access port. A one-inch piece of capillary tubing (Cole-Palmer PTFE no. 06417–31) was inserted into the connector forming the RCI. The RCI was placed at the output of the separation channel and access channel (Fig. 2f). UV curable acrylamide solution was prepared using 500 mL 25% acrylamide/bisacrylamide (37.5:1), 250 mL 1.5 M Tris-HCl at pH 8.8, 2 mL riboflavin (0.2%), 50 ml TEMED (10%) and 448 mL of DI water to yield a final concentration of 12.5%. Acrylamide gel solution was placed into the channel though the RCI (Fig. 2f). Once filled, the device was exposed to UV light for 10 min at 25 mW/cm2. This resulted in microchannels and RCIs with polymerized acrylamide gel. A platinum wire was placed in the RCI and acted as an electrode for the separation process (Fig. 2g). The input reservoir of the separation channel was in contact with a platinum wire © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim All experiments were conducted using 12.5% acrylamide gel looking at band separation and post separation sample collection using the RCI (Fig. 3). Figure 3a shows the top view of the device during operation. Figure 3b shows seven bands of TriChromRanger as it separates. The bands shown are myosin (210 K), phosphorylase B (110 K), BSA (80 K), ovalbumin (47 K), carbonic anhydrase (32 K), trypsin inhibitor (25 K), and lysozyme (16.5 K). Figure 3c shows an intersection with the separation and access channel. As the band approached the intersection, the electrodes in the collection channel were activated (electrodes in the separation channel were shut off) driving the band into the access channel towards the RCI for collection. 3.2 Band-broadening and leakage When the band reaches the intersection, some broadening in the shape occurs as the band is no longer confined within the separation channel. In addition, band www.electrophoresis-journal.com Electrophoresis 2006, 27, 3772–3778 leakage occurs into channels at the cross-section when it is not collected (Fig. 3d). These results are similar to that reported by Lin et al. [26] and more detail regarding this can be found in their publication. If a band of interest comes after a band that is not collected, most of the leaked material was removed from the path by activating the electrodes in the access channel and driving the Miniaturization 3777 material in the direction of a “waste” RCI directly opposite of the RCI used for sample extraction. This is done before the band of interest reaches the intersection. Occasional bubbles could show up in the gel during fabrication as seen in Fig. 3c. The bubbles did not have any major impact on the performance of the microelectrophoresis chip. Figure 3. Separation and collection of proteins with microelectrophoresis device. (a) Top view of a device during use. (b) Image of TriChromRanger proteins as separation is occurring. (c) Orthogonal intersection of separation channel and access channel. The band of interest approaches the intersection. Once it is in range of the access channel, the electrodes at the end of the access channel are activated directing the protein to the RCI where it can be collected. (d) Band leakage into the channel was observed if a band was not collected. (e) The RCI with integrated electrode inserted into a PDMS connector to interface with the device. (f) Acrylamide gel removed from the RCI after collecting the protein. The protein is stored in a cylindrical shaped gel. Dark portion of the gel is BlueRanger protein. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com 3778 S. K. Mohanty et al. Electrophoresis 2006, 27, 3772–3778 3.3 Sample removal off-chip 5 References Figure 3e shows an image of the RCI with the electrode coming out of the capillary tubing. The RCI is inserted into a PDMS connector to interface with the device. As mentioned above, acrylamide gel was polymerized in the RCI and a positive potential was applied at the electrode to drive the protein bands towards the RCI. After the sample was collected inside the tubing, it was removed from the chip by pulling the capillary out. The RCI provided a convenient package to transport the protein. The gel was removed from the capillary by applying pressure at one end leaving a cylindrical shaped gel. Figure 3f shows a protein (dark color) filled gel from a capillary lying on a microelectrophoresis device. [1] Ugaz, V. M., Elms, R. D., Lo, R. C., Shaikh, F. A. et al., Philos. Trans. R. Soc. Lond. A 2004, 362, 1105–1129. [2] Kan, C. W., Fredlake, C. P., Doherty, E. A. S., Barron, A. E., Electrophoresis 2004, 25, 3564–3588. [3] Lagally, E. T., Mathies, R. A., J. Phys. D 2004, 37, R245– R261. [4] Vesterberg, O., Electrophoresis 1993, 14, 1243–1249. [5] Guttman, A., Ronai, Z., Electrophoresis 2000, 21, 3952– 3964. [6] Chen, X. J., Ugaz, V. 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[27] Xu, X., Luo, G. A., Chu, X. H., Lin, B. C., Chin. Sci. Bull. 1995, 40, 482–487. 4 Concluding remarks The work shown here illustrates a microelectrophoresis chip with an integrated sample collection scheme that is constructed using a simple, inexpensive fabrication methodology. Expensive micromachining equipment associated with microtechnology is not needed. The only materials required are glass slides, a photo-mask transparency, PIBA, and a UV source. The cost of these materials is minimal and will allow researchers to take advantage of the benefits of microscale analysis. Devices are built in minutes and modifications to a design are made easily by changing a photo-mask transparency. The cost of such a mask is typically $20 US (as compared to standard chrome masks that cost $400–$500 US). One important point this device addresses is the issue of post separation sample collection without the use of expensive liquid handling instruments. The RCI presented here provides a sample recovery method that can interface with the macro world, allowing researchers to further process samples with traditional molecular biology techniques. For demonstration purposes, we have shown a simple device for 1-D single lane separation of biomolecules. It should be noted that parallel lanes can be easily integrated into this platform allowing for multiple sample analysis. As mentioned above, various microfluidic tools have been demonstrated using mFT. Electrophoresis and the RCI are additional tools that can be added to the mFT toolbox to create a versatile platform for electrophoretic and other biological analysis methods. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
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