An active microfluidic system packaging technology

Sensors and Actuators B 122 (2007) 337–346

An active microfluidic system packaging technology Ki-Ho Han a , Rachel D. McConnell b , Christopher J. Easley b , Joan M. Bienvenue b , Jerome P. Ferrance b , James P. Landers b , A. Bruno Frazier c,∗ a

School of Nano Engineering, Inje University, Republic of Korea Department of Chemistry, University of Virginia, VA 22904, USA c School of Electrical and Computer Engineering, Georgia Institute of Technology, 777 Atlantic Drive, Atlanta, GA 30332, USA b

Received 26 January 2006; received in revised form 14 June 2006; accepted 19 June 2006 Available online 8 September 2006

Abstract This paper presents the design, fabrication, and characterization of a microfluidic system interface (MSI) technology for integration of complex microfluidic systems containing multiple functionalities. The microfluidic system interface technology provided a simple method for realizing complex arrangements of on-chip/off-chip microfluidic interconnects, integrated microvalves for fluid control, and optical windows for on-chip optical processes. The microfluidic interconnects were designed to provide one-step plug-in fluid interconnection utilizing a post assembled o-ring to ensure a tight seal. The integrated microvalves were completed when the MSI was assembled atop of the microfluidic system. The microvalves have been designed for zero dead volume with a displaced channel volume of 24 nl in the closed state. The valve was pneumatically actuated up to a valve pressure of 450 kPa. The optical windows were designed to allow for analysis operations such as infrared polymerase chain reaction and conventional fluorescence detection. A microfluidic system for genetic sample preparation was used as the test vehicle to prove the usefulness of the MSI technology with respect to complex microfluidic systems containing multiple functionalities. The miniaturized genetic sample preparation system consisted of several functional compartments including cell purification, cell separation, cell lysis, solid phase DNA extraction, polymerase chain reaction and capillary electrophoresis. Use of the MSI technology to enable integration of this complex lab-on-a-chip system in a hybrid multi-chip format was demonstrated. Additionally, functional operation of the solid phase extraction and PCR thermocycling compartments was demonstrated using the MSI. © 2006 Published by Elsevier B.V. Keywords: Microfluidic system interface; Packaging; Stereolithography

1. Introduction For more than two decades, analytical microfluidic devices [1–3] have been developed for manipulating and analyzing samples. Microfluidic devices, when compared to macroscale devices, have advantages such as smaller geometrical size, shorter analysis times, less sample/reagent consumption, and disposability. Many researchers have successfully demonstrated singular functional microfluidic devices [4–6] and transducers [7–9] for fluid manipulation. However, the development of integrated complex microfluidic systems has shown relatively modest growth. The reasons for this are at least partially rooted in the fact that integration of sample processing steps and ana-

∗

Corresponding author. Tel.: +1 404 894 2030; fax: +1 404 385 0343. E-mail address: [email protected] (A.B. Frazier).

0925-4005/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.snb.2006.06.028

lytical functions into a complex microfluidic system has numerous technical difficulties, such as the need for more complex packaging, the possibility of biological/chemical cross contamination between functional compartments, and the possible need for different substrate materials for the individual compartment functionalities. One of the major technical difficulties in developing complex microfluidic systems is realizing complex arrangements of microfluidic interconnects for sample/reagent/buffer introduction, for interconnection between multiple chips, and for interconnection between the microsystem and macro world. Several fluidic interconnection technologies [10] have been demonstrated including adhesive bonding of miniaturized connectors [11–14], assembled interconnection blocks [15,16], silicone rubber o-ring couplers [17,18], silicon connectors fabricated by deep reactive ion etching (DRIE) [19,20], flanges [21], injection molded components [14], and socket-type multi-connectors

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[22]. Adhesive bonding of miniature fittings over through-holes in the substrate or coverplate has several inherent problems including a large dead volume, a large footprint, labor intensive assembly, and clogging/contamination by the adhesive. Assembled interconnection blocks are bulky and expensive. Therefore, these fluid interconnect schemes do not lend themselves to the development of disposable microfluidic systems. Socket-type interconnects provide a viable option for complex microfluidic system at the commercial level. However, sockets lack the on-the-fly design flexibility required at the research and development level. The microfluidic system interface (MSI) technology based on stereolithography (SLA) provides a practical method for realizing complex arrangements of microfluidic interconnects. The primary advantage of the SLA technology is that it allows for the fabrication of arbitrary three-dimensional (3D) structures with very few design constraints. In addition, it can be used to fabricate arbitrary 3D structures in minutes to a few hours with 25 ␮m vertical resolution and ∼1 ␮m horizontal pattern resolution [9]. Furthermore, it allows integration of other component functionalities such as the incorporation of electrical, mechanical, and optical components as part of the build process [9]. The MSI can easily be aligned onto complex microfluidic systems using tooling holes and/or edge alignment. Additionally, the use of o-rings to implement tight fluid seals avoids fluid leakage and prevents clogging of the microchannel by the bonding adhesive when the MSI is assembled to the underlying complex microfluidic system. One of the advantages of the MSI technology is the ability to integrate elastomeric microvalves into the MSI along with complex arrangements of microfluidic interconnects and optical windows. The integrated microvalves are essential fluid control elements for sequential processing and manipulation of fluids within the microsystems. A large number of microvalves have been reported using silicon/glass material [23]. While the microvalves have demonstrated good performance, many were realized using hybrid manufacturing approaches, leading to relatively large flow/dead volumes. To overcome this geometrical

scaling limitation, elastomeric microvalves have been realized. The microvalves were fabricated using an elastomer substrate of polydimethylsiloxane (PDMS) [8,24–26]. While the PDMSbased microfluidic devices with integrated microvalves were successfully demonstrated, the hydrophobicity and porosity of the PDMS remains an issue [27,28]. In addition, PDMS has significant auto-fluorescence [8], thus limiting its use in applications requiring high sensitivity fluorescence detection (e.g. laser-induced fluorescence detection). In contrast, glass-based microfluidic devices have demonstrated advantages in applications for which highly sensitive fluorescence detection was required [29–31]. Since microfluidic devices fabricated by glassto-glass bonding technology result in a permanent mechanical bonding between adjoining layers, the technology lends itself to microfluidic applications requiring mechanical rigidity, high fluid pressures, or relatively high operating temperatures. Furthermore, the development of integrated microvalves for glassbased microfluidic systems is critical as a method for controlling fluid flow within these microsystems. In this work, the MSI technology has been demonstrated to be a practical interface/packaging methodology for microfluidic systems requiring complex arrangements of microfluidic interconnects and fluidic control elements such as microvalves and optical interfaces for functions such as thermocycling and fluorescence detection. To demonstrate the usefulness of the MSI for complex microfluidic systems, an MSI has been created for a multi-chip miniaturized genetic sample preparation system. Operation of the functional compartments of the genetic sample preparation system (i.e., genomic DNA extraction in the solid phase extraction compartment, thermocycling in the PCR compartment) has been demonstrated utilizing the MSI technology. 2. Design and fabrication 2.1. Design Fig. 1 shows a schematic of the miniaturized genetic sample preparation system. In sequential order of function for genomic

Fig. 1. A schematic of the hybrid multi-chip genetic sample preparation system consisting of one chip for whole cell purification and a second chip for genetic DNA analysis.

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analysis, the first compartment was for whole cell purification of white blood cells (WBC’s) from whole blood; the second functional compartment was for extraction of DNA from the WBC lysate; the third compartment was for amplification of the extracted DNA; the fourth compartment was for capillary electrophoretic separation of the amplified DNA. A hybrid multichip approach was used to realize the total analysis system (TAS). The first glass chip, Chip 1, contained the functionalities for introduction of a whole blood sample, purification and separation the red/white blood cells, and cell lysing. The second glass chip, Chip 2, contained the functionalities for solid phase extraction (SPE), infrared mediated polymerase chain reaction (PCR), capillary electrophoresis (CE) and laser-induced fluorescence detection. Each of the glass chips in the two chips ␮-TAS was a packaged assembly consisting of a glass chip and the corresponding MSI. Associated with the genetic sample preparation system, we have previously shown solid phase extraction (SPE) of DNA from biological samples with single-functional microfluidic devices [29,32,33], but a number of technical difficulties in integrating this process with additional processing steps were discovered [33]. These include prevention of the solid phase matrix from forming in other regions of the device, contamination of the PCR compartment with SPE reagents inhibitory to the PCR process (i.e., guanidine and isopropyl alcohol), and isolation of the PCR coating reagent from the SPE compartment. Elimination of these problems in simple glass microfluidic systems is difficult and requires unusual fluid flow protocols, thus fluid valving elements are critical for these glass-based systems. In order to gain the functionality required for Chip 1, the MSI required four fluidic interconnects. The MSI of Chip 2

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required seven microfluidic interconnects, two microvalves and two optical windows, as shown in Fig. 1. Fig. 2 illustrates the fabrication and assembly process for the packaged glassbased microsystem including the microfluidic interconnects, the integrated microvalves, and the optical windows. The one-step plug-in microfluidic interconnects were designed with two orings. The first o-ring was to prevent clogging and contamination of the microchannels by the bonding adhesive when the MSI was bonded to the glass chip. The second o-ring provided a tight fluid seal when the capillary tubing was connected to the microfluidic interconnects. In this work, 1/16 in. (1.6 mm) capillary tubing was used. The microfluidic interconnects were 4.0 mm in diameter. The MSI microvalves was designed and fabricated with a dome shape to avoid fluid leakage from sharp corners and rough surfaces. The latex gasket surrounding the microvalves, shown in Fig. 2, was used to tightly hold the latex membrane on the glass chip and to avoid leakage of the bonding adhesive into the valve region during bonding of the MSI to the glass chip. Recent reports [4,8] by other groups using elastomeric valving technologies on microfluidic devices required integration of the flexible elastomeric membrane into the microfluidic chip as part of the chip fabrication process. The method reported in this work allows the glass microfluidic device to be fabricated independently, with the microvalve formed as the MSI is assembled. Furthermore, this approach provides the possibility of using various materials for the valve membrane since it does not need to be sealed within the device itself. The elastomeric membrane could be tailored for specific applications. To enable optical interfacing between the microfluidic system and external optical sources/detectors, optical windows can be

Fig. 2. Cross-section view of the one-step plug-in microfluidic interconnects and the dome-shaped monolithic microvalve: (a) when the microvalve is open and (b) when the microvalve is closed.

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integrated into the MSI, as shown in Fig. 1. Relevant applications of the optical windows in the packaged microsystem include on-chip infrared mediated PCR and laser-induced fluorescence detection. 2.2. Fabrication Stereolithography (SLA) was used as the underlying fabrication technology for the MSI. The diameter of the laser beam for the SLA system (Viper SI2, 3D Systems Corp., Valencia, CA) was 75 ␮m with 2 ␮m in-plane pattern resolution. The vertical resolution of the platform motion was 25 ± 1 ␮m. First, the MSI design was generated using Pro/Engineer® (Parametric Technology Co., Needham, MA). The drawing was imported into the SLA system, and then multiple copies of the design were built in an automated fashion with the photosensitive polymer (SL5510TM , 3D Systems Corp.). Once the pre-cured MSI was formed, it was removed from the platform and cleaned to remove the excess uncured photopolymer resin. A 5 min immersion in 100% isopropyl alcohol in an ultrasonic bath was used as the cleaning process, followed by post-curing using an ultraviolet source for 1.0 h. Fig. 3 shows the fabrication and assembly process used to realize the packaged microsystem including the glass-based microfluidic system and the microfluidic system interface. Chip 1 and Chip 2 were fabricated using the same glass etching and bonding technologies. For Chip 2, the bottom glass (BorofloatTM glass, 0.7 mm thick, Howard Glass Co., Worchester, MA), which contained the SPE, PCR, thermocouple microchamber and CE separation microchannel, was etched 60 ␮m deep using a 25% HF solution. The SPE compartment consisted of an etched microchannel 2.2 cm long with a width of 400 ␮m. The top glass containing the PCR chamber and thermocouple (T240C, Physitemp Instruments, Clifton, NJ) microchannel was etched 150 ␮m deep. The dome-shaped

microvalve holes were etched using 25% HF solution for approximately 10 h until the appropriate diameter hole was etched through the bottom side of the top glass (Fig. 3(a)). A mechanical drill was used along with a SLA alignment jig to fabricate the through-holes in the glass used for fluid interconnections, as shown in Fig. 3(b). The top and bottom glasses were then bonded together using glass-to-glass thermal bonding at 685 ◦ C for 3.5 h (Fig. 3(c)). A nitrile rubber o-ring (Size 001-1/2, McMASTER-CARR, Atlanta, GA) was used to avoid clogging of the microchannels by the low viscosity epoxy adhesive (#7377, Epoxylite Corp., Irvine, CA) for bonding the MSI to the underlying glass chip. Another nitrile rubber o-ring (Size-001, McMASTER-CARR) was used to provide a tight fluid seal between the external capillary tubing and the microfluidic interconnects. A 120 ␮m thick latex sheet was used to form the active element of the microvalve (Fig. 3(d)). The MSI and the glass chip were aligned and placed in a clamped jig. Subsequently, an epoxy adhesive was put into the ‘bonding’ vias designed into the MSI. Capillary forces pulled the adhesive into the gaps between the MSI and the glass chip. The epoxy adhesive was cured at room temperature for 24 h, completing fabrication of the packaged microfluidic system (i.e., the miniaturized genetic sample preparation system) as shown in Fig. 3(e). 3. Results and discussion 3.1. Multi-chip microfluidic analysis systems Fig. 4 shows the fabricated glass chips and the corresponding MSIs for the miniaturized genetic analysis system. The overall size of Chip 1 and Chip 2 were 2.5 cm × 7.5 cm and 5 cm × 10 cm, respectively. The o-rings (1.8 mm in inner diameter and 3.7 mm in outer diameter) were fitted into recesses located on the underside of the MSI’s fluid interconnects before

Fig. 3. (a–e) The fabrication and assembly process used to realize the packaged microsystem including the glass-based microfluidic system and the microfluidic system interface.

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Fig. 6. The pull-out pressure characteristics for the one-step plug-in interconnect and the capillary tubing used for micro-to-macro fluid interconnection.

Fig. 4. Top views of the glass-based microfluidic systems and bottom views of the microfluidic system interfaces of: (a) cell separation system and (b) DNA separation system.

the MSIs were bonded to the glass chips. The latex membranes (5.0 mm in diameter) used for the microvalves were attached to the underside of the valve hole using perfect-glueTM 2 (Macco, Cleveland, OH). Vacuum ports (0.8 mm in diameter) were designed close to the fluid and valve interconnects as a method for pulling the epoxy adhesive into the gaps between the MSI and the glass chip, as shown in Fig. 4. After assembly of the MSIs and the glass chips, a second o-ring (0.9 mm in inner diameter and 2.8 mm in outer diameter) was inserted into each fluid interconnect using a curved-pointed tweezers. Fig. 5 shows the miniaturized, two-chip genetic analysis system. The hybrid system includes eleven fluid interconnects, two pneumatic interconnects for the microvalves, and two optical windows for laser-induced fluorescence detection and for PCR infrared thermocycling.

Fig. 5. An optical micrograph on the two-chip hybrid total analysis system for genetic analysis. The hybrid system includes on-chip/off-chip microfluidic and pneumatic interconnections, microvalves, and windows for optical detection.

3.2. Integrated microfluidic interconnects The fluid interconnects were used to facilitate the movement of sample/reagent/buffer solutions in to and out of the microfluidic system. Fig. 6 shows the pull-out pressure of the external capillary tubings (TEPLON® FEP 1/16 in. tubing, Upchurch, Oak Harbor, WA) for varying capillary inner diameter. The inner diameters of the external capillary tubings were 0.25, 0.5, 0.75, and 1.0 mm, respectively. The experimental results show that the pull-out pressure of the capillary tubing with 1.0 mm inner diameter was slightly less than the other tubing sizes. This variation might be due to the elasticity of the thinner walled 1.0 mm capillary tubing when compared to the other capillaries. The pull-out pressures of the other three tubings were statistically equivalent and greater than 1.0 MPa. Other designs of SLA-based microfluidic interconnect are possible. As a first example, screw type interconnects with a stainless steel flushnut (e.g. F-385, Upchurch) can be fabricated and would be appropriate for applications requiring high fluid and pneumatic pressure. The principle disadvantages of this approach are the large required footprint (∼8 mm minimum diameter) and the expensive cost of the flushnut. As a second example, barbed or conical port type interconnects along with a silicone tubing (e.g. 694441, Helix Medical Inc., Carpinteria, CA) can easily be realized with small diameter of 2–3 mm. Additionally, the interconnection morphology of the integrated microfluidic interconnect can be designed to mate with an offchip socket-type connector leading to an external fluid control system [22]. This is an interesting extension of the current designs and allows for single point fluid connection for disposable cartridges and other quick connect/disconnect applications. Each of the interconnect designs that can be fabricated using the MSI technology, have their own merits, while these have the respective problems, as mentioned above. Therefore, these types of interconnect along with the proper external capillary tubings should be selected carefully depending on applications of individual functional compartments and microfluidic systems.

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3.3. Integrated MSI microvalve A critical part of the MSI technology is the ability to form microvalves directly into the interface. The integrated microvalve technology provides a mechanism for fluid control in the underlying microfluidic system. Microvalve #1 in Chip 2 was used to isolate the solid phase extraction (SPE) microchannel during filling and curing of the silica bead/sol–gel hybrid matrix. Microvalve #2 was designed to isolate the PCR microchamber while filling the CE separation microchannel with polymer, and for preventing the PCR solution from leaking into the CE separation and the injection microchannel during the PCR thermocycling. Fig. 7(a) shows the operation of the integrated microvalve. The depth and width of the underlying microchannel were 60 and 400 ␮m, respectively. The minimum diameter of the domeshaped valve hole was 0.9 mm. Therefore, the volume displaced by closing of the microvalve was 24 nl. Fig. 7(b) shows a graph of the fluid pressure required to cause leakage for a range of valve pressures. The experimental result shows a linear relationship between the fluid and the valve pressures for a valve pressure range from 0 to 450 kPa. Additionally, the experimental results show that the microvalve was in the closed state with a minimum required valve pressure of 150 kPa using the 120 ␮m thick latex sheet (Fig. 2(b)). With respect to external capillary connection for these pneumatically actuated microvalves, the results prove that the one-step plug-in interconnects were robust over an appropriate pressure range. This correlated well with the data presented in Fig. 6, since the pull-out pressure of the external capillary tubings was greater than 1 MPa. To demonstrate the fluid control aspects of the genetic analysis system, red dye was used during operation of each functional compartment (Fig. 8). The integrated microvalves were actuated using a valve pressure of 400 kPa. When microvalves #1 and #2 were closed, the red dye flowed through the SPE microchamber (Fig. 8(a)). Next, microvalve #1 was opened to elute the red dye from the SPE microchamber into the PCR microchamber (Fig. 8(b)). As a final procedure, microvalve #2 was opened, allowing the red dye in the PCR microchamber flows into the CE separation microchannel. Operation of the genetic analysis system using red dye demonstrates the efficacy of the integrated microvalves for sequential on-chip procedures and can be used to avoid biological/chemical cross contamination between the individual functional compartments. Our previous work [32] on packing of the silica beads in the SPE microchamber showed that frits had to be formed by filling the outlet reservoir with a silica bead/sol–gel matrix, which was subsequently gelled to form a porous solid. The silica beads were then packed against the frit and the sol–gel used to hold the beads in place. The extra steps require in forming these frits, the low success rate, and their instability after formation, suggested that this was not an ideal method. In addition, this method could not be used in an integrated microfluidic system because the frit mixture easily flowed into the adjacent regions of the microfluidic system, in particular the PCR microchamber. Additionally, if a piece of the frit broke off during the extraction process, this might prevent flow of

Fig. 7. (a) The monolithic microvalve in the open and closed state and (b) the fluid pressure required to cause leakage for a range of valve pressures.

the eluted DNA into the proper regions, or occlude the PCR microchamber and CE separation microchannel. In the miniaturized genetic analysis system, microvalve #1 allowed simpler packing of the silica bead/sol–gel hybrid matrix since a frit does not have to be formed. Microvalve #1 was closed to allow silica bead packing and sol–gel filling, and then the silica bead/sol–gel hybrid matrix was cured at 50 ◦ C for 6.0 h. The DNA extraction procedure in the SPE compartment consisted of load, wash and elution steps. The load step was performed using a syringe pump (Model 101i, World Precision Instruments, Sarasota, FL), with a 250 ␮l gas-tight syringe (Hamilton, Las Vegas, NV) connected to the SPE microchamber inlet. Using a flow

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Fig. 8. Red dye flowing through the microfluidic compartments in the DNA separation system with microvalve #1 and microvalve #2: (a) closed, closed; (b) open, closed; (c) open, open, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

rate of 4.2 ␮l/min, 21 ␮l of the load solution was passed through the SPE microchamber. Proteins and other cellular material that adsorbed on the SPE matrix during the load step were removed by washing the extraction bed with 20 ␮l of an isopropanol/water 80/20 (v/v) solution. The captured DNA in the SPE microchamber was then eluted in 1× PCR buffer; fractions were collected every 2.5 min (10.5 ␮l). Each fraction was PCR amplified using

a Perkin-Elmer Thermocycler (Santa Clara, CA). All amplified samples and the remaining non-amplified 3 ␮l aliquot from each fraction were analyzed on the Bio-Analyzer 2100 (Agilent Technologies, Palo Alto, CA) using the DNA 1000 kits according to the manufacturer’s instruction. The amount of PCR amplified product generated using the eluted DNA in each fraction as template is plotted, as shown in Fig. 9. The PCR amplified product

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Fig. 9. The DNA amplification profile from the SPE elution fractions.

was seen in all fractions after the fifth fraction, indicating DNA elution from the silica bead/sol–gel hybrid matrix occurred in this range. The experimental result for SPE of DNA shows that microvalve #1 effectively isolated the SPE microchamber during filling and curing of the silica bead/sol–gel hybrid matrix.

Fig. 11. An electropherogram of the PCR product for the 275-bp invA gene from Salmonella typhimurium.

Additionally, microvalve #1 was successfully used for loading, washing, and elusion of the SPE process in the integrated genetic sample preparation system. 3.4. Optical windows The MSI optical window #1 was designed to allow optical interfacing with the PCR microchamber for infrared thermo-

Fig. 10. PCR thermocycling using the IR-mediated non-contact instrument and the glass-based microfluidic system.

Fig. 12. Robustness of the microvalve technology was demonstrated by the containment of the thermocycled red dye in the PCR microchamber after 90 cycles (a) chip underside view and (b) chip top view.

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cycling. The MSI optical window #2 was designed as part of the packaging for the CE separation microchannel, to allow laser-induced fluorescence detection of target DNA fragments. To show the utility of integrating PCR with other functional domains in the miniaturized genetic sample preparation system, genomic DNA was amplified using primers for amplification of the 275-bp invA gene from Salmonella typhimurium. The PCR thermocycling was performed between 58, 72, and 94 ◦ C with the IR-mediated non-contact heating instrumentation for 30 cycles, as shown in Fig. 10. Once the PCR thermocycling began, closing microvalve #2 served to prevent leakage of the solution out of the PCR microchamber and served to isolate the expanding PCR solution between the closed microvalve #1 and microvalve #2. Fig. 11 shows an electropherogram of the invA gene amplified by 30 PCR thermocycles. Interestingly, an initial version of this system failed due to delamination of the MSI from the glass chip around the PCR microchamber during the PCR thermocycling. This problem was rectified by switching to a more thermally stable epoxy adhesive (#7377, Epoxylite Corp., Irvine, CA). Fig. 12 shows isolation of red dye in the PCR microchamber after 90 thermocycles, demonstrating that both of the integrated microvalves remained functional throughout the thermocycling process. 4. Conclusions In this paper, we presented the design, fabrication, and characterization of the microfluidic system interface (MSI) for the multi-functional miniaturized analytical systems. The MSI technology contained plug-in style microfluidic interconnects designed to enable simple connection of external capillary tubing, dome-shaped integrated microvalves to control sequential fluid processing, and optical windows to prevent heating of the MSI during PCR thermocycling and to avoid interference from the packaging during laser-induced fluorescence detection. The microfluidic interconnects have shown advantages such as easeof-use, a small footprint (less than 4.0 mm in diameter), tight fluid seals using o-rings, operation at fluid pressures up to 1 MPa, minimal exposure of the interconnect materials to reagents for the functional compartments, and the ability to mate with standard sockets for multi-fluid interconnections. The dome-shaped integrated microvalves, which have zero dead volume with the displaced channel volume of 24 nl in the closed state, were actuated pneumatically in the range of valve pressure from 0 to 450 kPa. The microvalve #1 placed between the SPE and PCR microchambers was used for packing and curing of the SPE silica bead/sol–gel hybrid matrix. The microvalve #2 located between the PCR microchamber and the CE separation microchannel was used to prevent contamination of the PCR microchamber during filling of the CE separation microchannel with sieving polymer, and for preventing the PCR solution from leaking into the CE separation microchannel during the PCR thermocycling. Optical window #1 prevented heating of the MSI during the PCR thermocycling. The SPE extraction and the PCR thermocycling operations were performed on the integrated genetic

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sample preparation system to demonstrate the efficacy of the MSI technology. Acknowledgements This work was supported by the National Institutes of Health (NIH)/National Institute for Environmental Health Sciences (NIEHS) under Grant ES10846. References [1] S.C. Terry, J.H. Jerman, J.B. Angell, IEEE Trans. Electron Devices ED-26 (1979) 1880. [2] O. Bakajin, T.A.J. Duke, J. Tegenfeldt, C.-F. Chou, S.S. Chan, R.H. Austin, E.C. Cox, Anal. Chem. 73 (2001) 6053. [3] Y. Huang, S. Joo, M. Duhon, M. Heller, B. Wallace, X. Xu, Anal. Chem. 74 (2002) 3362. [4] A.Y. Fu, C. Spence, A. Scherer, F.H. Arnold, S.R. Quake, Nat. Biotechnol. 17 (1999) 1109. [5] K.S. Chang, S. Tanaka, C.L. Chang, M. Esashi, Tech. Dig. 12th Int. Conf. Solid-State Sensors and Actuators Workshop (Transducers’03), Boston, USA, 2003, pp. 635–638. [6] K.-H. Han, A.B. Frazier, Lap Chip 6 (2006) 265. [7] P.F. Man, D.K. Jones, C.H. Mastrangelo, Proc. IEEE Micro Electro Mechanical Systems Conference (MEMS’97), Nagoya, Japan, 1997, pp. 311–316. [8] W.H. Grover, A.M. Skelley, C.N. Lui, E.T. Lagally, R.A. Mathies, Sens. Actuators B 89 (2003) 315. [9] A. Han, O. Wang, M. Graff, S.K. Mohanty, T.L. Edwards, K.-H. Han, A.B. Frazier, Lab Chip 3 (2003) 150. [10] C.K. Fredrickson, Z.H. Fan, Lab Chip 4 (2004) 526. [11] T.B. Stachowiak, T. Bohr, E.F. Hilder, D.S. Peterson, M. Yi, F. Svec, J.M.J. Fr´echet, Electrophoresis 24 (2003) 3689. [12] A.V. Pattekar, M.V. Kothare, J. Micromech. Microeng. 13 (2003) 337. [13] H. Chen, D. Acharya, A. Gajraj, J.-C. Meiners, Anal. Chem. 75 (2003) 5287. [14] B.L. Gray, D. Jaeggi, N.J. Mourlas, B.P. van Drieenhuizen, K.R. Williams, N.I. Maluf, G.T.A. Kovacs, Sens. Actuators A 77 (1999) 57. [15] V. Nittis, R. Fortt, C.H. Legge, A.J. de Mello, Lab Chip 1 (2001) 148. [16] D. Verlee, A. Alcock, G. Clark, T.M. Huang, S. Kantor, T. Nemcek, J. Norlie, J. Pan, F. Walsworth, S.T. Wong, Tech. Dig. of Solid-State Sensor and Actuator (Hilton Head 1996), Hilton Head, USA, 1996, pp. 9–14. [17] T.-J. Yao, S. Lee, W. Fang, Y.-C. Tai, Proc. IEEE Micro Electro Mechanical Systems Conference (MEMS 2000), Miyazaki, Japan, 2000, pp. 624–627. [18] E. Meng, S. Wu, Y.-C. Tai, Proceedings of the ␮TAS 2000 Symposium (␮TAS), Enschede, Netherlands, 2000, pp. 41–44. [19] C. Gonz´alez, S.D. Collins, R.L. Smith, Sens. Actuators B 49 (1998) 40. [20] B.L. Gray, S.D. Collins, R.L. Smith, Sens. Actuators A 112 (2004) 14. [21] A. Puntambekar, C.H. Ahn, J. Micromech. Microeng. 12 (2002) 35. [22] Z. Yang, R. Maeda, J. Chromatogr. A 1013 (2003) 29. [23] G.T.A. Kovacs, Micromachined Transducers Sourcebook, McGraw-Hill Co., 1998. [24] K. Hosokawa, R. Maeda, J. Micromech. Microeng. 10 (2000) 415. [25] Y.-C. Wang, M.H. Choi, J. Han, Anal. Chem. 76 (2004) 4426. [26] S. Lee, W. Jeong, D.J. Beebe, Lab Chip 3 (2003) 164. [27] X. Ren, M. Bachman, C. Sims, G.P. Li, N. Allbritton, J. Chromatogr. B 762 (2001) 117. [28] S. Hu, X. Ren, M. Bachman, C.E. Sims, G.P. Li, N. Allbritton, Anal. Chem. 74 (2002) 4117.

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K.-H. Han et al. / Sensors and Actuators B 122 (2007) 337–346

[29] K.A. Wolfe, M.C. Breadmore, J.P. Ferrance, M.E. Power, J.F. Conroy, P.M. Norris, J.P. Landers, Electrophoresis 23 (1997) 727. [30] D.J. Harrison, K. Fluri, K. Seiler, Z. Fan, C.S. Effenhauser, A. Manz, Science 261 (1993) 895–897. [31] B.C. Giordano, L. Jin, A.J. Couch, J.P. Ferrance, J.P. Landers, Anal. Chem. 76 (2004) 4705.

[32] M.C. Breadmore, K.A. Wolfe, I.G. Arcibal, W.K. Leung, D. Leung, B.C. Giordano, M.E. Powers, J.P. Ferrance, S. Feldman, P.M. Norris, J.P. Landers, Anal. Chem. 75 (2003) 1880. [33] J.P. Ferrance, Q. Wu, B.C. Giordano, C. Hernandez, Y. Kwok, K. Snow, S. Thibodeau, J.P. Landers, Anal. Chem. Acta 500 (2003) 233.