Copyright 2009 Year IEEE. Reprinted from IEEE TRANSACTIONS ON ADVANCED PACKAGING. Such permission of the IEEE does not in any way imply IEEE

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1 Copyright 2009 Year IEEE. Reprinted from IEEE TRANSACTIONS ON ADVANCED PACKAGING. Such permission of the IEEE does not in any way imply IEEE endorsement of any of Institute of Microelectronics products or services. Internal of personal use of this material is permitted. However, permission to reprint/republish this material for advertising or promotional purposes or for creating new collective works for resale or redistribution must be obtained from the IEEE by writing to

2 528 IEEE TRANSACTIONS ON ADVANCED PACKAGING, VOL. 32, NO. 2, MAY 2009 Development of a Disposable Bio-Microfluidic Package With Reagents Self-Contained Reservoirs and Micro-Valves for a DNA Lab-on-a-Chip (LOC) Application Ling Xie, C. S. Premachandran, Senior Member, IEEE, Michelle Chew, and Ser Choong Chong Abstract A disposable self-contained microfluidic package has been developed and tested for on-chip DNA extraction from human blood for practical lab-on-a-chip platform. The microfluidic package has been customized to allow easy interface between the microscale sample injection to the Si-based microscale sample preparation chip. For precise sample dispensing and to minimize dead volume and/or sample lost conical-shaped reservoirs have been employed. Reservoirs filled with reagents are sealed by a highly elastic thin rubber membrane. Automated actuation system has been designed and implemented for programmable sample/reagent dispensing using thin rubber membrane-plunger mechanism. The packaged DNA chip has been tested using blood sample and the testing protocol has been optimized to meet the requirements for DNA extraction. Index Terms Disposable package, lab-on-a-chip (LOC), microfluidic dispense, microfluidic package, self-contained reagent. I. INTRODUCTION I NFLUENCE of microelectronics into life science has introduced many developments in bio-applications. One of the main impacts on bio-systems is the miniaturization that has seen clearly on microelectronics industry. Micro- and nano-level of interaction in bio-systems has emerged new field of research and new application products. Microfluidics is one of the fast emerging fields where fluid flows in microchannels and very little dead volumes are present which eliminate contamination and mixing among the fluids. Convergence of microfluidics and microelectronics result in a new kind of devices, microfluidic chips. They require channels, reservoirs, filters to process the fluid [1] [10]. Microfluidic structures are formed on silicon/polymer substrate by micromachining or microstamping method. Microfluidic chip offers the ability to work with shorter reaction time, smaller fluid sample/reagent volumes, and promises of parallel processes. Manuscript received August 28, 2006; revised November 07, 2007, May 16, First published May 15, 2009; current version published May 28, This work was recommended for publication by Associate Editor L. Nguyen upon evaluation of the reviewers comments. The authors are with Institute of Microelectronics, A-star, Singapore Science Park 2, Singapore ( xieling@ime.a-star.edu.sg; prem@ime.astar.edu.sg; chewbr@ime.a-star.edu.sg; scchong@ime.a-star.edu.sg; serchoong@yahoo.com.sg). Color versions of one or more of the figures in this paper are available online at Digital Object Identifier /TADVP Fig. 1. Schematic view of an integrated bio-microfluidic package for DNA LOC application. Major applications of microfluidics are on the clinical diagnostics, drug discovery, bio-terrorism monitoring, and therapeutics devices. Fluidic components required for clinical diagnostics is different from the fluidic components required for drug discovery. In most of the cases a common platform can be used to combine the basic modules in order to implement a microfluidic device for diagnostic applications or drug discovery for pharmaceutical applications. The lab-on-a-chip (LOC) concept is to realize the functions of bio-laboratory in a silicon chip (Fig. 1). The miniaturized bio-laboratories are fabricated by photolithographic process developed in the microelectronics industry to form circuits, chambers, valves, and channels in quartz or silicon substrate [11] [20]. Fluidic samples can be manipulated by placing valves, pumps in the chip and fluid can be diluted, mixed with other reagents or separated by other process on the same chip. In this paper, a microfluidic chip for DNA extraction and amplification is described. Silicon substrate is used to form microfluidic components in the chip. Different techniques have been used for extraction of DNA/RNA from the body fluid. Liu et al. have developed a microfluidic assay for sample preparation, polymer chain reaction (PCR) amplification, and microarray detection for DNA [6]. In this approach magnetic beads are used for cell capture. A magnet is activated when the target cells labeled with antibody coated super paramagnetic beads pass through a separation column and in the presence of strong magnetic field the labeled cells are retained on the chip. In this method efficient binding happen. However, additional requirement such as a magnet is required to magnetize and demagnetize the beads /$ IEEE

3 XIE et al.: DEVELOPMENT OF A DISPOSABLE BIO-MICROFLUIDIC PACKAGE WITH REAGENTS SELF-CONTAINED RESERVOIRS 529 Fig. 3. DNA microfluidic chip. TABLE I PROTOCOL REQUIREMENT FOR DNA EXTRACTION Fig. 2. Schematic view of an integrated bio-microfluidic package for DNA LOC application. Cho et al. has explained a fully integrated, pathogen specific DNA extraction device utilizing centrifugal microfluidics on a polymer based compact disc (CD) platform [11]. In this study DNA extraction is done on CD based microfluidic assay where a laser is used to shine the ferrowax microvalve together with cell lysis using irradiation on magnetic particles. In an another approach by Ahn et al., a microfluidic assay with a plastic chip is used for LOC application for biochemical detection of parameters such as blood gas concentration and glucose and lactate concentrations [12]. In this LOC system a disposable plastic chip incorporating smart passive microfluidics with embedded on chip power sources and integrated biosensor array for applications in clinical diagnostics and point of care testing is described. Packaging of microfluidic chip is an important factor that supports the chip function by dispensing and controlling fluidic flow in order to realize particular bio-protocol (Fig. 2). Fluidic control includes controlling of the fluids flow sequence, flow duration, flow direction, and flow rate. The package needs to have a mechanism to control each fluid/reagent to follow the protocol and, at the same time, to prevent reagents from cross mixing and contamination. The package also completes the connection between the microfluidic chip and other systems such as fluidic source, electrical source, optical sensor, etc. In the microfluidic packaging, polymers are generally used for encapsulation. Packaging material is based on the biocompatibility and to the reagents used for the extraction the key parameters of the package development [15]. II. MICROFLUIDIC PACKAGE The fluidic chip in this package is a DNA chip which has micromachined inlet and outlet holes. The chip consists of a filter, binder, and a polymer chain reaction (PCR) chamber (Fig. 3). The DNA LOC is fabricated in silicon. The filter, binder, and PCR chamber are formed by bulk micromachining method. An 8-in silicon DNA LOC wafer is bonded to a glass wafer by anodic bonding method. The bonding process is conducted at the wafer level by using a wafer bonder. The main parameter for anodic bonding is the temperature and force. The temperature of about 350 C is used for bonding with a force of 3.5 kn. The bonding strength and quality are verified by the fluidic testing. A syringe pump with maximum pressure of 700 kpa is used to test the chip for any fluidic leakage after bonding. The required pressure for the chip to withstand current application is only about 30 kpa based on the load analysis done using an Instron machine. Based on the test the bond interface can withstand up to 100 kpa pressure which is much higher than the required pressure for the current application. The glass wafer is used for analyzing the sample by optical detection method. The filter is used to separate the blood cell which contains DNA from the blood sample and a binder is used to bind the DNA onto the chip. The bound DNA particles are removed from the chip by a process called elution. The collected elution sample containing DNA particles is mixed with the primer and injected into the PCR chamber of the chip for amplification and further analysis. Extraction of DNA starts with mixing of lysis buffer with bio-fluid, for example blood. During lysis the cell membrane breaks and the DNA release out from the cell. When the lysed blood flows through the microchannels on silicon chip, the DNA will bind on the silicon surface. The subsequent reagents pushed to the chip helps to enhance binding of DNA and also remove the unwanted bio-particles. Finally the DNA is detached from chip and is collected in tube. The typical protocol is shown in Table I. The main role of the package here is to store the reagents in individual reservoirs before testing and to dispense them into the chip according to the protocol requirements during testing. Fig. 4 shows a microfluidic package designed for the DNA/RNA extraction application. The reagents used for DNA extraction is stored in four reservoirs. The reagents are dispensed into the cartridge one by one using an external actuator. Reagents are prestored in the package (self contained). Self contained package/cartridge makes fluidic circuit compact and eliminates the external storage bottles and tubing connections. Therefore it eliminates the possible contamination when connecting the package with external bottles. Storing of required

4 530 IEEE TRANSACTIONS ON ADVANCED PACKAGING, VOL. 32, NO. 2, MAY 2009 Fig. 4. Microfluidic package integrated with reservoir and valve (cross-section view). Fig. 6. External actuator acting on reservoir. Fig. 7. No dead volume after completing the actuation. Fig. 5. Top view of the microfluid package ( mm). volume of reagents inside the cartridge minimizes possible contamination by sharing reagents between samples. The package will be disposed after DNA extraction. The package is made with three layers of polydimethylsiloxane (PDMS) and is bonded with biocompatible double sided adhesive tape. The reservoirs are formed on the top layer of PDMS and the fluidic channels are formed in the bottom PDMS layer. Top view of the package is shown in Fig. 5. Different reagents are connected serially and it starts from injecting the bio sample, blood, and subsequently dispensing reagents, from reagent 1 to reagent 4. Reagents 1 3 are used to remove the unwanted particles from the chip keeping the bound DNA onto the chip. The last reagent helps to unbind the DNA from the chip. Each reagent flow with different flow rates to enhance the binding and unbinding process of the DNA onto the chip. In the package design the main points to be addressed are 1) the package should be disposable and biocompatible, 2) during actuation, the actuator should not get contact with reagents, 3) an integrated valve to start/stop liquid dispensing, 4) no cross mixing before reservoir dispensing liquid, 5) no back flow to reservoir after dispensing, and 6) flow rate controllable during dispensing. III. RESERVOIR DESIGN The reservoir has a conical shape cavity covered with a thin layer of flexible film as shown in Fig. 6. The actuator diameter is the same as the bottom diameter of reservoir. The volume of dispensed liquid is equal to the volume of film deformation as shown in the dash line area in Fig. 6. Consider the volume of the liquid inside the reservoir is, top radius of the reservoir is, height of the reservoir is and radius of the actuator is, then the relation between flow rate and actuator speed can be written as follows: Therefore the liquid flow rate is proportional to the actuator speed. The advantage of conical shape reservoir is to have a minimum dead volume and easy membrane deformation during the actuation (Fig. 7) compared with other shape reservoirs (Fig. 8). A differential flow of fluid can be met using an external actuation method on a conical shape reservoir. During the actuation of the fluid the speed of the actuation can be changed and hence a different flow rate can also be achieved Fig. 9(a) and (b). A. Pin Valve Design Pin valve is the main control component of the microfluidic package which controls the flow into the DNA chip. It looks like a thumbtack with a round cap. The main body is a hollow needle with a side hole and a slant tip. Liquid flows into the hollow needle from side hole to the tip. The slant tip is to pierce through the bottom of reservoir during initial stage of the actuation of the reservoir (Fig. 10). As shown in Fig. 11, the reservoir bottom has a blind via to locate pin valve. The blind via is facing the port of fluidic

5 XIE et al.: DEVELOPMENT OF A DISPOSABLE BIO-MICROFLUIDIC PACKAGE WITH REAGENTS SELF-CONTAINED RESERVOIRS 531 Fig. 10. Pin valve with side hole and a slant tip. Fig. 8. Cylindrical shape reservoir during actuation. (a) Cylindrical reservoir with small piston! large dead volume. (b) Cylindrical reservoir with big piston! large stress on membrane. Fig. 11. Pin valve close. Fig. 12. Pin valve open. Fig. 9. (a) Differential volume with respect to time. (b) Differential speed with respect to time. channel. The blind via is not connected to fluidic channel during reagent storage period. The blind via opens when the pin valve moves down and pocks through the reservoir bottom. The pin is tight fitted into the bottom of reservoir so that the pin tip is the only outlet of reservoir. The length of the pin is calculated based on distance traveled for pin to open the valve, close the valve and dispense required amount of reagent. B. Fluid Flow Control Mechanism During the reagent storage period, the pin valve is located at the reservoir bottom and hold firmly by the blind via hole in the reservoir. The reagent is filled in the reservoir and is sealed by a thin rubber membrane. At this storage period, the valve is closed and the fluid is not in the channel layer Fig. 11. The next step is the actuator goes down to the reservoir and push down the reservoir membrane and at the same time pushing the pin down. The pin starts move down and pock through the reservoir bottom and blind via opens to the port. The fluid starts to flow through the pin valve to channel port then to the chip (Fig. 12). As shown in Fig. 13, the fluid continuously flows through the channel. A constant flow rate is achieved by keeping a constant actuation speed. The actuator keeps pushing the pin until pin tip is inserted into the bottom PDMS layer, and at this point, the

6 532 IEEE TRANSACTIONS ON ADVANCED PACKAGING, VOL. 32, NO. 2, MAY 2009 Fig. 13. Pin valve closed by three levels of sealing. Fig. 15. Cartridge with multiple reservoirs. Fig. 14. Fluid flow in round about channel and no back flow. valve is closed. The pin valve bottom was blocked. The pin cap reaches the bottom point of the reservoir. The pin side hole is sealed in the bottom of reservoir. The three levels of sealing are shown in circles in Fig. 13. after the valve close completely, there is no path that the fluid can reenter into the reservoir from channel. When subsequent reagents are dispensed into the channel, this valve closure prevent cross mixing of reagents between the reservoirs. A round about channel design is implemented on the fluid channel to ease the fluid flow when the pin valve is closed at the end of actuation Fig. 14. The valve and round about port design can be used in multiple reservoir structure (Fig. 15). It controls fluidic dispensing sequence of multiple reservoirs where various types of reagents are needed. IV. FABRICATION The fabrication of the cartridge requires bonding of different layers of PDMS substrates. A mold in acrylic material is fabricated based on the reservoir and fluidic channel design. Fig. 16. Substrates (fabricated in PDMS) and tapes (size in millimeters). (a) Membrane. (b) Tape for membrane-reservoir bonding. (c) Reservoir. (d) Tape for reservoir-port layer bonding. (e) Tape for chip-port layer bonding. (f) Vertical port substrate. (g) Tape for port substrate-channel substrate bonding. (h) fluidic channel substrate. The PDMS is mixed in the ratio of 10:1 and is poured into the acrylic mold. The filled PDMS on the mold is cured in an inert oven. The cured PDMS is removed from the mold and is ready for assembly. The micro fluidic cartridge is comprised of three substrates: reservoir substrate, the vertical port substrate and channel layer substrate (Fig. 16).

7 XIE et al.: DEVELOPMENT OF A DISPOSABLE BIO-MICROFLUIDIC PACKAGE WITH REAGENTS SELF-CONTAINED RESERVOIRS 533 Fig. 19. Microfluidic package filled with liquid (dimension in millimeters). TABLE II FLOW RATE WITH RESPECT TO ACTUATOR SPEED Fig. 17. Process flow chart for microfluidic package. reservoir. Finally the reservoir is covered by a thin highly elastic membrane and is bonded using a double sided adhesive tape. The package is ready for microfluidic testing as Fig. 19. Fig. 18. Assemble DNA chip on PDMS substrate. The assembly process starts with cleaning of the substrate by oxygen plasma treatment process (Fig. 17) [17]. Since PDMS is a hydrophobic substrate, it is difficult to bond PDMS to another substrate. By oxygen plasma treatment process the PDMS substrate becomes hydrophilic nature and the bonding can be achieved either by direct bonding or by using an intermediate layer. The tape used for substrate bonding should be bio-compatible and PCR compatible. The adhesive tape used in this study has no reaction with fluids being used [15]. The bonding strength of the PDMS substrates has been quantified by pull test and fluidic injection test. The purified water with pressure 100 kpa was injected into fluidic channel and confirmed there was no leak. The chip is attached to the PDMS substrate using a pick and place machine (Fig. 18). The inlet and outlet holes are aligned with the PDMS substrate holes using a vision system with tolerance of alignment 50 m. A double sided adhesive tape is placed between the chip and the substrate and a force is applied on to the chip. Alignment of channel with the chip inlet and outlet is critical to avoid any leakage during the fluid test. The pin valves are inserted into the reservoirs. The volume of the reagents are measured using a pipette and is filled in the V. MICROFLUIDIC TESTING An actuator which can control the speed and meet the required load is used to push the reservoirs. According to the linear function between actuator speed and flow rate, the speed of each reservoir calculated is shown in Table II. The actual flow rate of each reservoir dispensing is also measured. VI. BIOLOGICAL TESTING ON BIO-SAMPLE Bio-sample and lysis buffer is mixed by certain ratio in a syringe. During the mixing process, cell membrane breaks and DNA is released. The mixture is then injected into developed package from sample port (Fig. 5). When the lysed bio-sample flows through microfluidic chip, the DNA attaches on the microchannel (Si surface). The rest flows out of chip as waste. A computer controlled actuator presses reservoirs as the sequence and speed as protocol (Table I). The last reagent is low salt solution, which detach DNA from Si surface and flush it out. The process is called elution. The product of elution is collected into five PCR tubes, which are marked as elution 0 4. All the elutions in this extraction showed certain amount of DNA being eluted and all were of sufficient quantity and quality to be amplified by PCR. The gel fluorescence graph shows elution 0 4 are all containing DNA (Fig. 20). VII. CONCLUSION A disposable micro fluidic package is developed for DNA/RNA extraction application. A self contained cartridge storing the reagents within the cartridge is developed.

8 534 IEEE TRANSACTIONS ON ADVANCED PACKAGING, VOL. 32, NO. 2, MAY 2009 Fig. 20. All the elutions (0 4) were positively amplified in the experiment (bp: base pair, unit to describe the length of a DNA/RNA molecule). Reservoirs with integrated valve are used in the self-contained cartridge to control the fluid within the cartridge. By using an external actuation method a relationship is established with fluid flow rate and actuation speed and is found that fluid flow rate is proportional to actuation speed. A conical shape reservoir is selected and found to have minimum dead volume during external actuation. A pin valve with a side hole and a slant tip is designed to control the fluid flow when dispensing from reservoir. A close-open-close valve method is used in the developed cartridge and is proved to be effective in prevent reagents from cross contamination and back flow. A round about on the fluidic channel under the port location is eased the over all fluid flow in the package. Actuation speed is calibrated and the flow rates required as per the protocol are achieved. ACKNOWLEDGMENT The authors would like to thank Dr. J. Lau for his support in publishing this paper and the graduate student E. Hui Ling (Nanyang Technological University, Singpapore) in helping the fabrication and testing micro-fluidic package. The authors would also like to thank U. Raghavan and his team from SiMEMS Bio Pte Ltd. and Dr. C. K. Heng from Department of Pediatrics and Biological Science, National University of Singapore, for the test results. REFERENCES [1] P. S. Dittrich and A. Manz, Lab-on-a-chip: Microfluidics in drug discovery, Natrure Rev. Drug Discovery, vol. 5, pp , Mar [2] C. D. Chin, V. Linder, and S. K. Sia, Lab-on-a-chip devices for global health: Past studies and future opportunities, Lab on a Chip, vol. 7, pp , [3] T. Vilkner, D. Janasek, and A. Manz, Micro total analysis system: Recent developments, Anal. Chem., vol. 74, pp , [4] T. Thorsen, S. Maerkli, and S. R. 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Iyer, Disposable polydimethylsiloxane package for microfluidic system, in Proc. 55th Electron. Compon. Technol. Conf., Jun. 2005, pp [16] L. Xie, S. C. Chong, C. S. Premachandran, M. Chew, and U. Raghavan, Development of an integrated microfluidic package with micro valves and reservoirs for a DNA lab on a chip application, in Proc. 56th Electron. Compon.Technol. Conf., May 2006, pp [17] L. Xie, S. C. Chong, C. S. Premachandran, D. Pinjala, and M. K. Iyer, Disposable bio-microfluidic pakcage with passive fluidic control, in Proc. 7th Electron. Packag. Technol. Conf., Singapore, Dec. 2005, pp [18] T. Pan, S. J. McDonald, E. M. Kai, and B. Ziaie, A magnetically driven PDMS micropump with ball check-valves, J. Micromechan. Microeng., vol. 15, pp , [19] L. Yobas et al., A flow through shear type microfilter chip for separating plasma and virus particles from whole blood, in Proc. 8th Int. Conf. Miniaturized Syst. Chemistry Life Sci., Sweden, Sep. 2004, vol. 2, pp [20] L. Yobas et al., Microfluidic chips for viral RNA extraction and detection, IEEE Sensors, pp , [21] L. Xie, C. S. Premachandran, S. C. Chong, and M. Chew, Design, integration and testing of fluidic dispensing control valve into a DNA/RNA sample preparation microfluidic package for Lab On a Chip (LOC) applications, in Proc. 57th Electron. Compon. Technol. Conf., Jun. 2007, pp Ling Xie received the M.Eng. degree in mechanical engineering from National Unviersity of Singapore, in Currently she is working in Microsystems, Modules and Components Laboratory of the Institute of Microelectronics, Singapore. Her research interest is in design and fabrication of microfluidic assays, design and simulation of thermal solutions for optical and IC packages. She has published more than 20 technical papers and holds two U.S. patents.

9 XIE et al.: DEVELOPMENT OF A DISPOSABLE BIO-MICROFLUIDIC PACKAGE WITH REAGENTS SELF-CONTAINED RESERVOIRS 535 C. S. Premachandran (SM 02) received the M. Tech degree in solid state technology from Indian Institute of Technology, Madras, India. He is currently working as a Member of Technical Staff in Institute of Microelectronics, Singapore. His research focuses are on the MEMS, bio and advanced packaging technologies. He has published more than 40 technical papers and holds six U.S. patents. Ser Choong Chong received the M. Eng. in material science from Nanyang Technological University, Singapore, in His main interest is in MEMS package that includes inertial devices such as accelerometer, microphone, and micro-relay; and bio-devices such as polymerase chain reaction (PCR) and sample preparation for DNA detection. Michelle Chew received the M.S. degree in materials science from National University of Singapore, in She joined in Microsystems, Modules and Components Laboratory of the Institute of Microelectronics, Singapore, in She has been involved in biopackage process development and characterization of bio-compatible material.