Copyright © 2009 Year IEEE. Reprinted from IEEE ... · The lab-on-a-chip (LOC) concept is to realize the functions of bio-laboratory in a silicon chip (Fig. 1). The miniaturized

Copyright © 2009 Year IEEE. Reprinted from IEEE TRANSACTIONS ON ADVANCEDPACKAGING. 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 [email protected].

528 IEEE TRANSACTIONS ON ADVANCED PACKAGING, VOL. 32, NO. 2, MAY 2009

Development of a Disposable Bio-MicrofluidicPackage With Reagents Self-Contained

Reservoirs and Micro-Valves for a DNALab-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 packagehas been developed and tested for on-chip DNA extraction fromhuman blood for practical lab-on-a-chip platform. The microflu-idic package has been customized to allow easy interface betweenthe microscale sample injection to the Si-based microscale samplepreparation chip. For precise sample dispensing and to minimizedead volume and/or sample lost conical-shaped reservoirs havebeen employed. Reservoirs filled with reagents are sealed bya highly elastic thin rubber membrane. Automated actuationsystem has been designed and implemented for programmablesample/reagent dispensing using thin rubber membrane-plungermechanism. The packaged DNA chip has been tested using bloodsample and the testing protocol has been optimized to meet therequirements for DNA extraction.

Index Terms—Disposable package, lab-on-a-chip (LOC), mi-crofluidic dispense, microfluidic package, self-contained reagent.

I. INTRODUCTION

I NFLUENCE of microelectronics into life science hasintroduced many developments in bio-applications. One

of the main impacts on bio-systems is the miniaturizationthat has seen clearly on microelectronics industry. Micro- andnano-level of interaction in bio-systems has emerged new fieldof research and new application products. Microfluidics is oneof the fast emerging fields where fluid flows in microchan-nels and very little dead volumes are present which eliminatecontamination and mixing among the fluids. Convergence ofmicrofluidics and microelectronics result in a new kind ofdevices, microfluidic chips. They require channels, reservoirs,filters to process the fluid [1]–[10]. Microfluidic structures areformed on silicon/polymer substrate by micromachining ormicrostamping method. Microfluidic chip offers the ability towork with shorter reaction time, smaller fluid sample/reagentvolumes, and promises of parallel processes.

Manuscript received August 28, 2006; revised November 07, 2007, May 16,2008. First published May 15, 2009; current version published May 28, 2009.This work was recommended for publication by Associate Editor L. Nguyenupon evaluation of the reviewers comments.

The authors are with Institute of Microelectronics, A-star, Singapore SciencePark 2, Singapore 117685 (e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]).

Color versions of one or more of the figures in this paper are available onlineat http://ieeexplore.ieee.org.

Digital Object Identifier 10.1109/TADVP.2009.2015282

Fig. 1. Schematic view of an integrated bio-microfluidic package for DNALOC application.

Major applications of microfluidics are on the clinical di-agnostics, drug discovery, bio-terrorism monitoring, and ther-apeutics devices. Fluidic components required for clinical di-agnostics is different from the fluidic components required fordrug discovery. In most of the cases a common platform can beused to combine the basic modules in order to implement a mi-crofluidic device for diagnostic applications or drug discoveryfor pharmaceutical applications.

The lab-on-a-chip (LOC) concept is to realize the functionsof bio-laboratory in a silicon chip (Fig. 1). The miniaturizedbio-laboratories are fabricated by photolithographic processdeveloped in the microelectronics industry to form circuits,chambers, valves, and channels in quartz or silicon substrate[11]–[20]. Fluidic samples can be manipulated by placingvalves, pumps in the chip and fluid can be diluted, mixedwith other reagents or separated by other process on the samechip. In this paper, a microfluidic chip for DNA extraction andamplification is described. Silicon substrate is used to formmicrofluidic components in the chip.

Different techniques have been used for extraction ofDNA/RNA from the body fluid. Liu et al. have developed a mi-crofluidic 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. Amagnet is activated when the target cells labeled with antibodycoated super paramagnetic beads pass through a separationcolumn and in the presence of strong magnetic field the labeledcells are retained on the chip. In this method efficient bindinghappen. However, additional requirement such as a magnet isrequired to magnetize and demagnetize the beads.

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XIE et al.: DEVELOPMENT OF A DISPOSABLE BIO-MICROFLUIDIC PACKAGE WITH REAGENTS SELF-CONTAINED RESERVOIRS 529

Fig. 2. Schematic view of an integrated bio-microfluidic package for DNALOC application.

Cho et al. has explained a fully integrated, pathogen—spe-cific DNA extraction device utilizing centrifugal microfluidicson a polymer based compact disc (CD) platform [11]. In thisstudy DNA extraction is done on CD based microfluidic assaywhere a laser is used to shine the ferrowax microvalve togetherwith cell lysis using irradiation on magnetic particles.

In an another approach by Ahn et al., a microfluidic assaywith a plastic chip is used for LOC application for biochemicaldetection of parameters such as blood gas concentration and glu-cose and lactate concentrations [12]. In this LOC system a dis-posable plastic chip incorporating smart passive microfluidicswith embedded on chip power sources and integrated biosensorarray for applications in clinical diagnostics and point of caretesting is described.

Packaging of microfluidic chip is an important factor thatsupports the chip function by dispensing and controlling fluidicflow in order to realize particular bio-protocol (Fig. 2). Fluidiccontrol includes controlling of the fluids’ flow sequence, flowduration, flow direction, and flow rate. The package needs tohave a mechanism to control each fluid/reagent to follow theprotocol and, at the same time, to prevent reagents from crossmixing and contamination. The package also completes the con-nection between the microfluidic chip and other systems such asfluidic source, electrical source, optical sensor, etc. In the mi-crofluidic packaging, polymers are generally used for encapsu-lation. Packaging material is based on the biocompatibility andto the reagents used for the extraction the key parameters of thepackage development [15].

II. MICROFLUIDIC PACKAGE

The fluidic chip in this package is a DNA chip which has mi-cromachined 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, andPCR chamber are formed by bulk micromachining method. An8-in silicon DNA LOC wafer is bonded to a glass wafer by an-odic bonding method. The bonding process is conducted at thewafer level by using a wafer bonder. The main parameter foranodic bonding is the temperature and force. The temperatureof about 350 C is used for bonding with a force of 3.5 kN. Thebonding strength and quality are verified by the fluidic testing.

Fig. 3. DNA microfluidic chip.

TABLE IPROTOCOL REQUIREMENT FOR DNA EXTRACTION

A syringe pump with maximum pressure of 700 kPa is used totest the chip for any fluidic leakage after bonding. The requiredpressure for the chip to withstand current application is onlyabout 30 kPa based on the load analysis done using an Instronmachine. Based on the test the bond interface can withstand upto 100 kPa pressure which is much higher than the required pres-sure for the current application.

The glass wafer is used for analyzing the sample by opticaldetection method. The filter is used to separate the blood cellwhich contains DNA from the blood sample and a binder isused to bind the DNA onto the chip. The bound DNA particlesare removed from the chip by a process called elution. The col-lected elution sample containing DNA particles is mixed withthe primer and injected into the PCR chamber of the chip foramplification and further analysis.

Extraction of DNA starts with mixing of lysis buffer withbio-fluid, for example blood. During lysis the cell membranebreaks and the DNA release out from the cell. When the lysedblood flows through the microchannels on silicon chip, the DNAwill bind on the silicon surface. The subsequent reagents pushedto the chip helps to enhance binding of DNA and also removethe unwanted bio-particles. Finally the DNA is detached fromchip and is collected in tube. The typical protocol is shown inTable I.

The main role of the package here is to store the reagents inindividual reservoirs before testing and to dispense them intothe chip according to the protocol requirements during testing.Fig. 4 shows a microfluidic package designed for the DNA/RNAextraction application. The reagents used for DNA extraction isstored in four reservoirs. The reagents are dispensed into thecartridge one by one using an external actuator.

Reagents are prestored in the package (self contained). Selfcontained package/cartridge makes fluidic circuit compact andeliminates the external storage bottles and tubing connections.Therefore it eliminates the possible contamination when con-necting the package with external bottles. Storing of required



Fig. 4. Microfluidic package integrated with reservoir and valve (cross-sectionview).

Fig. 5. Top view of the microfluid package (70 � 50 mm).

volume of reagents inside the cartridge minimizes possible con-tamination by sharing reagents between samples. The packagewill be disposed after DNA extraction.

The package is made with three layers of polydimethyl-siloxane (PDMS) and is bonded with biocompatible doublesided adhesive tape. The reservoirs are formed on the top layerof PDMS and the fluidic channels are formed in the bottomPDMS layer.

Top view of the package is shown in Fig. 5. Different reagentsare connected serially and it starts from injecting the bio sample,blood, and subsequently dispensing reagents, from reagent 1to reagent 4. Reagents 1–3 are used to remove the unwantedparticles from the chip keeping the bound DNA onto the chip.The last reagent helps to unbind the DNA from the chip. Eachreagent flow with different flow rates to enhance the binding andunbinding 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) duringactuation, the actuator should not get contact with reagents, 3)an integrated valve to start/stop liquid dispensing, 4) no crossmixing before reservoir dispensing liquid, 5) no back flow toreservoir after dispensing, and 6) flow rate controllable duringdispensing.

III. RESERVOIR DESIGN

The reservoir has a conical shape cavity covered with a thinlayer of flexible film as shown in Fig. 6. The actuator diameteris the same as the bottom diameter of reservoir. The volume ofdispensed liquid is equal to the volume of film deformation as

Fig. 6. External actuator acting on reservoir.

Fig. 7. No dead volume after completing the actuation.

shown in the dash line area in Fig. 6. Consider the volume ofthe liquid inside the reservoir is , top radius of the reservoiris , height of the reservoir is “ ” and radius of the actuator is“ ,” then the relation between flow rate and actuator speed canbe written as follows:

Therefore the liquid flow rate is proportional to the actuatorspeed. The advantage of conical shape reservoir is to have a min-imum dead volume and easy membrane deformation during theactuation (Fig. 7) compared with other shape reservoirs (Fig. 8).

A differential flow of fluid can be met using an external actu-ation method on a conical shape reservoir. During the actuationof the fluid the speed of the actuation can be changed and hencea 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 microfluidicpackage which controls the flow into the DNA chip. It looks likea thumbtack with a round cap. The main body is a hollow needlewith a side hole and a slant tip. Liquid flows into the hollowneedle from side hole to the tip. The slant tip is to pierce throughthe bottom of reservoir during initial stage of the actuation of thereservoir (Fig. 10).

As shown in Fig. 11, the reservoir bottom has a blind viato locate pin valve. The blind via is facing the port of fluidic



Fig. 8. Cylindrical shape reservoir during actuation. (a) Cylindrical reservoirwith small piston� large dead volume. (b) Cylindrical reservoir with big piston� large stress on membrane.

Fig. 9. (a) Differential volume with respect to time. (b) Differential speed withrespect to time.

channel. The blind via is not connected to fluidic channel duringreagent storage period. The blind via opens when the pin valvemoves down and pocks through the reservoir bottom.

The pin is tight fitted into the bottom of reservoir so that thepin tip is the only outlet of reservoir. The length of the pin iscalculated based on distance traveled for pin to open the valve,close the valve and dispense required amount of reagent.

Fig. 10. Pin valve with side hole and a slant tip.

Fig. 11. Pin valve close.

Fig. 12. Pin valve open.

B. Fluid Flow Control Mechanism

During the reagent storage period, the pin valve is located atthe reservoir bottom and hold firmly by the blind via hole in thereservoir. The reagent is filled in the reservoir and is sealed by athin rubber membrane. At this storage period, the valve is closedand the fluid is not in the channel layer Fig. 11.

The next step is the actuator goes down to the reservoir andpush down the reservoir membrane and at the same time pushingthe pin down. The pin starts move down and pock through thereservoir bottom and blind via opens to the port. The fluid startsto flow through the pin valve to channel port then to the chip(Fig. 12).

As shown in Fig. 13, the fluid continuously flows through thechannel. A constant flow rate is achieved by keeping a constantactuation speed. The actuator keeps pushing the pin until pin tipis inserted into the bottom PDMS layer, and at this point, the



Fig. 13. Pin valve closed by three levels of sealing.

Fig. 14. Fluid flow in round about channel and no back flow.

valve is closed. The pin valve bottom was blocked. The pin capreaches the bottom point of the reservoir. The pin side hole issealed 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 fluidcan reenter into the reservoir from channel. When subsequentreagents are dispensed into the channel, this valve closure pre-vent cross mixing of reagents between the reservoirs. A roundabout channel design is implemented on the fluid channel to easethe fluid flow when the pin valve is closed at the end of actuationFig. 14.

The valve and round about port design can be used in mul-tiple reservoir structure (Fig. 15). It controls fluidic dispensingsequence of multiple reservoirs where various types of reagentsare needed.

IV. FABRICATION

The fabrication of the cartridge requires bonding of differentlayers of PDMS substrates. A mold in acrylic material isfabricated based on the reservoir and fluidic channel design.

Fig. 15. Cartridge with multiple reservoirs.

Fig. 16. Substrates (fabricated in PDMS) and tapes (size in millimeters). (a)Membrane. (b) Tape for membrane-reservoir bonding. (c) Reservoir. (d) Tapefor reservoir-port layer bonding. (e) Tape for chip-port layer bonding. (f) Ver-tical 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 theacrylic mold. The filled PDMS on the mold is cured in an inertoven. The cured PDMS is removed from the mold and is readyfor assembly. The micro fluidic cartridge is comprised of threesubstrates: reservoir substrate, the vertical port substrate andchannel layer substrate (Fig. 16).



Fig. 17. Process flow chart for microfluidic package.

Fig. 18. Assemble DNA chip on PDMS substrate.

The assembly process starts with cleaning of the substrate byoxygen plasma treatment process (Fig. 17) [17]. Since PDMSis a hydrophobic substrate, it is difficult to bond PDMS to an-other substrate. By oxygen plasma treatment process the PDMSsubstrate becomes hydrophilic nature and the bonding can beachieved either by direct bonding or by using an intermediatelayer.

The tape used for substrate bonding should be bio-compatibleand PCR compatible. The adhesive tape used in this study hasno reaction with fluids being used [15]. The bonding strengthof the PDMS substrates has been quantified by pull test andfluidic injection test. The purified water with pressure 100 kPawas injected into fluidic channel and confirmed there was noleak.

The chip is attached to the PDMS substrate using a pick andplace machine (Fig. 18). The inlet and outlet holes are alignedwith the PDMS substrate holes using a vision system with tol-erance of alignment 50 m. A double sided adhesive tapeis placed between the chip and the substrate and a force is ap-plied on to the chip. Alignment of channel with the chip inletand outlet is critical to avoid any leakage during the fluid test.

The pin valves are inserted into the reservoirs. The volumeof the reagents are measured using a pipette and is filled in the

Fig. 19. Microfluidic package filled with liquid (dimension in millimeters).

TABLE IIFLOW RATE WITH RESPECT TO ACTUATOR SPEED

reservoir. Finally the reservoir is covered by a thin highly elasticmembrane and is bonded using a double sided adhesive tape.The package is ready for microfluidic testing as Fig. 19.

V. MICROFLUIDIC TESTING

An actuator which can control the speed and meet the re-quired load is used to push the reservoirs. According to the linearfunction between actuator speed and flow rate, the speed of eachreservoir calculated is shown in Table II. The actual flow rate ofeach reservoir dispensing is also measured.

VI. BIOLOGICAL TESTING ON BIO-SAMPLE

Bio-sample and lysis buffer is mixed by certain ratio in a sy-ringe. During the mixing process, cell membrane breaks andDNA is released. The mixture is then injected into developedpackage from sample port (Fig. 5). When the lysed bio-sampleflows through microfluidic chip, the DNA attaches on the mi-crochannel (Si surface). The rest flows out of chip as waste. Acomputer controlled actuator presses reservoirs as the sequenceand speed as protocol (Table I). The last reagent is low salt so-lution, which detach DNA from Si surface and flush it out. Theprocess is called elution. The product of elution is collected intofive PCR tubes, which are marked as elution 0–4.

All the elutions in this extraction showed certain amount ofDNA being eluted and all were of sufficient quantity and qualityto be amplified by PCR. The gel fluorescence graph shows elu-tion 0–4 are all containing DNA (Fig. 20).

VII. CONCLUSION

A disposable micro fluidic package is developed forDNA/RNA extraction application. A self contained car-tridge storing the reagents within the cartridge is developed.



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-containedcartridge to control the fluid within the cartridge.

By using an external actuation method a relationship is estab-lished with fluid flow rate and actuation speed and is found thatfluid flow rate is proportional to actuation speed.

A conical shape reservoir is selected and found to have min-imum dead volume during external actuation. A pin valve with aside hole and a slant tip is designed to control the fluid flow whendispensing from reservoir. A close-open-close valve method isused in the developed cartridge and is proved to be effectivein prevent reagents from cross contamination and back flow.A round about on the fluidic channel under the port locationis eased the over all fluid flow in the package. Actuation speedis calibrated and the flow rates required as per the protocol areachieved.

ACKNOWLEDGMENT

The authors would like to thank Dr. J. Lau for his supportin publishing this paper and the graduate student E. Hui Ling(Nanyang Technological University, Singpapore) in helpingthe fabrication and testing micro-fluidic package. The authorswould also like to thank U. Raghavan and his team fromSiMEMS Bio Pte Ltd. and Dr. C. K. Heng from Departmentof Pediatrics and Biological Science, National University ofSingapore, for the test results.

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Ling Xie received the M.Eng. degree in mechanicalengineering from National Unviersity of Singapore,in 2002.

Currently she is working in Microsystems, Mod-ules and Components Laboratory of the Institute ofMicroelectronics, Singapore. Her research interest isin design and fabrication of microfluidic assays, de-sign and simulation of thermal solutions for opticaland IC packages. She has published more than 20technical papers and holds two U.S. patents.



C. S. Premachandran (SM’02) received the M. Techdegree in solid state technology from Indian Instituteof Technology, Madras, India.

He is currently working as a Member of TechnicalStaff in Institute of Microelectronics, Singapore. Hisresearch focuses are on the MEMS, bio and advancedpackaging technologies. He has published more than40 technical papers and holds six U.S. patents.

Michelle Chew received the M.S. degree in mate-rials science from National University of Singapore,in 2008.

She joined in Microsystems, Modules and Compo-nents Laboratory of the Institute of Microelectronics,Singapore, in 2005. She has been involved in bio-package process development and characterization ofbio-compatible material.

Ser Choong Chong received the M. Eng. in mate-rial science from Nanyang Technological University,Singapore, in 2008.

His main interest is in MEMS package thatincludes inertial devices such as accelerometer,microphone, and micro-relay; and bio-devices suchas polymerase chain reaction (PCR) and samplepreparation for DNA detection.


Copyright © 2009 Year IEEE. Reprinted from IEEE ... · The lab-on-a-chip (LOC) concept is to realize the functions of bio-laboratory in a silicon chip (Fig. 1). The miniaturized

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