A Polymer Microfluidic Chip With Interdigitated Electrodes Arrays for ...

IEEE SENSORS JOURNAL, VOL. 8, NO. 5, MAY 2008

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A Polymer Microfluidic Chip With Interdigitated Electrodes Arrays for Simultaneous Dielectrophoretic Manipulation and Impedimetric Detection of Microparticles Zhiwei Zou, Student Member, IEEE, Soohyun Lee, and Chong H. Ahn, Member, IEEE

Abstract—This paper presents the design, fabrication, and characterization of a polymer microfluidic biochip with integrated interdigitated electrodes arrays (IDAs) used to simultaneously separate, manipulate, and detect microparticles using dielectrophoresis (DEP) and electrochemical impedance spectroscopy (EIS) methods. The DEP response of silica microspheres has been characterized, and microspheres of different sizes (1.8 and 3.5 m in diameter) have been DEP flow separated and individually trapped in different microchambers by IDAs in a single run. Simultaneously, the impedance change caused by microspheres captured on IDAs has been analyzed for quantification. High-throughput polymer microfabrication techniques such as micro injection molding were used in this work, so that the polymer microfluidic chip can be produced in a low-cost, disposable platform. This low-cost microfluidic chip provides a generic platform for developing multifunctional lab-on-a-chip devices that require the ability to handle and sense microparticles. Index Terms—Dielectrophoresis (DEP), electrochemical impedance spectroscopy (EIS), interdigitated electrodes arrays (IDAs), microparticles.

I. INTRODUCTION N life science research and clinical diagnostics, lab-on-achip devices and micro total analysis systems (microTASs) have become particularly interesting for their reduced sample consumption, shorter assay time, mass production, and ease of use [1]–[4]. Among the various lab-on-a-chip applications, one significant area is the on-chip controlled handling and monitoring of biological microparticles and nanoparticles (e.g., cells, bacteria, molecules) and synthetic microbeads and nanobeads (e.g., latex beads, silica spheres).

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Manuscript received June 1, 2007; revised July 16, 2007; accepted July 17, 2007. This work was supported in part by the National Science Foundation (NSF) under Grant NSF-0622036. The associate editor coordinating the review of this paper and approving it for publication was Dr. Richard Fair. Z. Zou and S. Lee are with the MicroSystems and BioMEMS Laboratory, Department of Electrical and Computer Engineering, University of Cincinnati, Cincinnati, OH 45221-0030 USA (e-mail: [email protected]; [email protected]). C. H. Ahn is with the MicroSystems and BioMEMS Laboratory, Department of Electrical and Computer, and also with the Department of Biomedical Engineering, University of Cincinnati, Cincinnati, OH 45221-0030 USA (e-mail: [email protected]). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/JSEN.2008.918907

Dielectrophoresis (DEP), which arises from the interaction of a nonuniform AC electric field with the dipoles induced in the particles, has been used as one of the most efficient ways to handle neutral particles [5]–[8]. Among different electrode designs used for DEP, microfabricated interdigitated electrodes arrays (IDAs) are preferable because they can generate high electric field strength and desirable field gradients with low voltage and heat generation, and because it is easy to be arrayed and implemented. Various biological and nonbiological microparticles have been reported as the targets for the on-chip DEP separation and manipulation. Typically, DEP is most readily observed for particles with a diameter range of around 1–1000 m. Since cells [9]–[17], bacteria [18]–[20], microbeads [21]–[24], and nanowires [25] fall within this range of mean particle diameter, they can be very effectively manipulated in fluidics using DEP. By scaling down the IDAs to nanoscale, it is also possible to manipulate nanoparticles such as the biomolecule [26], [27] and nanoscopic latex beads [28], [29]. On the other hand, micro and nano IDAs are widely used in on-chip electrochemical detection of biological targets as well. Many researchers have reported that IDAs have an improved signal-to-noise ratio for reversible or quasi-reversible charge transfer response because of the redox cycling arising from the close proximity of the interdigitated electrodes [29]–[34]. IDAs are also very prominent as the impedimetric biosensor for the direct detection of impedance change including capacitance, dielectric constant, and bulk conductivity in fluids. Electrochemical impedance spectroscopy (EIS) using micro and nano IDAs has demonstrated a remarkable potential for the analysis of various biochemical or biological events especially because it can do this in a label-free manner [35]–[42]. Pioneering works have reported using positive DEP forces to concentrate particles along the microelectrodes to assist impedimetric measurement of the particle concentration [11], [18], [19]. Apparently, IDAs have both the actuating and sensing capabilities for the microparticles in fluidics. Both DEP manipulation and EIS sensing take advantage of differences in conductivity and permittivity between the particles and the medium [12]. In this regard, it is of great interest to integrate IDAs on microfluidic chips to make devices capable of simultaneously performing sample preparation by various DEP manipulation (e.g., filtration, isolation, separation, and concentration) and sample characterization by EIS detection. This simple scheme can further increase the effectiveness

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of microparticles analysis by lab-on-a-chip devices, and is especially important for the future development of multianalyte bead-based immunoassay and on-chip cell total analysis systems. Batch microfabrication and polymer micromachining techniques [3] have been employed to produce a low-cost polymer microfluidic chip that performs DEP manipulation and EIS analysis in a single step. In this experiment, silica microspheres were used as the target particles because silica microspheres have been extensively utilized as probe beads in bead-based immunoassay and in flow cytometry applications. Furthermore, the silica microsphere is a good model to develop this methodology and extend the results from this work to different microbeads and biological particles such as cells and bacteria which have similar shapes and sizes. II. WORKING PRINCIPLE AND DESIGN A. Dielectrophoresis (DEP) DEP is a phenomenon in which a force is exerted on a dielectric particle when it is subjected to a nonuniform electric field. The force does not require the particle to be charged. All particles exhibit dielectrophoretic activity in the presence of electric fields. However, the strength of the force strongly depends on the medium and electrical properties of the particles, shape and size of the particles, as well as the frequency of the electric field. Consequently, fields of a particular frequency can manipulate particles with great selectivity [1], [5], [6]. The time-averaged DEP force is given by [1], [5], [6] (1) where is the radius of particles, is the permittivity of the is the Clausius–Mossoti factor, is the root medium, is the real part mean square value of the electric field. , which can be represented as follows [1], [5], [6]: of the (2) where is the complex permittivity ; is the conductivity, is the electric field frequency. Subscripts and represent particles and the surrounding medium, respectively. means that particles show positive DEP means negative DEP response. response, while The particles experiencing positive DEP forces are attracted to the maximum electric field regions while the particles experiencing negative DEP forces are repelled to minimum electric field regions. Using the difference between DEP forces exerted on different particles in nonuniform electric fields is known as DEP separation. Finite-element analysis (FEA) simulation was performed in this work to analyze the electric field distribution in the aqueous solution around the IDA using CFD-ACE+ software (CFD Research Corporation, AL, USA). As shown in Fig. 1(a), is generated the nonuniform electric field energy close to two pairs of IDA electrodes, and the maximum electric field regions (A) can be found at the electrode edges and the minimum electric field regions (B) are at the spacing between the electrode pairs. In the IDA generated electric field, particles

Fig. 1. Electrical behavior close to the IDA. (a) Simulation of the electric field energy ( E ) around the IDA. A is the maximum field regions and B is the minimum field regions. (b) Simplified electrical modeling and equivalent circuits for the IDA in the aqueous medium.

experiencing repulsive and weak attractive DEP forces (usually negative DEP at the spacing between electrode pairs) are eluted by fluid flow, whereas particles experiencing strong attractive DEP forces are trapped at electrode edges against flow drag. B. Electrochemical Impedance Spectroscopy (EIS) EIS is a rapidly developing electrochemical technique because it directly provides detailed information on bioaffinityevent induced capacitance and resistance changes at an electrode or substrate’s surface. This enables label-free detection of biomolecules, cells, and microparticles. EIS studies the electrodes system response to the application of a periodic small amplitude AC signal. These measurements are carried out at different frequencies. EIS that uses IDAs in an aqueous solution can be reduced to a simple electrical circuit for modeling electrical behaviors [35]. Fig. 1(b) shows that two parallel branches are in the equivalent circuit. Contributions to the impedance value from each partial circuit and the total impedance could be expressed by [41] (3) (4) (5) where is the excitation frequency, is the solution resisis the double layer capacitance, is the impedance tance, and , is the solution dielectric capacitance of the and is the impedance of , and is the total impedance and , as shown in Fig. 1(b) [41]. of the parallel becomes essentially resistive at the lowBy analyzing (4), frequency range, offering the major contribution to . When increases, the significance of in the total decreases and

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Fig. 3. Summary of fabrication processes of the polymer microfluidic chip with integrated IDAs.

removal through the outlet. Each kind of silica sphere is separated at the different working chamber and concentrated over the IDA, so the separated microspheres can be retained and quantified by measuring the corresponding impedance change through the IDA in real time. III. EXPERIMENTAL Fig. 2. Schematic illustrations. (a) The polymer biochip. (b) Flow separation and trapping of different sized microbeads at different IDAs using DEP. (c) Sequential impedance sensing of the captured microbeads at IDAs due to resistance (R ) change between electrodes.

becomes stable and mainly depends on which reflects the ionic strength of the solution. When the frequency is high instead of . Thereenough, the current passes through dominates the total impedance, and and can fore, be ignored in this case [35], [41]. These equations are consistent with the three regions observed in the impedance spectra in the Section IV-B. When the microparticles were trapped by the DEP at the IDA electrode, particles of complex permittivity replace an equal volume of the suspending medium having complex permittivity . As a result, the impedance between the electrodes will vary with changes in the average complex permittivity of the medium that separates them [43]. This phenomenon would be observed in the intermediate frequency range as discussed dominates, before. At the low-frequency range where the trapped microparticles act as insulating particles, and the impedance change at this range is dominated by the change in capacitance caused by the trapped particles. C. Device Design A prototype microfluidic device has been designed to perform flow separation of two kinds of silica microspheres with different diameters and simultaneous measurement of the impedance change after trapping the spheres at the IDAs. As described in Fig. 2, two IDAs are located and confined in two independent working microchambers which are in sequence and connected by microchannels. The microsphere mixture and buffer solution can be continuously applied through the different inlets to the working microchambers with wastes

A. Device Fabrication The fabrication process is illustrated in Fig. 3. The IDA chip was fabricated on the polymer substrate (cyclic olefin copolymer, COC) using standard photolithography and liftoff techniques. The 3-inch blank COC substrate with a polished surface was made by plastic injection molding using a highly thick S1818 (MicroChem polished nickel mold-disk. 2 Corporation, MA, USA) photoresist was first patterned on the COC substrate, and then a 100-nm-thick gold layer was deposited on the patterned substrate by an e-beam metal evaporator (Temescal FC1800, BOC Edwards Temescal, CA, USA). The chip was then placed in acetone for liftoff; resulting in gold IDA. Mass production and low-cost fabrication is one of the principal objectives of this work. A high-throughput polymer micromachining technique has been developed in our group for polymer microfluidic chip fabrication [3]. As shown in Fig. 3, the OmniCoat and SU-8 2075 photoresist (MicroChem Corporation, MA, USA) was first spin-coated on the 3-inch nickel disk to achieve 100 m thickness, followed by a prebake process. After the photoresist layer was exposed to a UV source, it was baked again for SU-8 cross-linking. After developing, electroplating was performed in a nickel electroplating bath, using a two-electrode system with a nickel anode and the patterned nickel disk cathode. Finally, a nickel mold with a 100- m-thick plating nickel microstructure was obtained after removal of the residual SU-8. The microfluidic chip was then replicated from this mold in a COC substrate by a high-throughput injection molding machine (BOY 22A, BOY Machines Inc., PA, USA). After drilling holes for fluidic interconnection at inlets and outlets using 800- -diammter micro drill bit, the microfluidic chip was bonded with the IDA chip using thermal fusion bonding method by a MTP-10 versatile bench-top precision press (Tetrahedron Associates Inc., CA, USA) to make the final device.

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Fig. 4. The photograph of the entire device and magnified working chambers with IDAs.


measurement. Sinusoidal voltage with the frequency up to 20 MHz and amplitude up to 5 Vpp (peak-to-peak) were applied to the IDA to excite the electric field inside the working chambers and EIS was conducted by measuring the impedance response. Impedance amplitude and phase angle were recorded for frequencies between 20 Hz and 1 MHz with continuously scanned spectra using the LCR meter. The images of the DEP behaviors at the working chamber were captured by means of a Nikon Optiphot microscope (Nikon, Inc., NY, USA) with a Javelin Chromachip II CCD camera (Javelin Electronics Company, CA, USA) and a PVR-Plus image capture system (Honest Technology, Inc., TX, USA) installed in a laptop computer. 1.8 and 3.5 m silica spheres (Bangs Laboratories, Inc., IN, USA) were diluted to 0.05% solids in DI water, and the solution conductivity was adjusted by adding KCl as needed (1–10 S cm ). IV. RESULT AND DISCUSSION A. Dielectrophoretic Separation and Manipulation of Microparticles

Fig. 5. Experiment setups for the simultaneous DEP separation, manipulation, and EIS analysis of microparticles using polymer microfluidic chip.

Fig. 4 shows photographs of the final device and the magnified view of the working chamber. The entire chip size is 1.5 cm 2 cm. Each IDA consists of 100 electrode fingers of 2 mm length, 5 m width, and 10 m spacing. All working chambers have length, width and depth of 3 mm, 1 mm, 100 m, respectively, for a volume of 0.3 L. The width and depth of the inlet and outlet microchannels are 250 and 100 m, respectively. The depth of the microchannel can be further reduced, forcing the particles to flow near the IDA electrodes and thus increase the separation efficiency. By considering the possible deformation of the microchamber during the thermal fusion bonding and the relative high density of the silica spheres, microchannels were designed with a 100 m depth. B. Experimental Setup Fig. 5 shows the experimental setup for characterizing the performance of the proposed device. Flows containing microparticles mixture and cleaning buffer were injected into microchannel by a dual syringes pump (pump 33, Harvard Apparatus, MA, USA) at controlled flow rates. The flow rate was adjusted to find the threshold flow rate at which the particles captured with DEP can remain captured against the fluid flow. Agilent 33220A function waveform generators and 4284A precision LCR meter (Agilent Technologies, Inc., CA, USA) were electrically connected to the IDAs through the contact pads, and were used for the DEP separation and impedance

A major strength of DEP is that the frequency-dependent dielectric properties of particles can be used to manipulate the particles. In our prototyping device, the DEP responses of two silica spheres with different diameters were characterized, and the crossover frequency is summarized in Table I. The 3.5 m spheres change from positive DEP to negative DEP at 5 kHz, whereas the crossover frequency for 1.8 m spheres is tenfold higher at 50 kHz. 1.8 m spheres encounter anomalous phenomenon at frequency lower than 1 kHz, which cannot be solely attributed to the DEP response, and the details will be discussed later. Another important factor for the DEP flow separation is the capturing threshold flow velocity which reflects the strength of the DEP force at a certain voltage and frequency. Here, the capturing threshold flow velocity is defined as the maximum flow velocity at which the moving silica spheres can be captured and retained at the electrode by the DEP force. The relationship between the threshold flow velocity and the applied voltage for the two silica spheres has been studied and the result is shown in Fig. 6. It is clearly found that the higher applied voltage provides higher DEP force to counteract the stronger imposed dragging force by the fluidic flow. It is also noticed that when the applied voltage becomes high (i.e., 5 V in this experiment), bubbles were generated due to the Joule heating at the electrode. Based on the analysis of Table I and Fig. 6, various manipulations can be achieved to sort, isolate, and trap microparticles by controlling the flow velocity, applied frequency, and voltage. In the first experiment, the 1.8 m silica spheres were separated from the 3.5 m silica spheres and trapped at the center of the electrodes. It was found at frequency range lower than 1 kHz, 1.8 m silica spheres were strongly trapped at the center of electrodes, and the corresponding threshold velocity was greatly increased compared with the threshold velocity at 20 kHz, as shown in Fig. 7(a). However, for the 3.5 m silica spheres, there was no such phenomenon observed in our experiment. Thus, an extremely efficient and rapid filtering and trapping of 1.8 m spheres from the mixture has been achieved at a high flow velocity (2000 m/s), as shown in Fig. 7(b). In this case,

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TABLE I DEP RESPONSE AT DIFFERENT FREQUENCY RANGE FOR TWO PARTICLE SIZES

Fig. 6. Plots of the capturing threshold flow velocity versus applied voltage change for 1.8 and 3.5 m spheres undergoing positive DEP.

Fig. 7. (a) Threshold flow velocity dramatically increases for 1.8 m spheres for a low-frequency range (