An assembly disposable degassing microfluidic device using a gas-permeable hydrophobic membrane and a reusable microsupport array

Sensors & Actuators: B. Chemical 286 (2019) 353–361

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

An assembly disposable degassing microfluidic device using a gas-permeable hydrophobic membrane and a reusable microsupport array Hyungseok Cho, Jinho Kim, Ki-Ho Han

T

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Department of Nanoscience and Engineering, Center for Nano Manufacturing, Inje University, Gimhae, 50834, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Degassing Disposable Microfluidic device Silicone-coated release film Hydrophobic membrane Microsupport array

We introduce an assembly disposable degassing (ADD)-method as a simple and fast active degassing method. The ADD-method is achieved with a microstructured disposable superstrate and a reusable microsupport substrate, which can be assembled and disassembled simply by vacuum pressure. The reusable substrate can be equipped with functionalities to create an elaborately patterned energy field for active manipulation of substances in the microchannel. Degassing performance was evaluated for varying thicknesses of the polydimethylsiloxane (PDMS) membrane and heights of the microsupport array. Based on the ADD-method, an active bubble-trap structure was developed to prevent bubble injection into the microchannels. Deterministic lateral displacement and microvortex devices, as complex structured microfluidic devices that are easily suffered by bubble formation, were used to demonstrate the usefulness of the ADD-method. As a test vehicle of active functional microfluidic devices, an assembly-disposable degassing lateral magnetophoretic microseparator (CTC-addChip) was developed. For the CTC-addChip, a ferromagnetic wire array was inlaid on the reusable substrate to create a high-gradient magnetic field, which can transfer through the PDMS membrane and isolate circulating tumor cells with magnetic nanobeads in the microchannel without bubble interference.

1. Introduction Due to the advantages of microfluidic devices, such as small size, low sample consumption and fast response with high surface-to-volume ratio, they constitute very useful tools in the fields of biology, chemistry, medicine and pharmacology [1,2]. Current microfluidic devices, however, should be further improved to enhance convenient and automation [3,4]. To effectively advance microfluidic devices, critical challenges must be addressed. The most common and troubling problem is bubble formation in microchannels, which is typically caused by bubble injection during instrument setup, stiction of bubbles in complex microstructures and gas vaporization [5]. Bubbles in microchannels cause poor performance and malfunctions of microfluidic devices. For example, bubbles in microfluidic cell culture devices [6–8] threaten cell viability and culture efficiency. In addition, bubbles in polymerase chain reaction (PCR) microchamber may promote irregular thermal distribution and PCR reagent expulsion [9,10]. In drug screening microfluidic devices, bubbles may contribute to non-uniform cell loading and cause drug loss [11]. If trained personnel do not handle microfluidic devices with care, bubbles form easily in the microchannels and are very difficult to remove. Therefore, bubble formation is one of the critical hindrances to full automation of microfluidic ⁎

devices, and bubble blocking and degassing functionalities are essential for improving these useful devices. Existing degassing systems are independent products and are ineffective for microfluidic devices because they require additional tubing, which is more likely to allow bubbles to enter the microchannels. To prevent bubble formation, the most common and traditional method is flushing microchannels with low-polarity aqueous solutions [12,13], such as isopropanol, ethanol and methanol, before operating microfluidic devices. In addition, on-chip integrated bubbletrap structures located at the entrance of the microchannel [6] can be used to effectively block bubble injection. To avoid stiction of bubbles to the microchannel, the surface can be made hydrophilic by oxygen plasma treatment [10,14]. Although the microchannel is pre-flushed with low-polarity aqueous solutions, bubbles may still attach to complex microstructures and corners, which have high surface-to-volume ratios. The pre-flushing process is also a cumbersome extra step in the preparation of the microfluidic device for operation. On-chip integrated bubble-trap structures usually have a limited trapping volume due to their small size, so that if the bubbles exceed the trapping volume, they eventually invade into the microchannels. In addition, the plasmatreated hydrophilicity is temporal and lost gradually within a few hours [15].

Corresponding author at: Department of Nanoscience and Engineering, Inje University, 197, Inje-Ro, Gimhae, Gyongnam, 50834, Republic of Korea. E-mail address: [email protected] (K.-H. Han).

https://doi.org/10.1016/j.snb.2019.01.118 Received 13 September 2018; Received in revised form 22 December 2018; Accepted 24 January 2019 Available online 25 January 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.

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through the PDMS membrane and actively manipulate substances in the microchannel of the disposable superstrate. Degassing performance of the ADD-method is evaluated with varying thicknesses of the PDMS membrane and heights of the microsupport array. Burst and leakage tests were performed to measure bond stability of the PDMS membrane. Based on the ADD-method, a bubble-trap structure is proposed to simultaneously block bubble injection and remove bubbles. Deterministic lateral displacement (DLD) and microvortex devices, as complexstructured microfluidic devices that is often seriously disturbed by bubble interference, are used to show the usefulness of the ADDmethod. As a demonstration of active functional microfluidic devices implemented by the ADD-method, an assembly disposable degassing lateral magnetophoretic microseparator (CTC-addChip) is developed. For the CTC-addChip, a micropatterned ferromagnetic wire array is inlaid on the reusable substrate to generate a high-gradient magnetic field, which can transmit through the PDMS membrane and precisely isolate circulating tumor cells (CTCs) with magnetic nanobeads in the microchannel of the disposable superstrate.

To overcome the limitations of passive methods for bubble trapping and prevention, active degassing methods were developed. The active degassing methods usually capture bubbles upon entering the microchannels and simultaneously extract the trapped bubbles through a gaspermeable membrane [16–21]. The most active degassing methods require an additional structure to connect vacuum pressure. In addition, because the material for the gas extraction is usually dissimilar to the main material, it is assembled using an adhesive, which may decrease the reproducibility of the devices. To solve these problems, active degassing functionalities have been developed using polydimethylsiloxane (PDMS) as it is material most commonly used to fabricate microfluidic devices, being gas-permeable, strongly hydrophobic, biocompatible and optically transparent [22–25]. The active degassing method based on the gas permeability of PDMS has been used in many applications, including PCR [9,26], cell culture [6,11,27], micro-pumping, valving, mixing [28,29] and DNA analysis [30]. However, to create a gas degassing path connected to low pressure, active degassing functionalities were usually generated by multilayered structures, which are difficult to fabricate due to their complex structure. For this reason, integrating the active degassing functionality on-chip with other functionalities is difficult, thereby resulting in the presence of impractical devices on the market. Although the active degassing function [6,26] can be implemented in an in-plane structure, these structures have low degassing rates due to the thick degassing barrier of several hundred micrometers and a restricted degassing area. In this study, we introduce a simple and fast active degassing method, named the assembly disposable degassing (ADD)-method. The ADD-method was developed using a disposable superstrate, including microchannel networks with a micrometer-thick PDMS membrane and a vacuum trench, and a reusable substrate, containing a microsupport array (Fig. 1(a)). The disposable superstrate and reusable substrate can be simply assembled and disassembled by applying a vacuum pressure. For more advanced applications, the reusable substrate can be equipped with functionalities to generate energy field, which can transmit

2. Materials and methods 2.1. Design and working principles For the ADD-method, the disposable superstrate is fabricated by bonding a micrometer-thick PDMS membrane and a microstructured PDMS replica, containing a microchannel structure and a vacuum trench. The vacuum trench surrounds the microchannel for device assembly and is used with the microsupport array to remove bubbles within the microchannel by applying uniform low pressure to the bottom of the microchannel. The PDMS membrane has two functions: formation of the microchannel structure and gas transmission. The reusable substrate includes a uniformly distributed microsupport array on the top surface, which supports the microchannel during vacuum assembly and serves as the passage for air degassing. To keep the Fig. 1. (a) Illustration of the assembly disposable degassing (ADD)-method, using the disposable superstrate and reusable substrate. The disposable superstrate includes a microchannel network, a vacuum trench, a vacuum hole and a micrometer-thick PDMS membrane. The reusable substrate has a microsupport array structured on its top surface. (b) Illustration of the tight assembly of the disposable superstrate and reusable substrate, achieved by applying a vacuum pressure. (c) Cross-sectional view of the microchannel (along the A-A' cross-section in Fig. 1(b)), in which trapped bubbles are gradually degassed through the PDMS membrane and the microsupport array and finally dismissed.

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Fig. 2. (a) Photograph of the fabricated disposable superstrate. (b)−(c) Enlarged cross-sectional views of the microchannel (along the B-B' cross-section in Fig. 2(a)), including 14-μm-thick PDMS membrane. (d) Photograph of the fabricated reusable superstrate with the microsupport array. (e)−(f) Scanning electron microscope (SEM) images of the microsupports (2.6 μm in height). (g) Photograph of an assembly disposable degassing microfluidic device, in which the disposable superstrate and the reusable substrate are tightly assembled by applying a vacuum pressure.

Dow Corning), was poured into the SU-8 mold and cured for 1 h at 75 °C in an oven (Fig. S1(c)). After peeling the PDMS replica off from the SU-8 mold, the inlet and outlet reservoirs and the vacuum hole of the PDMS replica were generated using a punch (Fig. S1(d)). PDMS membranes of 9 ± 1.3, 14 ± 1.2, 19 ± 1.0 and 21 ± 1.1 μm in average thickness were respectively fabricated by spincoating of the liquid PDMS (10:1 resin and curing agent) at 5500, 4000, 2500 and 1000 rpm for 30 s on the polypropylene (PP) film (100-μmthick) and curing for 1 h at 75 °C (Fig. S1(e) and S1(f)). Then, the PDMS membrane on the PP film is bonded with the PDMS replica after oxygen plasma treatment for 60 s at 6.8 W RF power (PDC-32G-2; Harrick Plasma) (Fig. S1(g)). After cutting the PDMS membrane on the PP film along the edge of the PDMS replica, the disposable superstrate fabricated by bonding the PDMS replica and the membrane is detached from the PP film (Figs. 2(a) and S1(h)) and then, the residual PDMS membrane under the vacuum trench is teared off. Fig. 2(b) and (c) shows cross-sectional views of the microchannel, structured with the PDMS replica and the PDMS membrane. The fabrication process for the reusable substrate used a 0.7-mmthick glass slide (Borofloat33 Pyrex; Schott) with 1000 Å Cr deposited. A SU-8 3005 photoresist was spun (Fig. S1(i)) and patterned for creating the microsupport array on the glass substrate with various thicknesses, from 0.6 to 5 μm (Figs. 2(d) and S1(j)). The cylindrical microsupports are 10-μm in diameter and 3-μm apart (Fig. 2(e) and (f)). By applying a vacuum pressure, the disposable superstrate and the reusable substrate are tightly assembled with each other (Figs. 2(g) and S1(k)).

superstrate and substrate close to each other and prevent the microchannel from being deformed during vacuum assembly, the height of the microsupports was limited to less than 5 μm and the spacing was designed to be 3 μm. To improve the degassing performance, the microsupport array is patterned only inside and along the length of the vacuum trench. The disposable superstrate and the reusable substrate are tightly assembled by applying vacuum pressure to the vacuum hole (Fig. 1(b)). During vacuum assembly, when bubbles are trapped and form in the microchannel, they are quickly extracted through the micrometer-thick PDMS membrane and the microsupport array and finally evacuated along the vacuum trench to the vacuum hole (Fig. 1(c)). While bubbles can be extracted due to the gas permeability of the PDMS membrane, the solution remains in the microchannel due to the strong hydrophobicity of the PDMS membrane. After use of the device, the superstrate and reusable substrate are disassembled by vacuum and the superstrate is replaced for the next operation. Due to the simplicity and versatility of the ADD-method, various designs of the single-layered microchannel are available. 2.2. Fabrication process To fabricate the disposable superstrate, a 50-μm-thick layer of SU-8 3050 photoresist (MicroChem Corp.) was first spun and patterned for creating a SU-8 mold for the microchannel on a 0.7-mm-thick glass master with 1000 Å Cr evaporated to increase the adhesion of the SU-8 (Fig. S1(a)). To define the vacuum trench, acrylic square bars with a cross section of 2 × 2 mm2 were bonded adhesively around the microchannel mold patterned on the glass master (Fig. S1(b)). The SU-8 mold was completed by assembling the glass master and an aluminum frame, which is used to pour the liquid PDMS. Liquid-phase PDMS, prepared by mixing resin and curing agent in a 10:1 ratio (Sylgard 184; 355

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the ambient pressure and temperature of the surrounding environment and solution. The gas permeability of PDMS is influenced by the solution-diffusion mechanism [31], which is associated with the free volume ratio in the cross-linked PDMS structure, in turn determined by curing temperature [32]. In this study, the curing temperature of PDMS was set at 75 °C to achieve high gas permeability [33]. In the ADDmethod, the range of vacuum pressures that can be used for device assembly and degassing is from −20 to −80 kPa. If the vacuum pressure is high, the degassing rate may be high, but if air is introduced, the high vacuum pressure (−60 to −80 kPa) varies within −7 kPa, which is a significant change over −2 kPa at low vacuum pressure (−20 to −40 kPa). If the vacuum pressure is low, the degassing rate becomes lower. Therefore, the vacuum pressure was fixed at −50 kPa in all experiments, for the purpose of relatively stable use in sudden air inflows along with a high degassing rate. The solution and the surrounding temperature were kept at room temperature (25 °C). To evaluate the degassing rate of the PDMS membrane, a closed meandering microchannel, shown in Fig. S2(a), was fabricated based on the ADD-method. The PDMS membranes were made with average thicknesses of 9, 14, 19 and 21 μm, while the microsupport arrays were fabricated with heights of 0.6, 1, 1.5, 2, 2.6, 3.2, 4.2 and 5 μm. After dropping a drop of solution onto the inlet reservoir, the time taken for the solution to fill 12 areas in the closed microchannel was measured using video images (Fig. S2(b)−(k)). The data points in Figs. S3 and S4 were obtained from four datasets, measured using deionized (DI) water and phosphate-buffered saline (PBS) solution. In the proposed ADD-method, the degassing rate, the same as the degassing flow rate per area, is mainly determined by gas flow resistance of the PDMS membrane and the microsupport array. Therefore, it can be assumed that the thickness of the PDMS membrane and the height of the microsupport array are the main parameters to determine the degassing rate. For the theoretical analysis, the degassing flow rate, dQ/dt, per area, A, can be expressed with the degassing rate, k., as

Fig. 3. Degassing rates (k), measured using (a) deionized (DI) water and (b) phosphate-buffered saline (PBS) solution, for varying thicknesses of the PDMS membrane and heights of the microsupport array.

1 ⎛ dQ ⎞ = −k [m s−1] A ⎝ dt ⎠

(1)

where A and Q are the non-wetted degassing area and gas volume in the closed microchannel, respectively, and t is time. The first-order ordinary differential Eq. (1) can be derived with the height of the microchannel, h (=50 μm), as

dQ k = −kA = − Q dt h

(2)

k

Q = Q0 e − h t

(3)

where Q0 is the initial gas volume, 6.85 mm (=6.85 μl), of the closed microchannel. According to the thickness of the PDMS membrane and the height of the microsupport array, the degassing rate, k, can be obtained by leastsquares fit of Eq. (3) to the measured gas volume, Q, over time, t, as shown in Fig. 3(a) and (b). The measured data show that the degassing rate proportionally increases as the PDMS membrane becomes thinner. The data also predict that the degassing rate increases with the height of the microsupport array and is eventually saturated at a microsupport height above 1.5–2.5 μm. Interestingly, the height of the microsupport array, associated with saturation, seems to increase as the thickness of the PDMS membrane decreases, because the gas can easily escape through the thinner membrane and the taller the microsupports. From the experimental results, 14-μm-thick PDMS membrane was used for subsequent microfluidic devices based on the ADD-method. Even though the degassing rate of 14-μm-thick PDMS membrane is about 20% less than that of 9-μm-thick membrane, its strength is 4 times higher, which allows more stability on handling. In addition, the height of the microsupport array was set at 4.2 μm to accommodate a high degassing flow rate. 3

Fig. 4. Measured burst pressure of the PDMS membranes (9 ± 1.3, 14 ± 1.2, 19 ± 1.0 and 21 ± 1.1 μm in thickness) used for an assembly-disposable degassing microfluidic device, having a microchannel with both sides in Y-shape (Fig. S5(a)) and a cross-section of 1 × 0.05 mm2.

3. Results and discussion 3.1. Degassing test The degassing capability of the PDMS membrane depends on its gas permeability and thickness, and on the experimental conditions, such as 356

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Fig. 5. (a) Illustration of the bubble-trap structure realized based on the ADD-method. It consists of main and bypass microchannels, each containing two bubblestoppers. Sequence photographs showing (b) small and (c) large bubbles, trapped by the bubble-stoppers and gradually removed within 1 min.

PDMS membrane endured a total flow rate of up to 300 ml h−1, at which point two injection syringe pumps (Legato 100, KD Scientific) were stopped due to high repulsive forces. This result confirmed that the assembly disposable degassing microfluidic device can operate reliably at high flow rates of at least 300 ml h−1.

3.2. Bond stability To measure the maximum pressure within the microchannel that the PDMS membrane can withstand, microchannels with both sides in Y-shape (Fig. S5(a)) and straight microchannels (Fig. S6(a)) were used and air pressure from 0 to 700 kPa was applied to the microchannels through a gas regulator. Then, a vacuum pressure of −50 kPa was used for assembly of the disposable superstrate and the reusable substrate. To confirm that the air pressure is applied uniformly to the overall microchannel, pressure sensors (XGZP6847001MPG; CFSensor) were connected to terminals of the microchannel, as shown in Fig. S5(a) and S6(a). As the air pressure increases, the PDMS membrane swells (Fig. S5(b), S5(c), S6(b) and S6(c)) and eventually bursts at a critical value of the air pressure. For the burst test, the air pressure increased by 20 kPa at 5-minute intervals until the PDMS membrane burst. The burst tests were repeated using five identical disposable superstrates (Figs. 4 and S7). The experimental results demonstrated that the maximum internal pressure that the microchannel can withstand increases proportional to the thickness of the PDMS membrane. Even the thinnest 9-μm-thick PDMS membrane can withstand internal pressure of more than 250 kPa, which is much higher than the 10–20 kPa required to drive fluid flow in typical microfluidic devices. As another bond stability test, the leakage test was performed using an assembly disposable degassing microfluidic device, having two inlets, two outlets and a microchannel with a cross-section of 1 × 0.05 mm2. The disposable superstrate was fabricated using a 14-μm-thick PDMS membrane and assembled with a reusable substrate, having a 4.2-μm-height microsupport array, by applying a vacuum pressure of −50 kPa. Red- and blue-colored solutions were injected into two inlets at several flow rates, of 20, 40, 60, 80, 100, 120, 140 and 150 ml h−1, for 1 min (see Video S1). The microfluidic device with a 14-μm-thick

3.3. Applications 3.3.1. Bubble-trap structure A bubble-trap structure based on the ADD-method is proposed to trap bubbles entering through inlet reservoirs and to remove the trapped bubbles. The bubble-trap structure consists of main and bypass microchannels, each containing two bubble-stoppers, as shown in Fig. 5(a). The bubble-stoppers are used for trapping bubbles in the inlet microchannel; the width and spacing of the stoppers were designed to be 60 and 52 μm, respectively. Consequently, the bubble-stoppers can also function as a strainer for biological samples. If the main microchannel is blocked by trapped bubbles, fluid detours through the bypass microchannel. Therefore, even if large bubbles are trapped by the bubble-stoppers in the main microchannel, the sample flow is not affected by the trapped bubbles. For the experimental test, human peripheral blood diluted with PBS at a ratio of 1:4 was used as a biological sample. As shown in Fig. 5(b) and (c), when bubbles are trapped at the bubble-stoppers in the main microchannel, the blood sample flows through the bypass microchannel (Fig. 5(b)). If the fluid driving pressure is large enough (> 5.3 kPa) such that bubbles pass through the first bubble-stopper, these are eventually trapped by the second bubble-stopper, as shown in Fig. 5(c). Then, the bypass microchannel also serves to reduce the pressure difference between the trapped bubble and the bubble-stopper, because the fluid detours through the bypass microchannel. All of the trapped bubbles 357

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Fig. 6. Sequence photographs show bubbles degassing in (a) deterministic lateral displacement (DLD) with a cylindrical micropost array and (b) microvortex device with sudden expansion-contraction reservoirs; all devices are fabricated based on the ADD-method. In these devices, bubbles form easily in the micropost array and corners of the sudden expansion-contraction reservoirs, as complex structures. For the experimental test, blood diluted 1:9 with PBS was used as a biological sample.

are degassed and removed within 1 min; then, the sample fluid gradually flows through the main microchannel again. In this current design, the maximum trapping volume is 25 nl. Larger trapping volume can be attained by changing the dimension of the main microchannel and inserting the bubble-stoppers more deeply into the main microchannel. Due to the simplicity of the proposed bubble-trap structure, it is suitable for integration with the assembly disposable degassing microfluidic devices.

on the ADD-method, they are completely removed within 15 s on average, with the exact time depending on the bubble size in the microchannel (Fig. 6(a)). The result can be compared with the experimental result (Fig. S9(a)) of the DLD device fabricated using 19-μmthick polyethylene terephthalate (PET) film instead of 14-μm-thick PDMS membrane. The microvortex device (Fig. S8(c)) is another example of a complex microfluidic device that is associated with frequent bubble formation. This device has sudden expansion-contraction reservoirs within the microchannel, which can trap cells over a critical size due to the shear gradient lift force in the expansion reservoirs. At the time of sample filling, bubbles usually form at the corner in the expansion reservoirs, as shown in Fig. 6(b). Because large-sized cells are hydrodynamically captured at the bay of the expansion reservoirs, bubbles trapped at the corner seriously impair isolation. In the case of the microvortex device fabricated by the ADD-method, trapped bubbles disappeared within 20 s. The result can be compared with the experimental result (Fig. S9(b)) of the microvortex device fabricated using 19-μm-thick PET film instead of 14-μm-thick PDMS membrane. Consequently, the experiment showed that the ADD-method is a very effective approach to quickly remove bubbles trapped in complex structured microfluidic devices.

3.3.2. Complex structured microfluidic devices To demonstrate the usefulness of the ADD-method for complex structured microfluidic devices, deterministic lateral displacement (DLD) [34] (Fig. S8(a) and (b)) and microvortex devices [35] (Fig. S8(c) and S8(d)) were fabricated based on the ADD-method. The key component of DLD is their micropost arrays with slanted angles in microchannel (Fig. S8(b)). Cells smaller than a critical size flow between the micropost gaps, while larger cells move laterally and follow the micropost arrangement and thereby, cells are isolated depending on their size [36]. Although the micropost array is a key component for isolation of cells from biological samples, its complex structure results in bubble formation, which may degrade the isolation performance. However, when bubbles are trapped in the DLD micropost array fabricated based 358

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Fig. 7. Fabricated (a) disposable superstrate and (b) reusable substrate of the CTC-addChip, which includes the bubble-trap structure in the disposable superstrate, the microsupport array and the inlaid ferromagnetic wire array on the reusable substrate. (c) Vacuum assembled CTC-addChip. (d) Bubble-trap structure showing a CTC flowing through the main or bypass microchannels depending on bubbles trapped at the bubble-stopper; and (e) sequence images of bubble degassing. Photographs of CTCs flowing through the microchannel of (f) the assembly disposable lateral magnetophoretic microseparator fabricated using 19-μm-thick PET film, in which the artificially injected bubbles are trapped, and (g) the CTC-addChip fabricated using 14-μm-thick PDMS membrane and 4.2-μm-height microsupport array, from which all the injected bubbles are removed.

the disposable superstrate. As a test vehicle for active functional microfluidic devices, the previously developed assembly disposable lateral magnetophoretic microseparator [37] was advanced using the ADDmethod. To isolate CTCs from blood, the developed assembly disposable degassing lateral magnetophoretic microseparator (the CTC-addChip) involves the bubble-trap structure in the sample inlet microchannel (Fig. 7(a)), and the microsupport array and inlaid ferromagnetic wire array on the reusable substrate (Fig. 7(b)). In the vacuum-assembled

3.3.3. Assembly-disposable degassing lateral magnetophoretic microseparator Another advantage of the ADD-method relates to the functionality of the reusable substrate, which can be equipped with various functionalities that generate elaborately patterned energy fields, such as acoustic, electric, magnetic and thermal fields. Due to the ultrathin thickness, the energy field can effectively transmit through the PDMS membrane and thereby manipulate substances in the microchannel of 359

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and microvortex, in which bubbles form easily and seriously affect its performance. In addition, the most important advantage of the ADDmethod is that the reusable substrate can be equipped with functionalities that can generate energy fields, such as acoustic, electric, magnetic and thermal fields. Due to the micrometer thickness of the PDMS membrane and the micrometer height of the microsupport array, the microchannel of the disposable superstrate can be located within 20 μm of the energy source on the reusable substrate. Therefore, the energy field is effectively transferred into the microchannel and can actively manipulate substances without any bubble interference. The vacuum assembly also contributes to effective transmission of the energy field, because it keeps the disposable superstrate and the reusable substrate in close proximity, without an air gap. Consequently, the proposed ADDmethod has unlimited potential to be used in numerous microfluidic applications, such as particle manipulation based on hydrodynamics and/or elaborately patterned energy fields, as well as for cell culture, drug screening and microreactors, such as those valving and mixing. Furthermore, the ADD-method allows users to utilize microfluidic devices easily. Taken together, these features indicate that the ADDmethod will serve as a key technology to solve the chronic problem affecting many microfluidic devices, i.e., difficult automation due to bubble formation.

CTC-addChip (Fig. 7(c)), CTCs and blood cells detour through the bypass microchannel when a bubble is trapped at the bubble-stopper in the main microchannel (Fig. 7(d)). The trapped bubble is removed within 1 min (Fig. 7(e)) and then the blood sample flows into the main microchannel again. By artificially injecting bubbles with blood samples, the usefulness of the CTC-addChip was assessed and compared to the previous assembly disposable lateral magnetophoretic microseparator, fabricated using a silicone-coated release PET film instead of the PDMS membrane. When bubbles are trapped within the microchannel of the previous device using a PET film, the trapped bubbles disturb the flow of CTCs in the microchannel (Fig. 7(f)), thereby decreasing the isolation performance. Meanwhile, in the case of the CTC-addChip, the injected bubbles are first blocked and removed by the bubble-trap structure. Even if bubbles infiltrate the microchannel, they disappear within 1 min. Consequently, the CTC-addChip could be used to isolate CTCs from blood samples without any bubble interference (Fig. 7(g)). Because the energy field generated from the reusable substrate can be acoustic, electrical, thermal, etc., as well as magnetic as in this study, we believe that the ADD-method can be widely used for various applications involving microfluidic devices. 4. Conclusions

Acknowledgments This paper introduced the ADD-method, which can be applied to most single-layered microfluidic devices due to its simplicity that cannot be realized by multi-layered degassing microfluidic devices [9,28,29]. The ADD-method was implemented using a microstructured disposable superstrate with a micrometer-thick PDMS membrane and a reusable substrate with microsupport array. By vacuum assembly of the disposable superstrate and reusable substrate, bubbles in the microchannel were quickly extracted through the gas-permeable PDMS membrane and microsupport array. However, due to the superhydrophobicity of the PDMS membrane, the solution in the microchannel was not affected by the bubble degassing. As expected, the degassing rate increased as the thickness of the PDMS membrane decreased. The degassing rate also increased with the height of the microsupport array and was eventually saturated at microsupport heights above 1.5–2.5 μm. The degassing rate of the 14-μm-thick PDMS membrane was measured as about 0.35 μl s−1 cm−2, which means that a bubble of 1 μl formed in a 50-μm-height microchannel can be completely removed within 1 min. This degassing speed is really fast compared to in-plane degassing structures, which require more than 10 min to remove bubbles of 0.1 μl [6], 0.15 μl [26], and 0.002 μl [11], respectively, due to thick PDMS degassing barriers of more than 100 μm. Due to high flexibility of PDMS, it becomes more difficult to handle as thickness is reduced. However, because the micrometer-thick PDMS membrane used in the ADD-method is fixed along the edge of the microchannel, this did not cause any problems during use. The burst test showed that the bond stability between the PDMS membrane and the PDMS replica increases with the thickness of the PDMS membrane and even the thinnest 9-μm-thick membrane was able to withstand internal pressure in the microchannel higher than 300 kPa. The leakage test demonstrated that microfluidic devices with a 14-μm-thick PDMS membrane could be used with a flow rate of at least up to 300 ml h−1. Both the burst and leakage tests indicated that the ADD-method is available even to microfluidic devices requiring high flow rates. Because the ADD-method can be accomplished simply, it can be used for various applications wherein single-layered microfluidic devices are used. The bubble-trap structure developed using the ADDmethod is an effective method for bubble trapping and active degassing, since it does not require a complex structure or fabrication process. Its degassing rate can be adjusted simply by varying the thickness of the PDMS membrane and the bubble trapping volume can be changed by altering the position of the bubble-stopper. The ADD-method is especially useful for complex structured microfluidic devices, such as DLD

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