Technical Note pubs.acs.org/ac
Rotatable Reagent Cartridge for High-Performance Microvalve System on a Centrifugal Microfluidic Device Takayuki Kawai,* Nahoko Naruishi, Hidenori Nagai, Yoshihide Tanaka, Yoshihisa Hagihara, and Yasukazu Yoshida Stress Signal Research Group, Health Research Institute, National Institute of Advanced Industrial Science and Technology, MOL205, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan S Supporting Information *
ABSTRACT: Recently, microfluidic lab-on-a-CD (LabCD) has attracted attentions of researchers for its potential for pumpless, compact, and chip-inclusive on-site bioassay. To control the fluids in the LabCD, microvalves such as capillary, hydrophobic, siphon, and sacrificial valves have been employed. However, no microvalve can regulate more than one channel. In a complicated bioassay with many sequential mixing, washing, and wasting steps, thus, an intricate fluidic network with many microchannels, microvalves, and reservoirs is required, which increases assay costs in terms of both system development and chip preparation. To address this issue, we developed a rotatable reagent cartridge (RRC), which was a column-shaped tank and has several rooms to store different reagents. By embedding and rotating the RRC in the LabCD with a simple mechanical force, only the reagent in the room connected to the following channel was injected. By regulating the angle of the RRC to the LabCD, conservation and ejection of each reagent could be switched. Our developed RRC had no air vent hole, which was achieved by the gas-permeable gap between the bottle and cap parts of the RRC. The RRC could inject 230 nL−10 μL of reagents with good recoveries more than 96%. Finally, an enzymatic assay of L-lactate was demonstrated, where the number of valves and reservoirs were well minimized, significantly simplifying the fluidic system and increasing the channel integratability. Well quantitative analyses of 0−100 μM L-lactate could easily be carried out with R2 > 0.999, indicating the practical utility of the RRC for microfluidic bioanalysis. mong microfluidic “lab-on-a-chip” devices,1−10 a centrifugal platform is called “lab-on-a-CD (LabCD)” and has attracted much attention of researchers.3−15 Unlike other microfluidic devices, LabCD does not require a connection with a syringe pump for the fluidic control, permitting a rapid assay of a small-volume sample in a compact instrument. Thus, the LabCD is a promising technique for point-of-care diagnoses.3 Both fundamental and application researches have already been carried out for the assays of nucleic acid,4,5 proteins,6−9 and small compounds;10 however, there is still no versatile platform that is applicable to many bioassays easily. It is because many functions are inflexible and limited significantly by the chip design,1 and then almost a tailor-made LabCD is required for each assay.4−15 For a diagnosis with a small market, therefore, the cost of the system development would often be too much compared with the expected income, preventing a development of a new LabCD for minor diseases. Therefore, simplification of system development for reducing the cost is a quite important challenge. One of the main reasons of the complicated design, inflexible function, and high cost is in a current microvalve system on the fluidic network. As in the case of enzyme-linked immunosorbent assay (ELISA), most bioanalyses are based on multistep reactions including washing, mixing, and wasting processes using many solutions. Hence, microvalves such as capillary, hydrophobic, siphon, and sacrificial valves have been developed
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© XXXX American Chemical Society
and applied to LabCDs.11−15 In most cases, however, one valve is usually required for controlling one solution. Thus, many microvalves are necessary on the fluidic network,8−10 where the design and control of the fluidic network becomes complicated. The preparation of microvalves is also problematic. In the case of laser irradiated ferrowax microvalves,15 for example, precise introduction of ferrowax on the determined points and its oneby-one operation by a delicate LED irradiation would require expensive high-performance instruments. This kind of complexity makes the system development, chip manufacturing, and analytical procedure laborious, increasing the cost of system development and LabCD fabrication as well as decreasing the analytical throughput and accuracy. Therefore, development of simple, inexpensively preparable, and easily operable microvalve is required to accelerate the practical LabCD research around the world. Here we introduce our idea for the simplification: integration of several microvalves and reservoirs into one cartridge,16 which is operable with a simple mechanical force.17 We designed the integrated cartridge as a column-shaped tank which can be embedded and rotated in the LabCD. The cartridge contains several rooms with an ejection hole to store and inject reagents. Received: March 6, 2013 Accepted: June 7, 2013
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We named it as “rotatable reagent cartridge” (RRC). The detailed designs and working mechanisms of the RRC are shown in the Results and Discussion with Figures 1−3. The
was purchased from Cayman Chemical (Ann Arbor, MI), where the concrete composition of each solution was not disclosed. Fluorescein was purchased from Wako Chemical Industries (Tokyo, Japan). The microendmills NSME 230 (diameter = 0.05 mm), MHR 230 (diameter = 0.5 mm, l = 6 mm), and MSES 230P (diameter = 4 mm) were purchased from Nisshin Tool Corporation (Tokyo, Japan). A poly(ethylene terephthalate) (PET) plate seal was purchased from ASONE Corporation (Osaka, Japan). The fluorinated coating reagent XINT QC333 was purchased from Noda Screen (Komaki, Japan). All solutions were prepared with deionized water purified with Elix and Milli-Q gradient system (Nihon Millipore, Tokyo, Japan). Fabrication of Microchips and RRCs. LabCDs and RRCs were fabricated by using numerically controlled (NC) endmilling machine (Micro MC-3-ATC-SAN, PMT Corporation, Fukuoka, Japan). The detailed fabrication condition is explained in the Supporting Information. The LabCD substrate was fabricated from a PMMA panel with 1.85 mm or 5 mm for “high-recovery” or “large-volume” RRCs, respectively. The fabricated substrate was coated with XINT QC333 by simply putting drops of the polymer solution on the lid by a micropipet. Before the air dryness, the solution was spread throughout the channel network automatically by the capillary effect. The coated substrate was then sealed with a PET plate seal prior to use.20 After the assay, the plate seal was removed and the substrate was washed with deionized water and then with 70% ethanol. The remaining ethanol drops were then blown by an air duster, followed by air dryness at room temperature for the next use. Cap and bottle parts of the RRCs were fabricated from 5mm-thick PMMA and PTFE panels, respectively. For the “large-volume” prototype, the ejection holes with diameters of 0.5 mm were made on the RRC bottle by a drill for releasing reagents (Figure 1). In the “high-recovery” version, the holes were automatically produced as a gap between the bottle and cap. The reagents were manually loaded into the bottle with a micropipet (Eppendorf, Hamburg, Germany) before capping the bottle. The RRC containing the reagents was then embedded in the LabCD prior to every assay. After the assay, the RRC was removed from the LabCD and separated from the bottle and cap, followed by the same washing of the LabCD substrate. Procedure. A laboratory-built spinning instrument was employed to add centrifugal force to the developed LabCD.9 Before centrifugation, RRCs were manually embedded in the LabCD from the unfabricated side of the LabCD substrate, while the fabricated side was sealed with a PET plate seal. The LabCD-RRC composite was then placed on the centrifugal stage; RRC was manually rotated by a flat-head screwdriver to connect the appropriate room to the following channel. To release the reagents from the RRC, the LabCD-RRC composite was spun at 2 000 rpm for 5 s. The rotation of RRCs and centrifugation of the LabCD-RRC were repeated to inject all the focused reagents in the RRC to the following chamber. For mixing the reactor solutions, the LabCD was manually oscillated with around 500 rpm until the obtained fluorescence signal became stable. Fluorescent detection in the L-lactate assay was carried out with FLE-1000 (Nippon Sheet Glass, Tokyo, Japan) equipped with a LED diode at excitation/ detection wavelengths of 530/590 nm. To estimate the injection volume, a scaled channel of 0.5 or 2 mm width was employed. The channel was first analyzed with
Figure 1. Design of the “large-volume” RRC prototype: (a) a photograph of the fabricated RRCs with four, six, and eight rooms, (b) a sketch design of the RRC with four rooms, and (c) a cross-sectional design of the RRC embedded in the LabCD. The bottle and cap parts were fabricated from PTFE and PMMA panels, respectively. The gaps between the cap-bottle and RRC-socket were set at 5 and 10 μm on the NC program, respectively.
novel characteristics of the RRC are the following two: simply by rotating a single RRC on a LabCD, several kinds of solutions can be injected to the following channels one by one; the cartridge-style introduction of the reagents into the LabCD can facilitate the chip preparation and storage. It was also interesting that our RRC system is based on two unique mechanisms: rotation of a RRC on a LabCD changes the relative direction of the centrifugal force to the RRC, effectively reducing the leaking force of the untargeted reagents; the gap between two components of the RRC is impermeable to liquid but permeable to gas, so that simple structure without any air vent holes can be achieved. Our aim of this study is to develop a simple valving system in the LabCD with the RRC that can significantly reduce the numbers of valves, channels, and reservoirs. We first investigated the material and design of the RRC system to achieve the highest performance. It was checked if the focused solution was successfully injected without a leakage of other untargeted solutions. The recovery rate of the released solution was evaluated to show the potential of the RRC to eliminate “metering” reservoirs.18 Finally a basic enzymatic assay using Llactate dehydrogenase19 was demonstrated to show the performance of our RRC system.
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EXPERIMENTAL SECTION Materials and Chemicals. Panels of 1.85 and 5 mm thickness made from poly(methyl methacrylate) (PMMA) and poly(tetrafluoroethylene) (PTFE) were purchased from RS Components Japan (Kanagawa, Japan). The lactate assay kit B
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holes. To our experience, the best gap between the bottle and cap was 5 μm. When the gap was set at 0 μm on the NC fabrication program, releasing the reagents was often difficult probably due to the too narrow gap for the air to pass through. At 10 μm or more, there are sometimes observed leakages of reagents in the RRC. Since 2-μm-precision fabrication is available in the injection molding method, RRCs will be provided with a reasonable price through the mass-production. Figure 2 shows the overview of the developed LabCD with RRCs. The microchannels were fabricated on the bottom side of a PMMA substrate of the LabCD. The detailed design of the entire LabCD substrate was shown in Figure S-3 in the Supporting Information. To prepare the RRC, reagents were loaded into the rooms of the bottle, which was then sealed with the cap. The prepared RRCs were manually embedded in the socket holes of the LabCD substrate, of which the diameter was larger than that of the RRC by 20 μm for balancing its smooth rotation and fixed positioning. A PET plate seal was attached on the fabricated side of the LabCD substrate.20 The obtained microchannel was strong enough to avoid the leakage of the employed solutions. To release a reagent, the RRC was rotated on the LabCD with a screwdriver to connect the focused room to the following microchannel. The entire LabCD-RRCs composite was then spun around the center hole to inject the focused reagent. To release the next reagent, the centrifugation of the LabCD was stopped and then the RRC was rotated again to an appropriate angle (Figure 3). So far, the rotation of the
the confocal laser scanning microscope to measure the depth correctly. A fluorescein solution was then injected into the channel and the photograph was then taken with a digital camera, (IXY 220F, Canon, Tokyo, Japan). From the obtained picture, the liquid surface was digitally estimated based on pixels, and the volume was calculated with the obtained depth information.
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RESULTS AND DISCUSSION Concept of RRC Valving System. Figure 1 shows the design of the most typical RRC with four rooms. The detailed design is available in the Supporting Information, Figure S-1. This RRC prototype is a column-shaped tank, which consists of several symmetric rooms. Each room has an ejection hole to release the reagent. There is a socket hole in the LabCD, where the RRC can be embedded and rotated (Figure 2). As shown in
Figure 2. Overview of a RRC-embedded LabCD. RRCs were embedded into the socket holes of the LabCD substrate made from PMMA. The microchannels fabricated on the bottom side of the substrate were sealed with PET plate seal with holes corresponding to the center and socket holes in the LabCD substrate.
Figure 3. Concept of reagent release using a RRC with a “semipermeable” gap. (a) A reagent in the room facing to the following channel is injected with an air supply through the gas-permeable gap, whereas the other reagents are conserved at the corner of each room by the gap’s liquid-impermeable behavior. (b) By rotating the RRC, another reagent can be injected.
the cross-sectional design (Figure 1c), the ejection hole of the focused RRC room could be connected to the channel on the LabCD by rotating the RRC to an appropriate angle. The RRC consists of bottle and cap parts, which were made from PTFE and PMMA, respectively (Figure 1b). PTFE has a low friction coefficient, allowing the smooth insertion and rotation of the RRC in the LabCD. To avoid the leakage of reagents, the PMMA cap with a hydrophilic surface was coated with a fluorinated reagent XINT QC333. Although a cap made from PTFE would be better to avoid the leakage, PMMA was employed in this study to visualize the inside of the RRC. The fluorinated surface decreases the contact angle for both water and polar organic solvent, preventing their penetration into the gap between the bottle and cap as a hydrophobic valve.12 The result in a leakage test for water, detergent solution, and methanol is shown in Figure S-2 in the Supporting Information. On the other hand, air can penetrate inside the RRC because of the following two factors: air already exists in the gap so that the hydrophobic valve does not work, and viscosity and density of air are much lower than those of liquids inside the RRC, reducing the hydrodynamic resistance of the gap. This selective permeability allows the RRC to be simple without any air vent
RRC must be carried out manually. However, such torque actuated rotation can easily be automated by a small motor. A gear system also seemed to have a great potential for highthroughput analysis by a simultaneous operation of many RRCs (Figure S-4 in the Supporting Information), which would be an advantage over the other microvalves.11−15 Figure 3 shows how the RRC works for releasing reagents. Our RRC system is based on a unique concept: switching valve on/off is not regulated by the simple increase/decrease of the channel resistance and/or centrifugal force but by the geometrical change of the RRC to the centrifugal force vector. The reagent in the room facing the following channel is released by the applied centrifugal force. It should be noted that the channel connected to the RRC must be parallel to the centrifugal force vector. If not, the recovery of the released reagent would be lower and the leakage risk of untargeted reagents would be higher. Again, air can be introduced into the room through the narrow gap between the bottle and cap parts C
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connecting channel, the edge on the room top and bottom could also be eliminated. In the prototype, the manual hole positioning by a drilling process was not accurate, so that a large space had to be prepared as a “buffer area” to connect the ejection hole to the following channel (Figure 1c). To eliminate the buffer area where drops of reagents were often trapped, we produced the ejection hole as a gap between the bottle and cap portions of the RRC (Figure S-1 in the Supporting Information), which could be precisely positioned with the NC end-milling machine. It was also quite beneficial that the cumbersome manual drilling process was not needed. It should be noted, however, that the shape of the round rooms and decrease in the room depth caused the reduction in the room volume. The ratio of the room volume to the RRC-occupying area was 0.19 μL/mm2, which was 15-fold smaller than that of the prototype, 2.8 μL/mm2. For the washing process in immunoassays requiring just plenty enough of the buffers, the large volume capacity has a priority to the high recovery. Therefore, effective placement of the “large-volume” prototype and “high-recovery” type RRCs should be important. The recovery of the released reagent with the “high-recovery” RRC was estimated for the volumes of 230 nL, 760 nL, 1.9 μL, 5.0 μL, and 10 μL. The results were summarized in Table 1,
of the RRC, so that the simple structure with no air vent hole can be achieved. Untargeted reagents in the other rooms are trapped in the room corners because their ejection holes were facing the other direction than that of the centrifugal force vector. It should be noted that the volume of the reagent should not be too much to avoid leakage, especially in the case of treating low-viscosity solutions such as organic solvents and detergents (see the Supporting Information, pages S7−S12). Recovery of the Injected Solution. For realizing a highperformance bioassay on a LabCD, it is usually important to introduce precise and accurate amount of reagents into the reactor. Hence, the actually injected volume was estimated by employing a scaled chamber or channel (Figures S-9 and S-10 in the Supporting Information). Volume of the reagent injected to the scaled chamber or channel (Vch) was estimated from the image data digitally, whereas the originally loaded volume to the RRC (VRRC) was evaluated by measuring the change in the total weight of the RRC bottle before/after loading the reagent. The recovery was calculated as the ratio Vch/VRRC. When 5.0 μL of fluorescein solution was loaded to the RRC with four, six, and eight rooms shown in Figures 1, the recovery was estimated to be 87.6% ± 6.1%, 88.2 ± 5.1%, and 92.4 ± 3.3%, respectively (n = 5). The imperfect recovery was caused by the remaining drops of reagents around the edge of the RRC and the connecting channel (Figure S-11 in the Supporting Information). Thus, this prototype design was not suitable for the precise and accurate control of reagents, especially in the small-volume range. To improve the precision, a “metering” reservoir18 with a waste tank should be employed, which would significantly increase the complexity of the channel network. To address this issue, we developed a “high-recovery” RRC by changing its design. The main problem of the prototype was that it has many edges where drops of solutions were trapped. To eliminate the edges, we employed round-shaped rooms and smoothened the edges between the room and ejection hole with r = 0.5 mm (Figure 4 and Figure S-1 in the Supporting Information). By decreasing the room depth to 0.5 mm, which is almost the same as the size of the ejection hole and
Table 1. Estimated Recovery Performance of the “HighRecovery” Type RRC VRRCa/RSD
Vchb/RSD
recoveryc/%
233 nL/10.0% 765 nL/2.5% 1.89 μL/2.5% 5.02 μL/0.7% 10.2 μL/1.0%
224 nL/10.2% 765 nL/5.6% 1.89 μL/5.9% 5.02 μL/2.3% 10.3 μL/0.9%
96.1 100.0 99.8 99.8 100.7
a
Average volume of reagent loaded on the RRC by a micropipet. The volume was estimated by the difference in the total weight and the density of the solution (n = 5). bAverage volume of reagent yielded in the scaled channel after the centrifugation. cRecovery was estimated according to the following equation, % recovery = Vch/VRRC × 100.
where almost 100% recovery was obtained for the volumes of 760 nL−10 μL, indicating accurate injection performance of the RRC. Only for 230 nL, slightly worse recovery of 96% was obtained, probably because a very small amount (∼10 nL) of the solution remained in the RRC and microchannel. As Vch and VRRC became smaller, their RSDs became worse. The high RSDs of VRRC represents the poor precision of the micropipet employed for injecting samples. The difference between RSDs of Vch and VRRC was small, indicating the precise injection performance of the RRC. Coupled with long-term storage technique,21 our developed RRC would provide a precise and accurate injection with simple channel network, which will significantly contribute to the cost reduction in developing bioassay systems. Demonstration of Enzymatic Assay. To demonstrate that the RRC could actually simplify the fluidic network with a good analytical performance, an enzymatic assay of L-lactate standard was carried out on our LabCD with RRCs. The design of the fluidic network and the analytical procedure was summarized in the Supporting Information (Pages S-16−S19), where reagent consumption was 1/20-scale compared to the original protocol of the employed kit. The lactate dehydrogenase catalyzed the oxidation of L-lactate to pyruvate, along with the concomitant reduction of NAD+ to NADH.
Figure 4. Design of the “high-recovery” type RRC employed for the enzymatic assay: (a) a photograph of the fabricated RRC consisting of four rooms with i.d. of 2.4, 2.4, 3.6, and 5.2 mm, (b) the sketch design of the RRC, and (c) the cross-sectional design of the RRC embedded in the LabCD. The materials of the bottle, cap, and substrate and the gaps of their composite were the same as those in the prototype. The width and the depth of the microchannel were 500 and 600 μm, respectively. D
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NADH reacted with the fluorescent substrate to yield a highly fluorescent product. The reactant was analyzed with a fluorescent detector equipped with LED and a photomultiplier. In this assay, four reagents were introduced to the reactor chamber with a single “high-recovery” RRC. Compared to a conventional fluidic design with four reservoirs, channels, and valves, the present design was well simplified. Unlike the reagents, the sample was injected to the reactor chamber without using a RRC, because it is not realistic to load a precise amount of sample on the RRC with a micropipet on the diagnosis site. It is more reasonable to load an imprecise but sufficient amount of sample, where only the required amount is automatically collected in the “metering” reservoir with a waste tank (Figures S-13a,b in the Supporting Information). The collected sample can then be introduced to the reactor chamber. Oppositely, it is ideal that accurate amounts of reagents are already stored in the RRC to reduce the workload. Just by embedding and rotating the prepared RRC in the LabCD, users can introduce accurate amount of reagents and would obtain precise results easily. The obtained fluorescence intensities were summarized in Figure 5. It showed good linearity against the sample
between the bottle and cap parts. Two types of RRCs were introduced, a “large-volume” prototype with deep sectorshaped rooms and a “high-recovery” type with low circular rooms, which should be placed according to the required performance. For the “high-recovery” RRC, good recoveries up to 100% were obtained, showing the potential of a precise and accurate reagent supply without employing a sample “metering” system. Finally, the enzymatic assay of L -lactate was demonstrated, where good quantification performance was shown with R2 higher than 0.999. To develop a more practical bioassay method based on our developed RRC, automation of the ELISA process with many reaction steps is now investigated. For the high-throughput manipulation of RRCs, a gear system is also being studied to rotate many RRCs simultaneously (Figures S-4 and S-14 in the Supporting Information). Similarly, application of RRCs to the online metering and channel valving like Virtual Laser Valve (Spin-X Technologies, Switzerland) is also possible (Figures S-7 and S-8 in the Supporting Information). Our RRC system has been shown to have a great potential for simple, precise, and highthroughput analysis, which we believe will greatly contribute to the development of many practical bioassay systems in the LabCD researches.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-72-751-9953. Fax: +81-72-751-9950. E-mail: t.
[email protected]. Notes
The authors declare no competing financial interest.
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Figure 5. Enzymatic assay of L-lactate standards. The dotted line represents the calibration line for the obtained data with R2 > 0.999 (n = 3).
REFERENCES
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concentration with R2 larger than 0.999, which indicated the good utility of our developed RRC system. It is worth noting that the original protocol of mixing four kinds of reagents was easily traced by just connecting a single RRC and a reactor with a single channel, which significantly simplified the optimization in designing the channel network. A parallel assay of several samples was also achieved with a gear system (Figures S-4 and S-14 in the Supporting Information), indicating its potential for the high-throughput analysis. Although the demonstrated assay was not so complicated, our developed RRC system would be easily applicable to a multistep protocol because unlimited numbers of RRCs can be operated independently in theory. Studies along this line are now in progress in our laboratory focusing on simultaneous analyses of several stress markers.
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CONCLUSIONS A novel microvalve system using the RRC was developed. A fundamental working mechanism of the RRC was considered: the rotation of the RRC geometrically changed the centrifugal force to the RCR, effectively switching the conservation and ejection of the internal reagents; a vent-free structure was realized by the gas-permeable but liquid-impermeable gap E
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(15) Park, J.-M.; Cho, Y.-K.; Lee, B.-S.; Lee, J.-G.; Ko, C. Lab Chip 2007, 7, 557−564. (16) Kim, J.; Johnson, M.; Hill, P.; Sonkul, R. S.; Kim, J.; Gale, B. K. J. Micromech. Microeng. 2012, 22, 015007. (17) Godino, N.; del Campo, F. J.; Muñoz, F. X.; Hansen, M. F.; Kutter, J. P.; Snakenborg, D. Lab Chip 2010, 10, 1841−1847. (18) Steigert, J.; Grumann, M.; Brenner, T.; Mittenbühler, K.; Nann, T.; Rühe, J.; Moser, I.; Haeberle, S.; Riegger, L.; Riegler, J.; Bessler, W.; Zengerle, R.; Ducrée, J. J. Lab. Autom. 2005, 10, 331−341. (19) Gladden, L. B. J. Physiol. 2004, 558, 5−30. (20) Fuchiwaki, Y.; Nagai, H.; Saito, M.; Tamiya, E. Biosens. Bioelectron. 2011, 27, 88−94. (21) Hoffmann, J.; Mark, D.; Lutz, S.; Zengerle, R.; von Stetten, F. Lab Chip 2010, 10, 1480−1484.
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