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Centrifugal microfluidic system for a fully automated N-fold serial dilution
Accepted Manuscript Title: Centrifugal Microfluidic System for a Fully Automated N-fold Serial Dilution Authors: Tae-Hyeong Kim, Chi-Ju Kim, Yubin Kim, Yoon-Kyoung Cho PII: DOI: Reference:
S0925-4005(17)31998-6 https://doi.org/10.1016/j.snb.2017.10.096 SNB 23400
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
20-5-2017 14-9-2017 17-10-2017
Please cite this article as: Tae-Hyeong Kim, Chi-Ju Kim, Yubin Kim, Yoon-Kyoung Cho, Centrifugal Microfluidic System for a Fully Automated N-fold Serial Dilution, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.10.096 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Centrifugal Microfluidic System for a Fully Automated N-fold Serial Dilution Tae-Hyeong Kima, Chi-Ju Kima,b, Yubin Kimb, Yoon-Kyoung Choa,b1 a Center
for Soft and Living Matter, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
b Department
of Biomedical Engineering, School of Life Sciences, Ulsan National Institute of Science and Technology
(UNIST), Ulsan 44919, Republic of Korea
1
Corresponding author. Tel.: +82 52 217 2511; fax: +82 52 217 2509.
E-mail address: [email protected]
Tae-Hyeong Kima, Chi-Ju Kima,b, Yubin Kimb, Yoon-Kyoung Choa,b2 a Center
for Soft and Living Matter, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
b Department
of Biomedical Engineering, School of Life Sciences, Ulsan National Institute of Science and Technology
(UNIST), Ulsan 44919, Republic of Korea
Graphical abstract
N-fold serial dilution is fully automated on a centrifugal microfluidic system with a choice of N=2, 5, 10.
Highlights
Serial dilution is fully integrated and automated on a centrifugal microfluidic system.
Individually addressable, reversible diaphragm valves are utilized to enable the automatic control of the fluidic transfer and metering of the liquid.
N-fold serial dilution was performed automatically with a choice of N=2, 5, 10.
Abstract We present an innovative tool for a fully automated N-fold serial dilution with a choice of N=2, 5, 10 on a centrifugal microfluidic device. Individually addressable, reversible diaphragm valves are integrated on the disc for the automatic control of the fluidic transfer. Two metering zones are designed with three microfluidic chambers with the volume of 1 µL, 3 µL and 5 µL to meter the samples or the dilution buffer. The pre-programmed operation of the valves enabled the automatic serial dilution with the choice of the dilution factor of 2, 5, or 10 at each dilution steps. The samples prepared on the disc with various serial dilution factor showed excellent accuracy (R2 > 0.970). The automatic serial dilution disc could be an essential tool in the laboratory to replace the time-consuming and iterative pipetting especially when the sample volume is limited.
Keywords Centrifugal microfluidics; Serial dilution; Valves; Metering; Lab-on-a-disc
1. Introduction Serial dilution is one of the most common unit operations in biological and chemical experiments. Tedious and time-consuming manual pipetting is required to obtain the serially diluted solutions with desired concentration. In particular, accurate and precise pipetting of the solution is troublesome when the sample volume is in microliter scale or below. Microfluidics has facilitated the integration and automation of various fluidic unit operations on a miniaturized chip and it results in fast, accurate and highly efficient manipulation of fluids in microliter scale volume.[1-3] On-chip serial dilution with both linear and non-linear shape of the concentration
gradient have been also demonstrated.[4-9] However, most of the previous studies are to generate on-chip concentration gradient by diffusion between the adjacent channels filled with the flow of sample and buffer solution. The specific channel design depending upon the desired dilution ratio is required and the sample volume should be large enough to make the stable flow. New valving techniques enables versatile consecutive dilutions.[10-12] B. M. Paegel et al. introduced dilution circuit by utilizing the pneumatic valve on lamination structure of glass and PDMS layers.[10] This device allowed reversible flow control in circular loop and the dilution of the solution could be achieved on demand. However, only the final solution was possible to retrieve for the follow-up analysis and the intermediates had to be discarded. S. Ahrar et al. reported a serial dilution ladder to obtain all solution generated during the dilution process, however, only 1:1 dilution was possible.[12] Centrifugal microfluidic device is a microfluidic platform that utilizes the centrifugal force as a pumping source for the fluid transfer. Various kinds of unit operations such as pumping, mixing, metering, valving and switching of the fluid have been demonstrated.[13-19] Unique design of the fluidic networks and precise control of the rotation speed and spinning direction of the motor were demonstrated in order to integrate various biological and chemical assays on a disc.[15, 20-24] The previous effort to demonstrate on-disc generation of the dilution series without external active components could prepare 3- and 5-fold serial dilution from each specific design of the discs.[25] Adjustment of the spinning profile facilitates the fully automated preparation of the diluted samples. However, the repeatability was not excellent, which was attributed to the utilization of siphon as key unit operation and it could be critically influenced by surface properties and the tolerance of disc fabrication. In addition, other researches introduced systems for serial dilution by employing various additional components such as pneumatic pumping, dissolvable film and laser irradiated ferrowax microvalves (LIFM).[26-28] They enables a robust, batch-mode, serial dilution on a disc, however, only a fixed dilution ratio was possible using the specific design of the fluidic layout. In this paper, we present a fully automated centrifugal microfluidic chip for the N-fold serial dilution with the arbitral choice of N=2, 5, and 10. Here, we utilized recently reported, simple, reversible, and individually addressable diaphragm valves (ID valves) for the serial dilution on a disc. A single disc design can be used to offer several options for the dilution factor. The preloaded operation program can be used to perform automatic N-fold serial dilution with a good accuracy and repeatability.
2. Materials and methods 2.1 Disc design and fabrication Design of a disc for the serial dilution with the arbitral choice of dilution factor from N=2, 5, and 10 was conducted using 3D CAD software and milled with a CNC milling machine (smart 3530, PROTek, Korea). The pre-designed holes and chambers were milled on polycarbonate plates (polycarbonate, I-Components Co. Ltd, Korea). Two layers (top and bottom) were fabricated and they were laminated with a pressure sensitive adhesive layer (DFM 200 clear 150 POLY H-9V-95, FLEXcon, USA) which was prepared by a cutting plotter (Graphtec CE3000-60 MK2, 70 Graphtec Corporation, Japan) as previously reported.[29-30] Fig. 1a shows the detail design of the disc for N-fold serial dilution. It contains four storage chambers, two metering zones and five reservoirs for the storage of the diluted sample. Sample solution (10 µL) is loaded onto the sample loading chamber and transferred to the mixing chamber by spinning the disc. The buffer loading chamber contains buffer solution (100 µL) and about 15 µL of the buffer solution is transferred to the buffer metering zone when the disc is rotated when valve # 1 is in open and #2, #3, #4, and #5 are closed state. After metering 9 µL of the buffer solution, the excess amount of the buffer is discarded to the buffer waste chamber by spinning the disc after closing valve #1 and opening valve #5. Once the pre-programmed amount of the buffer is transferred to the mixing chamber by controlling valves #2, #3, and #4, the diluted sample is transferred to the diluted sample reservoirs (R1 ~ R5). The sequence of the sample transfer, buffer transfer, mixing, and transfer of the diluted sample is repeated until it fills the diluted sample reservoirs. The diluted sample could be retrieved for the downstream analysis by using a pipette through a hole prepared in each reservoir, which was kept sealed by an adhesive tape during the operation. In order to have the reversible actuation of the multiple valves, recently reported individually addressable diaphragm valve (ID valve) were utilized.[31] For the fabrication of the ID valves, elastic epoxy (Super-X, Cemedine, Japan) was inserted into grooves on the top layer of the disc in order to form a diaphragm. After curing the epoxy, 3D printed parts for the actuation of the valves were bonded on the top plate. Simple push and twist action allowed squeeze and release of the diaphragm to close and open the microfluidic channel on demand (Fig. 1b). Two metering zones were designed to trap and release the pre-programmed amount of solution in each micro-chambers as shown in Fig. 1c and Fig. 1d. Each metering zone consists of three sections with the chamber volume of 5 µL, 3 µL and 1 µL, respectively. The combination of the valve actuation allowed to
meter pre-determined volume of sample or buffer to prepare the N-fold serially diluted samples. ID valves allowed selective trap and release of the solution, which enables the arbitrary fold dilution on demand. The detail operation principle is described in the following sections.
2.2 Automatic actuation of multiple valves Operation of ID valve is actuated automatically using customized operating machine. Position of each ID valve is coordinated with radial position and angle with respect to the specific origin. Coordination is inserted to operating program and it recognizes the positions. To actuate the valve, spinning of a disc is stopped and angular position is aligned first depending on inserted coordination. Then, an automatic driver (KX050T2-01H1, Nitto Seiko Co., Japan) moves to the valve actuator by consecutive motion through x and z axes. Driver head is combined with groove on the top of ID valve and rotates it by 90° with clockwise direction to open the valve. Counter clockwise rotation can induce the close of valve and such operation can be conducted reversibly with automatic manner.
2.3 Operation of valve The automatic actuation of the multiple ID valves could be performed by using a customized disc operating machine.[31] The positions of each ID valves are coordinated with the radial position and the angle with respect to the specific origin. With the input of the coordination of the valves and the program for the actuation of the valves to open or close the valves, the operating system automatically perform the sequence of actuation. To actuate the valve, the disc is stopped, the angle is aligned, and the automatic driver (KX050T2-01H1, Nitto Seiko Co., Japan) aligns to the valve position utilizing two linear motors moving in x and z axes. The groove on the head of the ID valve can be selected from two positions in order to open or close the channels. The multiple actuation for the reversible control of the valves are fully automated.
2.4 Visualization of the fluid transfer on the spinning disc Images of the disc while it is rotating were acquired using the customized disc visualization module. A CCD camera (IK-TF5C, Toshiba Corp., Japan) and a strobe lamp (BUB0641-1A, B&B Corporation, Korea) were synchronized with the spinning speed and the series of images from specific position on a rotating disc were taken with a desired frame rate. The details of the visualization module have been previously described.[32]
3. Result and discussion 3.1 Principle of a fully automated serial dilution on a disc Two metering zones designed on the serial dilution disc play important roles for the arbitral choice of the dilution factor among N=2, 5, and 10. Fig. 2 illustrates the principle of the automated dilution process on the disc. The operation process for the dilution consists of three steps, i) metering of sample and buffer volume, ii) transfer of sample and remove of the excess amount of the buffer, and iii) transfer of the metered amount of the buffer to the mixing chamber and mix with the sample solution. We demonstrate the 5 serial steps of N-fold dilution with the arbitral choice of N from 2, 5, and 10 starting with 10 µL of the initial sample stock. First, the sample solution (red colour) is metered in the sample metering zone. Depending on the desired dilution ratio, metered volume to transfer and remaining volume of the sample can be adjusted by choosing the open or closed state of the ID valves of #7, #8, and #9. For example, in order to have 2X dilution, half of the initial volume, 5 µL in this case, can be prepared in the sample metering zone by closing valve #7 and the rest volume of the sample solution (5 µL) remains in the mixing chamber. Using the similar principle, 8 µL and 9 µL of the sample solution are transferred to the sample metering zone for 5X and 10X fold dilution by closing valve #8 or #9, respectively (Fig. 2a-i, 2b). Simultaneously, a buffer solution (light yellow colour in Fig 2a) is transferred to the chambers in the buffer metering zone by spinning at the condition of the open and closed states of the valve #1 and valve #5, respectively, as shown in Fig. 2a-i. Second, the metered sample solution is transferred to the sample reservoir and the excess amount of the buffer in the upper channel and the buffer loading chamber is discarded to the buffer waste chamber by opening the valve #5 (Fig. 2a-ii). It results in the metering of the buffer solution with 5 µL, 3 µL and 1 µL of volume, respectively. Finally, a buffer solution with the previously metered volume is transferred to the mixing chamber and mixed with the sample solution remaining in the mixing chamber. Depending upon the desired dilution ratio, released buffer volume from the buffer metering zone can be easily selected by the choice of the proper combination of the valve actuation steps. As shown in Fig. 2a-iii and Fig. 2b the buffer volume of 5 µL, 8 µL, and 9 µL could be selected by opening the valve #2 only, #2 and #3, all of #2, #3, and #4, respectively, to achieve 2X, 5X, and 10X dilution, respectively.
The same procedures given above for a single stage dilution can be repeated up to 3 additional times with an arbitral choice of the dilution factor among 2X, 5X, and 10X and the diluted solution at each stage can be stored in the corresponding sample reservoir sequentially (R1 ~ R5 as shown in Fig. 1a).
3.2 Automated serial dilution with constant dilution ratio To evaluate the accuracy of the serial dilution on the disc, we first tested the device with a fixed dilution ratio. Operation procedure during the four stages of 2X fold dilution was visualized and images are illustrated in Fig. 3. For the clear visualization purpose, food dyes of red colour and yellow colour were utilized as a sample solution and a buffer solution, respectively. The disc was rotated for 10 s at 3600 rpm for the pumping of solutions at each step. The alignment of the groove on the head of the ID valves indicate the open or closed state of the channel. If the groove is aligned in parallel to the radial direction, the valve is in open state. On the other hand, the valve is in the closed state if the groove is perpendicularly aligned to radial direction. The valves are operated by automatic actuation of the driver after disc is stop and positioned in the pre-programmed angle with respect to the specific origin. First, 10 µL of sample solution is injected in the sample loading chamber. Upon spinning the disc, the sample is transferred to the mixing chamber and the sample metering zone. 5 µL of solution is measured in the sample metering zone because the valve #7 was at closed state and the rest 5 µL of a sample solution remains in the mixing chamber. Also, buffer fills all the area of the buffer metering zone by spinning the disc at the open state of the valve #1 and the closed state of the valve #2, #3, #4, and #5 (Fig. 3a). Next, the metered 5 µL of a sample solution is transferred to the first sample reservoir (R1) by spinning the disc after closing valve #6 and opening the valves #7 and #10.
Therefore, 5 µL of the original
concentration of the sample stock is stored in the first sample reservoir (R1) as shown in Fig. 3b. The sample volume in the reservoir can be different depending on the dilution ratio, e.g. 8 µL and 9 µL for 5X and 10X dilution, respectively. In order to prepare the second round 2X dilution, 5 µL of buffer is transferred to the mixing chamber stored with remaining 5 µL of sample by opening the valve #2 to dilute the sample with 1:1 ratio as shown in Fig. 3c. In the centrifugal microfluidic device, efficient mixing could be easily achieved by utilizing high acceleration rate.[13-14] In this study, the following spinning program was used to expedite the mixing process. The unit module which takes total 3s is composed of an acceleration step (0.5s) to reach 3600 rpm,
spinning at 3600rpm (2s), and deceleration step (0.5s) to 0 rpm, was repeated for 6 times to achieve a complete mixing. Then, the buffer solution is refilled to the empty part of the buffer metering zone for the second step of the dilution by spinning the disc after opening valve #1 and at the closed state of valve #2, #3, #4, and #5. The excess amount of the buffer is removed to the waste chamber by spinning the disc after closing the valve #1 and opening the valve #5. Repeating the same protocol given in Fig. 3a, 5 µL of now 1:1 diluted solution is metered in the sample metering zone by transferring the solution by spinning the disc at the open and the closed state of the valve #6 and #7, respectively (Fig. 3d). Next, the metered 5 µL of the diluted sample solution is transferred to the second sample reservoir (R2) by opening #7, #11, #12 and closing #6 as shown in Fig. 3e. The similar dilution procedures are repeated 3 more times and the result is shown in Fig. 3f. Samples from C0 to 1/16 C0 is sequentially stored in the sample reservoirs from R1 to R5. The total time to prepare 5 samples with 2X fold serial dilution in a fully automated manner was less than 10 min and the details of the disc operation protocol for the fully automated operation is described in Table S1.
3.3 Fully automated serial dilution with an arbitral choice of the dilution factor among 2, 5, and 10 We next demonstrate a fully automated N-fold dilution with an arbitral choice of N among 2, 5, and 10 for each step utilizing the same disc design. As a proof of concept experiment, we diluted a sample stock solution with 5-, 10-, 2-, and 5-fold in each serial steps and the corresponding images of the disc are given in Fig. 4. Similar to the previous cases, metering of the sample and the buffer solution, removal of the excess buffer to the waste chamber and transfer of metered buffer to the mixing chamber stored with the remaining sample solution was performed in a sequential manner. As shown in Fig. 4a, 4b, valve #8 was closed to meter 8 µL of a sample solution and remaining 2 µL of sample solution was mixed with 8 µL of buffer solution transferred by opening of the valves #2 and #3 for the first 5X fold dilution. Subsequently, 10X (Fig. 4c,d), 2X (Fig. 4e,f), and 5X (Fig. 4g,h) dilution were performed by operating the corresponding valves with previously uploaded operation protocols. The detailed protocols are described in Table S2. As a result of the serial dilution with the arbitral choice of the dilution factors among 2-, 5-, and 10-fold, the diluted solutions with the concentrations of C0, 1/5 C0, 1/50 C0, 1/100 C0 and 1/500 C0 are stored from R1 to R5, respectively (Fig. 4i).
3.4 Performance evaluation with fluorescent dye solution The dilution accuracy and repeatability of the serial dilution disc were evaluated using the fluorescent solution (FITC, 1 mg/mL). 10 µL of FITC solution was injected into the sample loading chamber and an automated serial dilution was performed with a constant dilution factor, 2-, 5-, 10-fold, as shown in Fig. 5a, 5b, 5c, respectively. Furthermore, a serial dilution with N-fold dilution where the dilution factor could be freely selected from 2, 5, and 10 at each step of the dilution was performed as shown in Fig. 5d. Here, the dilution factor was 5-, 10-, 2-, and 5-fold to demonstrate the serial dilution with multiple kinds of dilution factors using the same disc design. The log-scale plot of each serial dilution showed excellent linearity. In the case of 10X dilution, dynamic range of the signals from the diluted samples was out of our measurement system and therefore samples from the first three steps were measured. From all of the experiments, the excellent correlation between the measured and estimated values with R2 >0.995 were demonstrated. It confirms that the automated serial dilution on the disc provides accurate and reliable performance.
3.5 Discussions The serial dilution which is one of the most common and important unit operation conducted in the laboratory has been integrated and automated on a centrifugal microfluidic system utilizing the robust and reversible ID valves. Compared to the conventional microfluidic devices which are often connected with several tubings and syringe pumps, the centrifugal microfluidic platform require only a single motor and relatively simple peripherals such as an automatic driver and the control system. The automatic control of the reversible operation of the ID valves were essential in the serial dilution process presented in this study. Though an additional external actuating system was necessary to operate the valves, it enabled accurate and repeatable operation. The facile control of the reversible valve actuation allows not only a constant but also arbitral choice of the dilution factor using a fixed design of the disc. In previous reports, most of the microfluidic chips for miniaturized serial dilution is designed for a fixed dilution ratio. The modification of channel dimension to control the flow resistance and re-fabrication of a device was prerequisite to adjust different dilution ratio. Here, various kinds of dilution factors could be selected and used with a fixed design of the disc. The current disc is designed for 2-, 5-, and 10-fold dilution. However, a simple modification of the chamber design could offer different dilution factors or more steps of serial dilution. In the current designs of the disc, the pipetting precision of the sample loading step is directly related to the accuracy of the automated serial dilution. To reduce the effect of the manual pipetting error, an
additional sample metering chamber could be added to the sample injection chamber so that the sample could be introduced without prior metering and the first portion of the sample could be also automatically measured and transferred to the next serial dilution steps. In addition, the metering precision is highly influenced by the manufacturing precision of the disc. In this study, the metering volume was determined by the channel dimension of the double adhesive tape which was prepared by a cutting plotter (repeatability precision: 100 µm) and the bonding with PC layers (CV% of the adhesive layer thickness after bonding: 5%). Therefore, in the worst case scenario, the metering channel designed for 1 µL, 1 mm width, 10 mm length, and 0.1 mm thickness, could have 10%, 1%, and 5% errors for the channel width, length, and thickness, respectively, which results in 12% total error. The precision could be further improved when more precise manufacturing methods such as injection molding and ultrasonic welding are employed for the large-scale mass production.
4. Conclusions Here, centrifugal microfluidic device for an automated arbitrary serial dilution is demonstrated. The ID valves were employed here to play an important role for the reversible valve actuation. Two metering zones are designed to transfer a metered volume of the sample and the dilution buffer easily selected from preprogrammed value; e.g. 5 µL, 8 µL, and 9 µL for 2-, 5-, and 10-fold dilution. The accuracy of the diluted samples prepared using the disc was excellent (R2 ≥ 0.970). Our device has intuitive design that can be easily modified to cover more various volume and concentration ranges. We expect this device to be powerful tool to perform efficient serial dilution by removing manual and mundane pipetting steps.
Acknowledgements This research was supported by a grant from IBS-R020-D1 and the Korean Health Technology R&D Project of the Ministry of Health & Welfare (A121994).
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Biographies Tae-Hyeong Kim received his Ph.D. in Biomedical Engineering from UNIST in 2014. Currently, he is a research fellow in the Center for Soft and Living Matter at the Institute for Basic Science (IBS), Republic of Korea. His research interests include development of centrifugal microfluidic devices for point of care tests, and fabrication of ultrasensitive sensors using nanomaterials.
Chi-Ju Kim received his BS in Nano-Bioscience and Chemical Engineering from UNIST in 2013 and he is a Ph.D. candidate in Biomedical Engineering from UNIST. His research interests cover novel microfluidic platforms for clinical diagnostics, and synthesis of nanomaterials for biological applications.
Yubin Kim received his BS in Nano-Bioscience and Chemical Engineering from UNIST in 2013 and he is a Ph.D. candidate in Chemical Engineering from UNIST. His research interests include lab-on-a-disc for pointof-care tests in environmental and biochemical applications.
Yoon-Kyoung Cho is a Professor in Biomedical Engineering at UNIST and a group leader in the Center for Soft and Living Matter at the Institute for Basic Science (IBS), Republic of Korea. She received her Ph.D. in Materials Science and Engineering from the University of Illinois at Urbana-Champaign in 1999, having obtained her M.S. and B.S. in Chemical Engineering from POSTECH in 1994 and 1992, respectively. She worked as a senior researcher (1999–2008) at Samsung Advanced Institute of Technology (SAIT), where she participated in the development of in vitro diagnostic devices for biomedical applications. Her research interests range from basic sciences to translational research in microfluidics and nanomedicine. Current research topics include a lab-on-a-disc for biomedical applications, quantitative analysis of single cells, and systems analysis of cellular communication.
Fig. 1. (a) A Photo image and the schematic diagram of the N-fold serial dilution disc. It consists of four chambers for sample loading, mixing, buffer loading, and buffer waste, two metering zones for the metering of the buffer and sample solution, five reservoirs (R1-R5) for diluted sample storage, and seventeen ID valves. Circles with light red colour indicate the positions of the ID valves. (b) Schematic diagram showing the operating principle of the ID valves. Diaphragm (Dark orange) is embedded on top layer and 3D printed valve actuator (Gray) manipulates the diaphragm to open or close the channel. Detail designs of the metering zones of the buffer (c) and sample (d) are shown. Each zone contains three sections with different volume (5 µL, 3 µL and 1 µL).
Fig. 2. (a) In order to achieve N-fold serial dilution with the arbitral choice of N=2, 5, and 10 using a single disc, a combination of the ID valves was actuated for the metering and transfer of previously programmed volume of the sample and the buffer. (i) Sample (red colour) and buffer (light yellow colour) is metered first at each metering zone. (ii) 5 µL, 8 µL, and 9 µL of the metered sample is transferred to the diluted sample reservoirs for 2X, 5X, and 10X dilution, respectively and the excess of buffer is removed to the buffer waste chamber. (iii) 5 µL, 8 µL, and 9 µL of the metered buffer solution is transferred to the mixing chamber containing 5 µL, 2 µL, and 1 µL of the sample for 2X, 5X, and 10X dilution, respectively. The arbitral dilution ratio could be selected among N=2, 5, and 10 at each steps of dilution by selecting the combination of the valve actuation. Green and orange colour circles with numbers indicate the open and closed state of the valves, respectively. The blue dot line illustrates flow path of each solution and red dot circle indicate the empty chamber in the buffer metering zone. (b) The summary of the metered volume of the sample and the buffer to achieve 2-, 5-, and 10-fold dilution and the corresponding state of the valves in the metering zone of the sample and buffer.
Fig. 3. Images taken during the operation of an automated serial dilution with constant dilution factor, N=2. (a) Spinning the disc when valve #7 is closed state can trap 5 µL of sample solution in the first metering section. At the same time, buffer is filled in the buffer metering zone because valve #1 is open. (b) Next spinning step after closing valve #6 and opening valve #7 and #10 moves 5 µL of sample solution to R1. At the same time, excess amount of buffer existing in upper part of buffer metering sections is removed because valve #1 and #5 are in closed and open state, respectively. (c) Spinning after opening valve #2 transfer 5 µL of buffer from the first buffer metering section to the mixing chamber preloaded with 5 µL of a sample solution resulting in 2X dilution. (d) Again, 5 µL of sample solution (2-fold diluted) is metered to perform the second round dilution by spinning after opening valve #6 and closing valve #7 while the buffer is refilled in the buffer metering section because valve #1 is changed to the open state again. (e) Similarly to the steps shown in (b), (c), the metered amount of sample solution, 5 µL, moves to R2 and desired volume of buffer, 5 µL, is mixed with remaining sample solution in the sample chamber. (f) These processes can be repeated to achieve four stages of 2X fold dilution and the serially diluted solution at each step with the concentration ranging from C0 to (1/2)4C0) is stored in diluted sample reservoirs from R1 to R5. Green colour and orange colour dot circles present open state and closed state of the ID valves, respectively.
Fig. 4. Images showing an automated arbitrary serial dilution on a disc with 5X, 10X, 2X, 5X fold dilution in each sequential steps. (a) 8 µL of a sample solution is metered by spinning the disc with the valve #8 in closed state, (b) the metered 8 µL of the sample is transferred to R1. The remaining 2 µL of sample solution is mixed with 8 µL of buffer (valves #2 and #3 are open) to do 5X dilution, (c) 9 µL of solution is metered to perform 10X dilution for the second round (valve #9 is closed), (d) 9 µL of 5X diluted sample is transferred to R2. 9 µL of buffer is transferred to the mixing chamber stored with the remaining 1 µL of the 5X diluted solution (valve #2, #3, and #4 are open to transfer 9 µL of buffer), (e) 5 µL of the 50 times diluted sample solution is metered (valve #7 is closed), (f) 5 µL of the 50 times diluted sample solution is aliquoted to R3. 2X dilution is performed by mixing with 5 µL of buffer (valve #2 is open to transfer 5 µL of buffer) (g) 8 µL of the 1/100 diluted sample solution is metered (valve #8 is closed), (h) The metered amount (8 µL) of the 1/100 diluted sample solution is transferred to R4. Finally, the remaining 1/100 diluted solution (2 µL) in the mixing chamber is diluted with 8 µL of the buffer (valve #2 and #3 are open). (i) the final solution of 500 times diluted solution is transferred to R5. Green and orange coloured circles illustrate the open and closed state of the valves, respectively.
Fig. 5. Log plots of fluorescent intensity at each step of the N-fold serial dilution. An automated serial dilution is performed on a disc with FITC solution and the samples at each reservoir are retrieved to measure the intensity. Five stepwise dilution with a constant dilution factor, (a) 2X, (b) 5X, and (c) 10X and (d) a choice arbitral N-fold dilution at each step of the serial dilution. As a proof of concept experiment, the stepwise dilution of 5-, 10-, 2-, 5-fold dilution was performed serially. The error bar is the standard deviation of three independent experiments conducted using three discs. Coefficient of determination (R2) is calculated from every run of experiment (n=3) and the worst value was presented in each figure.
S0925-4005(17)31998-6 https://doi.org/10.1016/j.snb.2017.10.096 SNB 23400
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
20-5-2017 14-9-2017 17-10-2017
Please cite this article as: Tae-Hyeong Kim, Chi-Ju Kim, Yubin Kim, Yoon-Kyoung Cho, Centrifugal Microfluidic System for a Fully Automated N-fold Serial Dilution, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.10.096 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Centrifugal Microfluidic System for a Fully Automated N-fold Serial Dilution Tae-Hyeong Kima, Chi-Ju Kima,b, Yubin Kimb, Yoon-Kyoung Choa,b1 a Center
for Soft and Living Matter, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
b Department
of Biomedical Engineering, School of Life Sciences, Ulsan National Institute of Science and Technology
(UNIST), Ulsan 44919, Republic of Korea
1
Corresponding author. Tel.: +82 52 217 2511; fax: +82 52 217 2509.
E-mail address: [email protected]
Tae-Hyeong Kima, Chi-Ju Kima,b, Yubin Kimb, Yoon-Kyoung Choa,b2 a Center
for Soft and Living Matter, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
b Department
of Biomedical Engineering, School of Life Sciences, Ulsan National Institute of Science and Technology
(UNIST), Ulsan 44919, Republic of Korea
Graphical abstract
N-fold serial dilution is fully automated on a centrifugal microfluidic system with a choice of N=2, 5, 10.
Highlights
Serial dilution is fully integrated and automated on a centrifugal microfluidic system.
Individually addressable, reversible diaphragm valves are utilized to enable the automatic control of the fluidic transfer and metering of the liquid.
N-fold serial dilution was performed automatically with a choice of N=2, 5, 10.
Abstract We present an innovative tool for a fully automated N-fold serial dilution with a choice of N=2, 5, 10 on a centrifugal microfluidic device. Individually addressable, reversible diaphragm valves are integrated on the disc for the automatic control of the fluidic transfer. Two metering zones are designed with three microfluidic chambers with the volume of 1 µL, 3 µL and 5 µL to meter the samples or the dilution buffer. The pre-programmed operation of the valves enabled the automatic serial dilution with the choice of the dilution factor of 2, 5, or 10 at each dilution steps. The samples prepared on the disc with various serial dilution factor showed excellent accuracy (R2 > 0.970). The automatic serial dilution disc could be an essential tool in the laboratory to replace the time-consuming and iterative pipetting especially when the sample volume is limited.
Keywords Centrifugal microfluidics; Serial dilution; Valves; Metering; Lab-on-a-disc
1. Introduction Serial dilution is one of the most common unit operations in biological and chemical experiments. Tedious and time-consuming manual pipetting is required to obtain the serially diluted solutions with desired concentration. In particular, accurate and precise pipetting of the solution is troublesome when the sample volume is in microliter scale or below. Microfluidics has facilitated the integration and automation of various fluidic unit operations on a miniaturized chip and it results in fast, accurate and highly efficient manipulation of fluids in microliter scale volume.[1-3] On-chip serial dilution with both linear and non-linear shape of the concentration
gradient have been also demonstrated.[4-9] However, most of the previous studies are to generate on-chip concentration gradient by diffusion between the adjacent channels filled with the flow of sample and buffer solution. The specific channel design depending upon the desired dilution ratio is required and the sample volume should be large enough to make the stable flow. New valving techniques enables versatile consecutive dilutions.[10-12] B. M. Paegel et al. introduced dilution circuit by utilizing the pneumatic valve on lamination structure of glass and PDMS layers.[10] This device allowed reversible flow control in circular loop and the dilution of the solution could be achieved on demand. However, only the final solution was possible to retrieve for the follow-up analysis and the intermediates had to be discarded. S. Ahrar et al. reported a serial dilution ladder to obtain all solution generated during the dilution process, however, only 1:1 dilution was possible.[12] Centrifugal microfluidic device is a microfluidic platform that utilizes the centrifugal force as a pumping source for the fluid transfer. Various kinds of unit operations such as pumping, mixing, metering, valving and switching of the fluid have been demonstrated.[13-19] Unique design of the fluidic networks and precise control of the rotation speed and spinning direction of the motor were demonstrated in order to integrate various biological and chemical assays on a disc.[15, 20-24] The previous effort to demonstrate on-disc generation of the dilution series without external active components could prepare 3- and 5-fold serial dilution from each specific design of the discs.[25] Adjustment of the spinning profile facilitates the fully automated preparation of the diluted samples. However, the repeatability was not excellent, which was attributed to the utilization of siphon as key unit operation and it could be critically influenced by surface properties and the tolerance of disc fabrication. In addition, other researches introduced systems for serial dilution by employing various additional components such as pneumatic pumping, dissolvable film and laser irradiated ferrowax microvalves (LIFM).[26-28] They enables a robust, batch-mode, serial dilution on a disc, however, only a fixed dilution ratio was possible using the specific design of the fluidic layout. In this paper, we present a fully automated centrifugal microfluidic chip for the N-fold serial dilution with the arbitral choice of N=2, 5, and 10. Here, we utilized recently reported, simple, reversible, and individually addressable diaphragm valves (ID valves) for the serial dilution on a disc. A single disc design can be used to offer several options for the dilution factor. The preloaded operation program can be used to perform automatic N-fold serial dilution with a good accuracy and repeatability.
2. Materials and methods 2.1 Disc design and fabrication Design of a disc for the serial dilution with the arbitral choice of dilution factor from N=2, 5, and 10 was conducted using 3D CAD software and milled with a CNC milling machine (smart 3530, PROTek, Korea). The pre-designed holes and chambers were milled on polycarbonate plates (polycarbonate, I-Components Co. Ltd, Korea). Two layers (top and bottom) were fabricated and they were laminated with a pressure sensitive adhesive layer (DFM 200 clear 150 POLY H-9V-95, FLEXcon, USA) which was prepared by a cutting plotter (Graphtec CE3000-60 MK2, 70 Graphtec Corporation, Japan) as previously reported.[29-30] Fig. 1a shows the detail design of the disc for N-fold serial dilution. It contains four storage chambers, two metering zones and five reservoirs for the storage of the diluted sample. Sample solution (10 µL) is loaded onto the sample loading chamber and transferred to the mixing chamber by spinning the disc. The buffer loading chamber contains buffer solution (100 µL) and about 15 µL of the buffer solution is transferred to the buffer metering zone when the disc is rotated when valve # 1 is in open and #2, #3, #4, and #5 are closed state. After metering 9 µL of the buffer solution, the excess amount of the buffer is discarded to the buffer waste chamber by spinning the disc after closing valve #1 and opening valve #5. Once the pre-programmed amount of the buffer is transferred to the mixing chamber by controlling valves #2, #3, and #4, the diluted sample is transferred to the diluted sample reservoirs (R1 ~ R5). The sequence of the sample transfer, buffer transfer, mixing, and transfer of the diluted sample is repeated until it fills the diluted sample reservoirs. The diluted sample could be retrieved for the downstream analysis by using a pipette through a hole prepared in each reservoir, which was kept sealed by an adhesive tape during the operation. In order to have the reversible actuation of the multiple valves, recently reported individually addressable diaphragm valve (ID valve) were utilized.[31] For the fabrication of the ID valves, elastic epoxy (Super-X, Cemedine, Japan) was inserted into grooves on the top layer of the disc in order to form a diaphragm. After curing the epoxy, 3D printed parts for the actuation of the valves were bonded on the top plate. Simple push and twist action allowed squeeze and release of the diaphragm to close and open the microfluidic channel on demand (Fig. 1b). Two metering zones were designed to trap and release the pre-programmed amount of solution in each micro-chambers as shown in Fig. 1c and Fig. 1d. Each metering zone consists of three sections with the chamber volume of 5 µL, 3 µL and 1 µL, respectively. The combination of the valve actuation allowed to
meter pre-determined volume of sample or buffer to prepare the N-fold serially diluted samples. ID valves allowed selective trap and release of the solution, which enables the arbitrary fold dilution on demand. The detail operation principle is described in the following sections.
2.2 Automatic actuation of multiple valves Operation of ID valve is actuated automatically using customized operating machine. Position of each ID valve is coordinated with radial position and angle with respect to the specific origin. Coordination is inserted to operating program and it recognizes the positions. To actuate the valve, spinning of a disc is stopped and angular position is aligned first depending on inserted coordination. Then, an automatic driver (KX050T2-01H1, Nitto Seiko Co., Japan) moves to the valve actuator by consecutive motion through x and z axes. Driver head is combined with groove on the top of ID valve and rotates it by 90° with clockwise direction to open the valve. Counter clockwise rotation can induce the close of valve and such operation can be conducted reversibly with automatic manner.
2.3 Operation of valve The automatic actuation of the multiple ID valves could be performed by using a customized disc operating machine.[31] The positions of each ID valves are coordinated with the radial position and the angle with respect to the specific origin. With the input of the coordination of the valves and the program for the actuation of the valves to open or close the valves, the operating system automatically perform the sequence of actuation. To actuate the valve, the disc is stopped, the angle is aligned, and the automatic driver (KX050T2-01H1, Nitto Seiko Co., Japan) aligns to the valve position utilizing two linear motors moving in x and z axes. The groove on the head of the ID valve can be selected from two positions in order to open or close the channels. The multiple actuation for the reversible control of the valves are fully automated.
2.4 Visualization of the fluid transfer on the spinning disc Images of the disc while it is rotating were acquired using the customized disc visualization module. A CCD camera (IK-TF5C, Toshiba Corp., Japan) and a strobe lamp (BUB0641-1A, B&B Corporation, Korea) were synchronized with the spinning speed and the series of images from specific position on a rotating disc were taken with a desired frame rate. The details of the visualization module have been previously described.[32]
3. Result and discussion 3.1 Principle of a fully automated serial dilution on a disc Two metering zones designed on the serial dilution disc play important roles for the arbitral choice of the dilution factor among N=2, 5, and 10. Fig. 2 illustrates the principle of the automated dilution process on the disc. The operation process for the dilution consists of three steps, i) metering of sample and buffer volume, ii) transfer of sample and remove of the excess amount of the buffer, and iii) transfer of the metered amount of the buffer to the mixing chamber and mix with the sample solution. We demonstrate the 5 serial steps of N-fold dilution with the arbitral choice of N from 2, 5, and 10 starting with 10 µL of the initial sample stock. First, the sample solution (red colour) is metered in the sample metering zone. Depending on the desired dilution ratio, metered volume to transfer and remaining volume of the sample can be adjusted by choosing the open or closed state of the ID valves of #7, #8, and #9. For example, in order to have 2X dilution, half of the initial volume, 5 µL in this case, can be prepared in the sample metering zone by closing valve #7 and the rest volume of the sample solution (5 µL) remains in the mixing chamber. Using the similar principle, 8 µL and 9 µL of the sample solution are transferred to the sample metering zone for 5X and 10X fold dilution by closing valve #8 or #9, respectively (Fig. 2a-i, 2b). Simultaneously, a buffer solution (light yellow colour in Fig 2a) is transferred to the chambers in the buffer metering zone by spinning at the condition of the open and closed states of the valve #1 and valve #5, respectively, as shown in Fig. 2a-i. Second, the metered sample solution is transferred to the sample reservoir and the excess amount of the buffer in the upper channel and the buffer loading chamber is discarded to the buffer waste chamber by opening the valve #5 (Fig. 2a-ii). It results in the metering of the buffer solution with 5 µL, 3 µL and 1 µL of volume, respectively. Finally, a buffer solution with the previously metered volume is transferred to the mixing chamber and mixed with the sample solution remaining in the mixing chamber. Depending upon the desired dilution ratio, released buffer volume from the buffer metering zone can be easily selected by the choice of the proper combination of the valve actuation steps. As shown in Fig. 2a-iii and Fig. 2b the buffer volume of 5 µL, 8 µL, and 9 µL could be selected by opening the valve #2 only, #2 and #3, all of #2, #3, and #4, respectively, to achieve 2X, 5X, and 10X dilution, respectively.
The same procedures given above for a single stage dilution can be repeated up to 3 additional times with an arbitral choice of the dilution factor among 2X, 5X, and 10X and the diluted solution at each stage can be stored in the corresponding sample reservoir sequentially (R1 ~ R5 as shown in Fig. 1a).
3.2 Automated serial dilution with constant dilution ratio To evaluate the accuracy of the serial dilution on the disc, we first tested the device with a fixed dilution ratio. Operation procedure during the four stages of 2X fold dilution was visualized and images are illustrated in Fig. 3. For the clear visualization purpose, food dyes of red colour and yellow colour were utilized as a sample solution and a buffer solution, respectively. The disc was rotated for 10 s at 3600 rpm for the pumping of solutions at each step. The alignment of the groove on the head of the ID valves indicate the open or closed state of the channel. If the groove is aligned in parallel to the radial direction, the valve is in open state. On the other hand, the valve is in the closed state if the groove is perpendicularly aligned to radial direction. The valves are operated by automatic actuation of the driver after disc is stop and positioned in the pre-programmed angle with respect to the specific origin. First, 10 µL of sample solution is injected in the sample loading chamber. Upon spinning the disc, the sample is transferred to the mixing chamber and the sample metering zone. 5 µL of solution is measured in the sample metering zone because the valve #7 was at closed state and the rest 5 µL of a sample solution remains in the mixing chamber. Also, buffer fills all the area of the buffer metering zone by spinning the disc at the open state of the valve #1 and the closed state of the valve #2, #3, #4, and #5 (Fig. 3a). Next, the metered 5 µL of a sample solution is transferred to the first sample reservoir (R1) by spinning the disc after closing valve #6 and opening the valves #7 and #10.
Therefore, 5 µL of the original
concentration of the sample stock is stored in the first sample reservoir (R1) as shown in Fig. 3b. The sample volume in the reservoir can be different depending on the dilution ratio, e.g. 8 µL and 9 µL for 5X and 10X dilution, respectively. In order to prepare the second round 2X dilution, 5 µL of buffer is transferred to the mixing chamber stored with remaining 5 µL of sample by opening the valve #2 to dilute the sample with 1:1 ratio as shown in Fig. 3c. In the centrifugal microfluidic device, efficient mixing could be easily achieved by utilizing high acceleration rate.[13-14] In this study, the following spinning program was used to expedite the mixing process. The unit module which takes total 3s is composed of an acceleration step (0.5s) to reach 3600 rpm,
spinning at 3600rpm (2s), and deceleration step (0.5s) to 0 rpm, was repeated for 6 times to achieve a complete mixing. Then, the buffer solution is refilled to the empty part of the buffer metering zone for the second step of the dilution by spinning the disc after opening valve #1 and at the closed state of valve #2, #3, #4, and #5. The excess amount of the buffer is removed to the waste chamber by spinning the disc after closing the valve #1 and opening the valve #5. Repeating the same protocol given in Fig. 3a, 5 µL of now 1:1 diluted solution is metered in the sample metering zone by transferring the solution by spinning the disc at the open and the closed state of the valve #6 and #7, respectively (Fig. 3d). Next, the metered 5 µL of the diluted sample solution is transferred to the second sample reservoir (R2) by opening #7, #11, #12 and closing #6 as shown in Fig. 3e. The similar dilution procedures are repeated 3 more times and the result is shown in Fig. 3f. Samples from C0 to 1/16 C0 is sequentially stored in the sample reservoirs from R1 to R5. The total time to prepare 5 samples with 2X fold serial dilution in a fully automated manner was less than 10 min and the details of the disc operation protocol for the fully automated operation is described in Table S1.
3.3 Fully automated serial dilution with an arbitral choice of the dilution factor among 2, 5, and 10 We next demonstrate a fully automated N-fold dilution with an arbitral choice of N among 2, 5, and 10 for each step utilizing the same disc design. As a proof of concept experiment, we diluted a sample stock solution with 5-, 10-, 2-, and 5-fold in each serial steps and the corresponding images of the disc are given in Fig. 4. Similar to the previous cases, metering of the sample and the buffer solution, removal of the excess buffer to the waste chamber and transfer of metered buffer to the mixing chamber stored with the remaining sample solution was performed in a sequential manner. As shown in Fig. 4a, 4b, valve #8 was closed to meter 8 µL of a sample solution and remaining 2 µL of sample solution was mixed with 8 µL of buffer solution transferred by opening of the valves #2 and #3 for the first 5X fold dilution. Subsequently, 10X (Fig. 4c,d), 2X (Fig. 4e,f), and 5X (Fig. 4g,h) dilution were performed by operating the corresponding valves with previously uploaded operation protocols. The detailed protocols are described in Table S2. As a result of the serial dilution with the arbitral choice of the dilution factors among 2-, 5-, and 10-fold, the diluted solutions with the concentrations of C0, 1/5 C0, 1/50 C0, 1/100 C0 and 1/500 C0 are stored from R1 to R5, respectively (Fig. 4i).
3.4 Performance evaluation with fluorescent dye solution The dilution accuracy and repeatability of the serial dilution disc were evaluated using the fluorescent solution (FITC, 1 mg/mL). 10 µL of FITC solution was injected into the sample loading chamber and an automated serial dilution was performed with a constant dilution factor, 2-, 5-, 10-fold, as shown in Fig. 5a, 5b, 5c, respectively. Furthermore, a serial dilution with N-fold dilution where the dilution factor could be freely selected from 2, 5, and 10 at each step of the dilution was performed as shown in Fig. 5d. Here, the dilution factor was 5-, 10-, 2-, and 5-fold to demonstrate the serial dilution with multiple kinds of dilution factors using the same disc design. The log-scale plot of each serial dilution showed excellent linearity. In the case of 10X dilution, dynamic range of the signals from the diluted samples was out of our measurement system and therefore samples from the first three steps were measured. From all of the experiments, the excellent correlation between the measured and estimated values with R2 >0.995 were demonstrated. It confirms that the automated serial dilution on the disc provides accurate and reliable performance.
3.5 Discussions The serial dilution which is one of the most common and important unit operation conducted in the laboratory has been integrated and automated on a centrifugal microfluidic system utilizing the robust and reversible ID valves. Compared to the conventional microfluidic devices which are often connected with several tubings and syringe pumps, the centrifugal microfluidic platform require only a single motor and relatively simple peripherals such as an automatic driver and the control system. The automatic control of the reversible operation of the ID valves were essential in the serial dilution process presented in this study. Though an additional external actuating system was necessary to operate the valves, it enabled accurate and repeatable operation. The facile control of the reversible valve actuation allows not only a constant but also arbitral choice of the dilution factor using a fixed design of the disc. In previous reports, most of the microfluidic chips for miniaturized serial dilution is designed for a fixed dilution ratio. The modification of channel dimension to control the flow resistance and re-fabrication of a device was prerequisite to adjust different dilution ratio. Here, various kinds of dilution factors could be selected and used with a fixed design of the disc. The current disc is designed for 2-, 5-, and 10-fold dilution. However, a simple modification of the chamber design could offer different dilution factors or more steps of serial dilution. In the current designs of the disc, the pipetting precision of the sample loading step is directly related to the accuracy of the automated serial dilution. To reduce the effect of the manual pipetting error, an
additional sample metering chamber could be added to the sample injection chamber so that the sample could be introduced without prior metering and the first portion of the sample could be also automatically measured and transferred to the next serial dilution steps. In addition, the metering precision is highly influenced by the manufacturing precision of the disc. In this study, the metering volume was determined by the channel dimension of the double adhesive tape which was prepared by a cutting plotter (repeatability precision: 100 µm) and the bonding with PC layers (CV% of the adhesive layer thickness after bonding: 5%). Therefore, in the worst case scenario, the metering channel designed for 1 µL, 1 mm width, 10 mm length, and 0.1 mm thickness, could have 10%, 1%, and 5% errors for the channel width, length, and thickness, respectively, which results in 12% total error. The precision could be further improved when more precise manufacturing methods such as injection molding and ultrasonic welding are employed for the large-scale mass production.
4. Conclusions Here, centrifugal microfluidic device for an automated arbitrary serial dilution is demonstrated. The ID valves were employed here to play an important role for the reversible valve actuation. Two metering zones are designed to transfer a metered volume of the sample and the dilution buffer easily selected from preprogrammed value; e.g. 5 µL, 8 µL, and 9 µL for 2-, 5-, and 10-fold dilution. The accuracy of the diluted samples prepared using the disc was excellent (R2 ≥ 0.970). Our device has intuitive design that can be easily modified to cover more various volume and concentration ranges. We expect this device to be powerful tool to perform efficient serial dilution by removing manual and mundane pipetting steps.
Acknowledgements This research was supported by a grant from IBS-R020-D1 and the Korean Health Technology R&D Project of the Ministry of Health & Welfare (A121994).
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Biographies Tae-Hyeong Kim received his Ph.D. in Biomedical Engineering from UNIST in 2014. Currently, he is a research fellow in the Center for Soft and Living Matter at the Institute for Basic Science (IBS), Republic of Korea. His research interests include development of centrifugal microfluidic devices for point of care tests, and fabrication of ultrasensitive sensors using nanomaterials.
Chi-Ju Kim received his BS in Nano-Bioscience and Chemical Engineering from UNIST in 2013 and he is a Ph.D. candidate in Biomedical Engineering from UNIST. His research interests cover novel microfluidic platforms for clinical diagnostics, and synthesis of nanomaterials for biological applications.
Yubin Kim received his BS in Nano-Bioscience and Chemical Engineering from UNIST in 2013 and he is a Ph.D. candidate in Chemical Engineering from UNIST. His research interests include lab-on-a-disc for pointof-care tests in environmental and biochemical applications.
Yoon-Kyoung Cho is a Professor in Biomedical Engineering at UNIST and a group leader in the Center for Soft and Living Matter at the Institute for Basic Science (IBS), Republic of Korea. She received her Ph.D. in Materials Science and Engineering from the University of Illinois at Urbana-Champaign in 1999, having obtained her M.S. and B.S. in Chemical Engineering from POSTECH in 1994 and 1992, respectively. She worked as a senior researcher (1999–2008) at Samsung Advanced Institute of Technology (SAIT), where she participated in the development of in vitro diagnostic devices for biomedical applications. Her research interests range from basic sciences to translational research in microfluidics and nanomedicine. Current research topics include a lab-on-a-disc for biomedical applications, quantitative analysis of single cells, and systems analysis of cellular communication.
Fig. 1. (a) A Photo image and the schematic diagram of the N-fold serial dilution disc. It consists of four chambers for sample loading, mixing, buffer loading, and buffer waste, two metering zones for the metering of the buffer and sample solution, five reservoirs (R1-R5) for diluted sample storage, and seventeen ID valves. Circles with light red colour indicate the positions of the ID valves. (b) Schematic diagram showing the operating principle of the ID valves. Diaphragm (Dark orange) is embedded on top layer and 3D printed valve actuator (Gray) manipulates the diaphragm to open or close the channel. Detail designs of the metering zones of the buffer (c) and sample (d) are shown. Each zone contains three sections with different volume (5 µL, 3 µL and 1 µL).
Fig. 2. (a) In order to achieve N-fold serial dilution with the arbitral choice of N=2, 5, and 10 using a single disc, a combination of the ID valves was actuated for the metering and transfer of previously programmed volume of the sample and the buffer. (i) Sample (red colour) and buffer (light yellow colour) is metered first at each metering zone. (ii) 5 µL, 8 µL, and 9 µL of the metered sample is transferred to the diluted sample reservoirs for 2X, 5X, and 10X dilution, respectively and the excess of buffer is removed to the buffer waste chamber. (iii) 5 µL, 8 µL, and 9 µL of the metered buffer solution is transferred to the mixing chamber containing 5 µL, 2 µL, and 1 µL of the sample for 2X, 5X, and 10X dilution, respectively. The arbitral dilution ratio could be selected among N=2, 5, and 10 at each steps of dilution by selecting the combination of the valve actuation. Green and orange colour circles with numbers indicate the open and closed state of the valves, respectively. The blue dot line illustrates flow path of each solution and red dot circle indicate the empty chamber in the buffer metering zone. (b) The summary of the metered volume of the sample and the buffer to achieve 2-, 5-, and 10-fold dilution and the corresponding state of the valves in the metering zone of the sample and buffer.
Fig. 3. Images taken during the operation of an automated serial dilution with constant dilution factor, N=2. (a) Spinning the disc when valve #7 is closed state can trap 5 µL of sample solution in the first metering section. At the same time, buffer is filled in the buffer metering zone because valve #1 is open. (b) Next spinning step after closing valve #6 and opening valve #7 and #10 moves 5 µL of sample solution to R1. At the same time, excess amount of buffer existing in upper part of buffer metering sections is removed because valve #1 and #5 are in closed and open state, respectively. (c) Spinning after opening valve #2 transfer 5 µL of buffer from the first buffer metering section to the mixing chamber preloaded with 5 µL of a sample solution resulting in 2X dilution. (d) Again, 5 µL of sample solution (2-fold diluted) is metered to perform the second round dilution by spinning after opening valve #6 and closing valve #7 while the buffer is refilled in the buffer metering section because valve #1 is changed to the open state again. (e) Similarly to the steps shown in (b), (c), the metered amount of sample solution, 5 µL, moves to R2 and desired volume of buffer, 5 µL, is mixed with remaining sample solution in the sample chamber. (f) These processes can be repeated to achieve four stages of 2X fold dilution and the serially diluted solution at each step with the concentration ranging from C0 to (1/2)4C0) is stored in diluted sample reservoirs from R1 to R5. Green colour and orange colour dot circles present open state and closed state of the ID valves, respectively.
Fig. 4. Images showing an automated arbitrary serial dilution on a disc with 5X, 10X, 2X, 5X fold dilution in each sequential steps. (a) 8 µL of a sample solution is metered by spinning the disc with the valve #8 in closed state, (b) the metered 8 µL of the sample is transferred to R1. The remaining 2 µL of sample solution is mixed with 8 µL of buffer (valves #2 and #3 are open) to do 5X dilution, (c) 9 µL of solution is metered to perform 10X dilution for the second round (valve #9 is closed), (d) 9 µL of 5X diluted sample is transferred to R2. 9 µL of buffer is transferred to the mixing chamber stored with the remaining 1 µL of the 5X diluted solution (valve #2, #3, and #4 are open to transfer 9 µL of buffer), (e) 5 µL of the 50 times diluted sample solution is metered (valve #7 is closed), (f) 5 µL of the 50 times diluted sample solution is aliquoted to R3. 2X dilution is performed by mixing with 5 µL of buffer (valve #2 is open to transfer 5 µL of buffer) (g) 8 µL of the 1/100 diluted sample solution is metered (valve #8 is closed), (h) The metered amount (8 µL) of the 1/100 diluted sample solution is transferred to R4. Finally, the remaining 1/100 diluted solution (2 µL) in the mixing chamber is diluted with 8 µL of the buffer (valve #2 and #3 are open). (i) the final solution of 500 times diluted solution is transferred to R5. Green and orange coloured circles illustrate the open and closed state of the valves, respectively.
Fig. 5. Log plots of fluorescent intensity at each step of the N-fold serial dilution. An automated serial dilution is performed on a disc with FITC solution and the samples at each reservoir are retrieved to measure the intensity. Five stepwise dilution with a constant dilution factor, (a) 2X, (b) 5X, and (c) 10X and (d) a choice arbitral N-fold dilution at each step of the serial dilution. As a proof of concept experiment, the stepwise dilution of 5-, 10-, 2-, 5-fold dilution was performed serially. The error bar is the standard deviation of three independent experiments conducted using three discs. Coefficient of determination (R2) is calculated from every run of experiment (n=3) and the worst value was presented in each figure.