Centrifugation-Controlled Thermal Convection and Its Application to

Nov 7, 2017 - (3) It has been highly desired to develop a point of care testing (POCT) tool, which can detect the DNA of a biotic target with fast res...
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Centrifugation-Controlled Thermal Convection and Its Application to Rapid Microfluidic Polymerase Chain Reaction Devices Masato Saito,*,† Kazuya Takahashi,† Yuichiro Kiriyama,† Wilfred Villariza Espulgar,† Hiroshi Aso,‡ Tadanobu Sekiya,‡ Yoshikazu Tanaka,‡ Tsuneo Sawazumi,‡ Satoshi Furui,§ and Eiichi Tamiya† †

Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita-shi, Osaka 565-0871, Japan ‡ Konica Minolta, Inc., JP Tower, 2-7-2 Marunouchi, Chiyoda-ku, Tokyo 100-7015, Japan § Food Entomology Unit, Food Research Institute, NARO, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan S Supporting Information *

ABSTRACT: Here, we report the developed cyclo olefin polymer (COP) microfluidic chip on a fabricated rotating heater stage that utilizes centrifugation-assisted thermal cycle in a ring-structured microchannel for polymerase chain reaction (PCR). The PCR solution could be driven by thermal convection and continuously exchanged high/low temperatures in a ring structured microchannel without the use of typical syringe pump. More importantly, the flow rate was controlled by the relative gravitational acceleration only. The platform enables amplification within 10 min at 5G and has a detection limit of 70.5 pg/channel DNA concentration (βactin, 295 bp). The current rotating system is capable of testing four different samples in parallel. The microfluidic chip can be preloaded with the PCR premix solution for on-site utility, and, with all of the features integrated to the system, the test can be conducted without the need for specialized laboratory and trained laboratory staff. In addition, this innovative chemical reaction technique has the potential to be utilized in other micromixing applications. The μ-TAS (Micro Total Analysis System) technique, which was proposed by A. Manz,6 is extremely useful for the miniaturization of devices. In a microchip device, fast reaction and high sensitivity from rapid thermal conduction and prevention of diffusion in a micro area are advantageous for PCR applications; in addition, minimization and simplification of the device are expected.7 A high-speed fluidic PCR by A. Manz8 was achieved by flowing the PCR solution in the micro channel placed on three heater plates having the exchange temperatures set as required for PCR. Similarly, we have also reported a rapid DNA amplification on a PDMS microfluidic chip.9,10 From the simulated thermal conduction in the microchannel fluid, a distance of approximately 50 μm is needed by the solution to reach the required temperature for PCR. Taking this into account, the rapid DNA amplification from a new strain of influenza within 10 min was successfully accomplished with good sensitivity. Still, these devices are not practical for on-site use because they require syringe and/or

S

ince the establishment of the polymerase chain reaction (PCR) by K. B. Mullis and co-workers in 1985,1,2 this DNA amplification method has assumed an important role not only in basic biological research but also in a wide range of areas (i.e., food, agriculture, medical diagnostics, forensic science, etc.). In principle, PCR can amplify the specific DNA fragment in an exponential manner from a single copy of DNA template, which promotes its high sensitivity that is ideal for assimilation with other sensing technology.3 It has been highly desired to develop a point of care testing (POCT) tool, which can detect the DNA of a biotic target with fast response, high sensitivity, and ease of use in on-site utility in cases for interdiction of pest infested agricultural export or import products at the border, for prevention of food poisoning in processed food factory and deterrence of manufacturing malpractices, for early detection of infectious disease in medical zone, for daily monitoring of health conditions and prevention of disease contagion, and for other cases where rapid turnaround of test results for critical decision making is needed related to public health.4,5 However, the PCR technique still has many issues for the on-site application, which conventionally requires complicated sample preparations, adept and trained users for operation, and long reaction time. © XXXX American Chemical Society

Received: August 3, 2017 Accepted: November 7, 2017 Published: November 7, 2017 A

DOI: 10.1021/acs.analchem.7b03107 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry pump for flowing the solution aside from the need for complex sample preparation. The thermal convection PCR, which was pioneered by M. Krishnan et al.,11−13 is performed in a thin cylinder structure where thermal convection was generated by heating from the bottom and cooling at the top at optimal temperatures. The miniaturization of the device system promotes the reduced required volume and quicker speed of temperature change without using a Peltier device. Calculation and analytical evaluation for elucidating thermal conduction and convection were done extensively.12,13 Additionally, further works had been done in multiple cylinder devices12 and in a microfluidic device.14,15 This natural convection known as Rayleigh−Bénard convection is governed by the Boussinesq equation. This was utilized in the study by replacing the gravity term to relative gravity, G, and read as ∂u 1 + (u·∇)u = − ∇p + v∇2 u − Gα(T − Tr) ∂t ρr

(1)

∂T + (u·∇)T = k∇2 T ∂t

(2)

∇·u = 0

(3)

where u is the velocity field, ρr is the standard density, p is the reference pressure, v is the kinematic viscosity, κ is the body expansion coefficient, α is the thermal diffusivity, and Tr is the reference temperature. By altering the relative gravity acceleration, it is thought that the thermal exchange speed can be controlled in further improving the PCR reaction time. A centrifugal microfluidic chip and a rotating heater stage as shown in Figure 1 were developed from this idea where the control of flow can be done by simply changing the rotation speed. A ring-structured microchannel chip was optimized on the basis of the balance between thermal cycle speed and DNA amplification. The chip has the feature of containing the PCR solution for later injection and mixing of DNA sample solution into the microchannel when the chip is spun. This simplifies the sample preparation and handling, which is beneficial for on-site testing. In addition, Coriolis force, that occurred under the effect of centrifugation,16 is effective in mixing the reaction solution, which could further improve the reaction time in this centrifugal-assisted thermal convection PCR device. This report discloses the optimization of the device operation, verification of the PCR amplification, and the elucidation of the flow dynamics in the microchannel that will be beneficial in realizing a platform for on-site use and developing this technology that can be utilized in other chemical reaction applications in a micro environment.

Figure 1. Schematic diagram of centrifugal-assisted thermal convection PCR and devices. (a) Schematic illustration of centrifugal-assisted thermal convection PCR (from top view). The ring-structured microchannel was placed on the two heaters, which were required for PCR, one at 95 °C for DNA denaturation and another one at 60 °C for annealing and DNA polymerization. This temperature difference has another important role to generate the thermal convection under spin. (b) Microfluidic chip design and fabricated COP chip. (c) Device configuration of centrifugal heater stage with temperature control and fabricated device. Chip stage head has two temperature heater blocks, which have built-in ceramic heaters in each, and are connected to a rotor. (d) Microfluidic chip on the heater stage. Ring structured microchannel is aligned on two heaters. (e) Chip is secured on the stage by a spring from a customized chip holder. This holder has neodymium magnet at the center and was fixed at the central shaft of the heater stage.

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EXPERIMENTAL SECTION Device Design. Figure 1 illustrates the PCR chip and the developed rotating heater stage. The PCR system needs two heat sources, one at 95 °C for DNA denaturation and another one at 60 °C for annealing and DNA polymerization. Separated block heater was placed at the same level on the stage. The ring-structured microchannel was placed on the two heaters as shown in Figure 1a. This generates the temperature difference needed for thermal convection. The chip and the heat sources were spun with a rotor, and the optimal speed for ideal increment of thermal convection speed was determined. Chip Fabrication. Microfluidic PCR chips were designed using AutoCAD2011 (AUTODESK) as shown in Figure 1b.

The ring-structured microchannel has 6 mm diameter, 500 μm width, and 300 μm depth, and the chip thickness was 2 mm. The chip is made of cyclo olefin polymer (COP) resin, which has low hygroscopicity, low self-fluorescence, high permeability, and good formability. For prototyping, the PCR chips were fabricated by NC micromilling from COP plate (Zeonor B

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rotational speed of the chip and the convection flow speed was calculated. Initially, 2.35 uL of the PCR mixture was filled into the ring structured microchannel. The PCR solution containing New Coccine dye (Wako Pure Chemical Industries, Ltd., Japan) was added into microchannel. The chip was incubated on the device for 30 s with optimal PCR temperature, and the chip was then spun at various rotation speed and observed under a high-speed camera. On-Chip DNA Amplification by Centrifugal Thermal Convection. The PCR mixture for on-chip PCR consisted of 1x Ampdirect Plus (241-08800-98, Shimadzu, Japan), 0.25 U/ μL SpeedSTAR HS DNA polymerase (RR070B, Takara Bio, Japan), 300 nM β-actin forward, reverse primers, 200 nM probe (FAM(5′)-TAMRATM(3′)) (TaqMan β-actin Detection Reagents, PN 401846, Applied Biosystems, U.S.), 0.1% (w/v) bovine serum albumin (SHIGMA Aldrich, U.S.), 0.01% (v/v) polyvinylpyrrolidone (Sigma Aldrich, U.S.), and human genomic DNA (PN 401846, Applied Biosystems, U.S., and Z6401N, Takara Bio, Japan). A concentration of 300 pg/uL of human genomic DNA concentration was used for all experiments, unless otherwise specified. For evaluation of the standard curve, 0−3 ng/chip reaction of human genomic DNA was applied. A volume of 2.35 μL PCR mixture was introduced into the PCR channel by centrifugation. Afterward, 2.35 μL of mineral oil (M5904-5 ML, Sigma) was added that stayed at the inlet of the PCR channel, which prevents the evaporation of the PCR mixture. Optical System for Measurement of Fluorescence with DNA Amplification. Figure S2 shows the optical setup to measure the fluorescence increments with DNA amplification. A 488 nm laser (emitting power 0.5 mW, OBIS488-30FP, Coherent Inc., U.S.) was applied to excite FAM probe in the PCR microchannel using an optics system consisting of objective lenses (EPL-5, SIGMAKOKI), a dichroic filter (67004-L, Edmund Optics), a fluorescence filter (67004-L, Edmund Optics), a plane-convex lens (SLB-25-30PM, SIGMA KOKI), and a condenser lense (EPL-5, SIGMAKOKI). The fluorescence signal at 520 nm wavelength was observed using a photomultiplier (PMT, operating voltage 0.65 V, H10720-110, Hamamatsu Photonics). The laser was scanned at the microchannel at 100 μm/s using an automatic positioning stage (SGSP26-100, SIGMAKOKI). The fluorescence signals from PMT were recorded at a rate of 20 Hz by a data logger (TR-V550, KEYENCE). Gel Shift Assay. After on-chip PCR, the amplified DNA was also estimated by gel electrophoresis analysis. A small hole was made on the ring-structured microchannel by micro drilling, and the PCR solution was extracted out by a micro pipet. A volume of 1 μL amplicon with loading buffer was applied into 4% NuSieve 3:1, which contained EtBr for DNA staining, and run at 100 V in the TBE running buffer. As standard DNA marker, 100 bp DNA ladder (BioRAD), was used. Simulation Analysis. Behaviors of the centrifugationassisted thermal convection in the microchannel were calculated using COMSOL Multiphysics Navier−Stokes and heat transfer modules (COMSOL AB, Sweden). A 3D structure of microchannel chip and heaters for simulation was drawn at actual size. Material of the microchannel chip and aqueous phase were COP (Zeonex 480R, Zeon Corp., Japan) and water, respectively. In the Navier−Stokes equation, water was assumed as incompressible aqueous with Boussinesq approximation. Condition of flow velocity at the microchannel wall

1020R, ZEON Co., Japan), which was outsourced to Konica Minolta, Inc. (Tokyo, Japan). The surface roughness (Ra) in the microchannel wall surface was less than 30 nm. Depth range of 100−400 μm of different microchannels was prepared and tested. After testing, the optimized PCR chips were fabricated by injection molding of COP (Zeonex 480, ZEON Co., Japan), which was outsourced to Nissei Technology Corp. (Kobe, Japan). This method enables the easy mass production of the chip. Molded COP chip was bonded to COP film (188 μm in thickness, ZF14-188, ZEON Co., Japan) by thermal compression.17 The molded COP chip and the COP film were cleaned with ultrasonic bath in IPA for 5 min and dried by blowing N2 gas. Contacting surfaces of the chips and the films were treated by oxygen plasma (RDC210, Yamato Scientific) and activated. Treatment conditions of O2 gas, RF time, and RF power were 100 cm3, 10 s, and 75 W, respectively. The surfaces were adhered to one another and placed in the thermal compression apparatus (SCiVAX, X300) for firm bonding. The press was operated at 133 °C with 1 MPa of press pressure in a −100 kPa vacuum for 30 min. The actual channel depth after thermal compression bonding was 100, 200, 250, 300, and 400 μm each for micromilling and was 250 μm for injection molding. Four fabricated chips can be set into the applicator as shown in Figure 1b. Inside walls of the microchannel were coated with 0.01% polyethylene glycol (PEG, molecular weight 20 000, 164-13801, Wako) diluted in distilled water for blocking and to promote a hydrophilic surface. After bonding the COP chip with the COP film, 0.01% PEG solution was introduced into the chip by centrifugation. Next, the chip was placed onto a hot plate at 100 °C to evaporate the water. This hydrolysis by coating also prevents generation of bubbles during PCR thermal cycling that can obstruct the flow. Centrifugal and Thermal Device. Figure 1c shows the arrangement of two heater blocks for maintaining the temperature. Two ceramic heater elements were inserted and affixed to each heater block. Thermocouples were also affixed with each heater block. These ceramic heater elements and thermocouples were connected to a thermal regulator (SCRSHQ-A, Sakaguchi E.H. VOC Corp., Japan), and the temperature was controlled with PID feedback control. Four protrusions on each heater block serve as contact plates for individual chips. These heater components were rotated by a DC motor (BLM230-A2, Oriental motor, Co., Ltd., Japan) and controlled by a speed control unit (NexBL US type, Oriental motor Co., Ltd., Japan). This heater unit can simultaneously operate four PCR chips. The PCR chips were pressed to the heater surface using contact probes. To evaluate the temperature of the ring-structured microchannel, thermometers (model 427-4 for 60 °C, model 427-5 for 95 °C, KN Laboratories Inc., Japan) were placed at the chip bottom, and this was set onto the heaters. Figure S1 shows the temperature distribution in the microchannel. Each heater temperature was confirmed to reach the required temperatures for denaturing and annealing/elongation. Thus, heater temperatures can be controlled at any desired value for other PCR conditions. Observations for Centrifugal-Assisted Thermal Convection. The motion of convection flow during centrifugation was observed by using a high speed camera (HX-3, NAC, Japan), which can take a shot of the same spot of the stage by synchronizing with a trigger signal. Obtained shots were reconstructed at real time motion, and the relationship between C

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10G was 45.9, 11.4, and 6.2 s/cycle, respectively. These findings show that the velocity of centrifugal convection can certainly be controlled by varying the relative gravitational acceleration. Additionally, we estimated the motion of thermal convection in this micro channel by a simulation model (Figure 2b), which is coherent with the experimental results. In addition, no thermal convection and unidirectional flow was observed at 10G without thermal control as shown in Figure S4. The effect of microchannel depth for convection velocity was investigated. The convection motions at 5G were observed in the chips with depths of 116−350 μm (Figure 2c). A faster low speed was observed in deeper microchannels; the flow cycle of 350 μm channel depth was 8.2 s/cycle, while the 116 μm depth was 53.3 s/cycle. Here, specific surface areas (surface area/ volume) of the chips with depths of 116 and 400 μm were 21.2 and 9.7 m−1, respectively. This decrement of velocity was thought to be caused by the increased friction between channel surface and solution with the increment of the specific surface area and by the increased hydraulic resistance with the decrease in cross-sectional area of the channel.19 Behavior of the Fluid under the Centrifugal Thermal Convection. Here, the Reynolds number in the centrifugal convection at 5G is 1.69, and is in the intermediate range flow (∼1 < Re < ∼100); therefore, inertial-effect-induced secondary flow cannot be ignored.20 Dean flow, which is a well-known secondary flow, induced by centrifugal force in curving channel, can be expected as the origin of secondary flow here. Faster flow in the center of the channel has a larger momentum directed outward, which produces two vortices in the lower and upper halves of the channel. The direction of Dean flow in the center of the channel is always from the center of the circular channel to the outside and not depending on the direction of angular velocity of the stage. Coriolis force is an inertial force when an object moves in a rotating system. The expression of Coriolis force per volume is as follows: Fc = −2ρω × v. In our device, angular velocity (ω) is perpendicular to centrifugal convection flow, so that Coriolisinduced secondary flow can be generated even if the fluid moves parallel to the direction of the spinning stage. The direction of Coriolis force in the device depends only on the angular vector of the stage because the direction of centrifugal convection does not change. Therefore, the direction of Coriolis-induced secondary flow in the center of the channel can be expected toward the inner periphery of ring-structured channel when the stage is rotated counterclockwise, and vice versa. Figure S3c illustrates the forces considered on the moving fluid in addition to the 3D model of the chip. Simulation of centrifugal convection applying two inertial forces, Dean force and Coriolis force, showed that vortices of secondary flow were generated in centrifugal convection (Figure 3a). These vortices were reproduced even if only Coriolis force was applied (Figure 3b); however, this pattern disappeared when only the Dean force was applied and when both were disregarded in the simulation (Figure 3c,d). When the opposite angular vector of the stage was applied in the simulation, the direction of secondary flow was reversed. Close inspection in Figure 3f,g of the motion of the flow front of the fluid in centrifugal convection shows that the position depended on the direction of the angular vector (clockwise and counterclockwise). The flow front got close to the inner side of the microchannel in counterclockwise rotation and to the outer side of microchannel in clockwise rotation. From these results, it was indicative that the Coriolis force

was set to “no slip”. High and low temperatures of the heaters were set at 95 and 60 °C, respectively. The basic equations for Bénard convection were solved at steady state and at various relative gravitational accelerations. The effect of gravity force for z axis was neglected. For further detailed analysis of centrifugal convection, secondary flow in the system was also simulated. Coriolis force and Dean force were applied to simulation of centrifugal convection for investigating the origin of secondary flow.18 Figure S3 shows the 3D model utilized for the simulation. The Coriolis force and the Dean force per volume related to the drag assumed for the x axis were 2ρωv and ρu2/x, and for the y axis were −2ρωu and ρv2/y, respectively (u and v are the components of the fluid velocity).

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RESULTS AND DISCUSSION Centrifugal Thermal Convection in the Microfluidic Device. To improve the reaction speed in the microfluidic PCR, control of thermal convection is necessary. To confirm that the thermal convection speed changed along with gravity acceleration change, the 250-μm-deep microchannel was operated on the rotating heater stage at various G values. For generating thermal convection in the microchannel, temperature-controlled heater stages were integrated as shown in Figure S1. The flow motion of the PCR solution with New Coccine dye (red color) was observed. As can be seen in Figure 2a and Supporting Information video 1, the speed of centrifugal convection increased with the relative gravitational acceleration. The flow velocity at the time for one flow cycle of 1G, 5G, and

Figure 2. Thermal convection in the microchannel under centrifugation with temperature control. (a) Motion of fluid at the various relative gravitational acceleration G was observed by high speed camera. (b) Cycle time in the ring-structured microchannel was plotted at various G values obtained from experimental results (diamond) and from simulation (square), respectively. (c) The relation between microchannel depth and fluid speed for on cycle at 5G. D

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However, the chip with a depth of 400 μm has an absence of the specific band indicating that no reaction for the specific DNA occurred. Figure 4c shows the temperature plots in one cycle in the microchannel at different angular positions based on the simulation. In the 400 μm depth, the temperature difference between the top and the bottom of the channel in the hot region was 1.6 °C. This depth-dependent inhomogeneous temperature at the same position of the PCR channel was attributed to the insufficient heat transfer in the deeper channel. The maximum temperature in the center of the 400 μm-deep-microchannel was 88.6 °C, which is 3 °C lower than that of the 250 μm-deep-microchannel. This temperature is lower than the usually prescribed temperature for denaturation to occur. In addition, the minimum temperatures in the center of 400 μm-deep-microchannel were 61.2 °C, which is 0.6 °C lower than that of the 250 μm-deep-microchannel. This depthdependent temperature inhomogeneous and divergence from the temperature of heater stage are the causes for a nonspecific amplification in the 400 μm-deep-microchannel at 5G. For these reasons, the 250 μm depth of microchannel was set as the optimum depth for the current device design. Relative Gravitational Acceleration versus PCR Amplification. To estimate the effect of various fluid velocity for DNA amplification, on-chip PCR was conducted at various relative gravitational accelerations (3−10G) for 10 min using the 250 μm-deep-microchannel. As shown in Figure 4d, the maximum fluorescence intensity after DNA amplification was observed at 5G. Amplicon band of β-actin (295bp) was also confirmed by gel electrophoresis, which verifies that the maximum amplification occurred at 5G (Figure 4e). The number of cycles by centrifugal convection at 5G for 10 min was approximately 58 (one flow cycle in ring-structured microchannel took 10.2 s/cycle, Figure 2b). At 3G, the number of cycles was approximately 33 (flow speed was calculated 17.7 s/cycle from curve fitting of experimental data). This insufficient number of PCR cycles resulted in the low fluorescence and the low amplified band. By contrast, the cycles at 10G were approximately 89 cycles; however, the amplification was lower than at 5G. Nonspecifically, a band under 100bp, which is amplified when the extension time is too short, was observed in the gel electrophoresis at the amplicon of 10G. This nonspecific amplification suggests that the cycle time of 6.7 s/cycle was too fast to conduct on-chip PCR reaction (e.g., annealing and extension). Moreover, the thermal distribution was analyzed by calculation (Figure 4f). The temperature difference between the heater and the microchannel center (125 μm deep) increases with the increment of relative gravitational acceleration. At 5G, the maximum and the minimum temperatures in the center of the channel were 91.6 and 60.6 °C, respectively. The computed maximum temperature in the center of the channel run at 10G is 1.4 °C lower than that at 5G. Moreover, the area where the fluid is at low temperature became narrower with increasing relative gravitational acceleration. Thus, no PCR amplification at 10G was attributed to not only the insufficient reaction time by high convection speed but also to the temperature divergence from ideal PCR condition. From these results, 5G was set as the optimum centrifugal condition for the reaction of β-act gene in the current device. Reaction Time versus PCR Amplification PCR. The time course of amplification reaction was estimated at 5G for 5−20 min (Figure 4g) using the 250 μm-deep-microchannel. The fluorescence intensity by DNA amplification was first observed

Figure 3. Simulation analysis of centrifugal thermal convection with consideration of secondary flow. Calculation was done with combinations of external force, that is, Dean force and Coriolis force. Cross-sectional view indicates fluid velocity along with microchannel direction (rainbow color), stream line (black line), and second flow vector (white arrow) perpendicular to microchannel direction: (a) with Dean and Coriolis force under clock rotation, (b) with Coriolis force, (c) with Dean force, (d) without external force. (e) The effect of rotation direction of stage for the fluid, anticlockwise rotation with consideration of Coriolis force was compared. Experimental results were observed with (f) clock rotation and (g) anticlock rotation.

generated the secondary flow in addition to centrifugal convection along the microchannel. Velocity of this secondary flow was approximately 100 μm/s. This Coriolis-induced secondary flow is expected to enhance the passive mixing of fluids in the microchannel, which is beneficial for PCR applications. Optimization for Centrifugal Thermal Convective PCR Amplification. The effect of channel depth to the chip PCR amplification was then tested. Centrifugal thermal convection PCR in-chip was conducted using the chips with a depth of 250, 300, and 400 μm, and the device was set at 5G for 15 min. A 295 bp DNA in the b-ACT gene21 was targeted and amplified from the human genomic DNA. The increment of fluorescence intensity was taken by scanning on the micro channel before and after PCR amplification (see device configuration for fluorescence detection in Figure S2). As shown in Figure 4a, the 250 μm depth chip has the maximum observed fluorescence with DNA amplification. The fluorescence intensity decreased with the increment of channel depth. Amplified DNA was confirmed by gel shift assay using 1 μL of reacted solution extracted from the chip. Specific DNA band of 295 bp amplicon was identified in 250 and 300 μm depth chip (Figure 4b). E

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Figure 4. Optimization for centrifugal-assisted thermal convection PCR. (a) Microchannel depth dependency for amplification reaction. Fluorescence intensity on microchannel measured from before and after reaction. (b) Gel shift assay for amplicons. Reacted solution was extracted from the chip and applied to electrophoresis. (c) Temperature distribution in various depths of ring-structured microchannel simulated and plotted per angular position. (d) Relative gravitational acceleration G dependency for amplification reaction in 250 μm depth chip. (e) Gel shift assay for amplicons. (f) Temperature distribution at various G values in ring-structured microchannel simulated and plotted per angular position. (g) Reaction time dependency for amplification. (h) Gel shift assay for amplicons.

ng/μL) was performed at 5G for 10 min using the 250 μmdeep-microchannel. Figure 5a shows the calibration curve of the centrifugal convective PCR. Increments of fluorescence intensities were clearly obtained from 70.5, 233, 705, and 7050 pg/channel. The gel electrophoresis confirmed the specific amplifications of β-actin (295bp) at 70.5−7050 pg/ channel (Figure 5b). The fluorescence and the specific amplified band increased with the increased concentration of DNA, and the detection limit was 70.5 pg/channel. With this condition, the human genomic DNA, which is 3.7 pg/ genome,22 could correspond to 19 genome/channel. These

at 10 min and reached the plateau by 15 min. The gel electrophoresis confirmed the specific amplifications of β-actin (295bp) at on-chip PCR for 10, 15, and 20 min (Figure 4h) in accordance with the result of fluorescence intensity. At on-chip PCR for 5 min, no fluorescence increase and no specific amplified band were observed. It was determined that the number of cycles at 5G for 5 min was 26, which was insufficient for DNA amplification. Sensitivity of Centrifugal Thermal Convective PCR and Application. To estimate the sensitivity, on-chip PCR from several diluted human genomic DNA samples (0.1−100 F

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the DNA sample was successfully performed using the centrifugal-assisted thermal convection PCR by simply injecting the sample solution and spinning the device. This result suggests that our device has the capability for on-site usage application.

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CONCLUSIONS The optimum parameters for the operation of the COP microfluidic chip on a rotating heater stage for PCR application were determined by empirical tests and verified by simulation and gel shift assay. A reaction time of 10 min with relative gravitational acceleration of 5G using the 250 μm-deepmicrochannel appeared to be the best condition for the current chip design. The utility for on-site testing of device was successfully demonstrated, which will allow tests to be conducted without the need of specialized laboratory. In addition, centrifugation is a common practice in general chemical and biological laboratories, and thus operation of the device is expected to pose minimal problem. Also, injection molding technique allows the easy mass production of the microfluidic chip. With the features of the developed device, a POCT PCR tool has been realized. Another significant contribution of this study is the introduction of the centrifugal-assisted thermal convection technique. This technique can be utilized in other micromixing applications for chemical reactions in confined volumes. Further exploitation of this technique can give fine-tuning of the temperature gradient control across the microchannel and modify the flow rate depending on the desired outcomes.

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ASSOCIATED CONTENT

* Supporting Information S

Figure 5. Performance of centrifugal-assisted thermal convection PCR and application. (a) Calibration curve of on-chip PCR for β-actin gene from initial human genomic DNA. (b) Gel shift assay of amplicons. (c) Operation scheme for on-site utility of PCR test and handling with rapid response. (d) On-chip sample mixing and amplification from the buccal cell solution without pretreatment needed. (e) Gel shift assay for amplicon.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b03107. Movie showing the speed of centrifugal convection increasing with the relative gravitational acceleration (MPG) Temperature distribution on the chip (Figure S1), fluorescence measurement system (Figure S2), 3D model and simulation of mixing (Figure S3), and flow motion without thermal gradient (Figure S4) (PDF)

results confirmed that the rapid DNA amplification in our PCR system was achievable and with high sensitivity. For on-site utility, convenience in PCR handling with rapid response is highly desired. Figure 5c illustrates the operation of the device using human buccal samples collected by cotton swabs. The PCR chips could already contain the prepared premix reaction solution without the template DNA in advance and be stored for future use. A volume of 1.69 μL of PCR premix solution without template DNA and 2.35 μL of mineral oil (M5904-5 ML, Sigma) for preventing evaporation of solution was filled inside the ring-structured microchannel in advance. A volume of 0.66 μL of buccal cell solution was injected into the PCR chip. The sample solution is transferred to the ring-structured microchannel by displacing the mineral oil when the chip is spun for 10 s. Afterward, the sample solution and PCR premix solution were mixed by centrifugal thermal convection at 5G for 30 s. The fluorescence was measured before amplification reaction to serve as background intensity reference, and then the on-chip amplification for βactin DNA was subsequently carried out for 9.5 min. Pure water without template DNA was used as negative control. After the reaction, the increment of fluorescence intensity and specific DNA band were clearly observed for the chip containing buccal cell (Figure 5d,e). Mixing and amplifying

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AUTHOR INFORMATION

Corresponding Author

*Fax: +81-6-6879-7840. E-mail: [email protected]. ac.jp. ORCID

Masato Saito: 0000-0003-4817-474X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was sponsored in part by the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry, Grants-in-Aid for Scientific Research (Kiban S, no. 15H05769).

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REFERENCES

(1) Mullis, K.; Saiki, R.; Horn, G.; Erlich, H. Cold Spring Harbor Symp. Quant. Biol. 1986, 51, 263−273.

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DOI: 10.1021/acs.analchem.7b03107 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.7b03107 Anal. Chem. XXXX, XXX, XXX−XXX