- Email: [email protected]
Valve-based microfluidic droplet micromixer and mercury (II) ion detection
Sensors and Actuators A 172 (2011) 546–551
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators A: Physical journal homepage: www.elsevier.com/locate/sna
Valve-based microfluidic droplet micromixer and mercury (II) ion detection Zhi-Xiao Guo 1 , Qian Zeng 1 , Meng Zhang, Long-Ye Hong, Yang-Feng Zhao, Wei Liu ∗ , Shi-Shang Guo ∗ , Xing-Zhong Zhao ∗ Key Laboratory of Artificial Micro- and Nano-structures of Ministry of Education and School of Physics and Technology, Wuhan University, Wuhan 430072, PR China
a r t i c l e
i n f o
Article history: Received 22 March 2011 Received in revised form 6 July 2011 Accepted 14 September 2011 Available online 19 September 2011 Keywords: Microfluidic Droplet Valve Micromixer
a b s t r a c t A valve-based microfluidic micromixer was developed for multiply component droplets generation, manipulation and active mixing. By integrating pneumatic valves in microfluidic device, droplets could be individually generated, merged and well mixed automatically. Moreover, droplet volume could be controlled precisely by tuning loading pressure or the flow rate of the oil phase, and certain droplets fusion conditions were also investigated by adjusting the droplet driving times and oil flow rates. In these optimized conditions, fluorescence enhancement of droplets was used to detect Hg (II) ions in droplet by mixing with probe droplets (Rhodamine B quenched by gold nanoparticle). This method would have powerful potential for tiny volume sample assay or real-time chemical reaction study. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The use of microfluidic device or microreactor to investigate chemical and biological reaction emerges great practical advantages to conventional methods [1–3]. Particularly, the droplet microfluidic technology, with characters such as economic sample consumption, high-throughput production, minimal cross-contamination, and adaption to different kinds of detections, arouses more and more interest for nanoliter or picoliter chemical or biological reagents handling and chemical reactions controlling [4–6]. Several studies about droplet microfluidics were developed to generate uniform droplet in regular sequences or manipulate them in passive or active ways [7–9]. For passive droplet manipulation, well designed microfluidic device with structured channels were developed to achieve various functions like droplet fusion, breaking, trapping and sorting [10–13]. Moreover, passive droplet actuators was applied widely for high throughput screening of gene assays, enzyme kinetic study, protein crystallization, cell assay and drug discovery according to certain capabilities of the droplets [14,15]. However, in order to control the individual droplet more accurately and enlarge the scale of parallelization of chemical or biological assay in microfluidic devices, active droplet manipulation technology was still of great need. So far, a variety of external actuators based on
optical, magnetic, acoustic, electric methods were integrated into microfluidic channel for effective and precise controlling of the movements of emulsion droplets [16–18]. Specially, mechanical droplet manipulation methods attract much attention for their robust operation and adaption to large scale integration [19–21]. For example, Abate et al. adopted mechanical methods to control emulsion flow or vary the size and the generation frequency of emulsion droplets by single side wall in single layer chips [22,23]. Zeng et al. demonstrated a mechanical valve actuated system for individual droplet generation and manipulation [24]. Here, we reported a valve-based microfluidic device with functions such as individual droplet generation, active mixing and trapping. The tiny volume reagents could be used to be encapsulated into the individual water-in-oil droplet with controllable volume. Then the droplets produced from different droplet generator could be actuated to merge together and with uniform mixing efficiency. Using this automatic microfluidic droplet micromixer, Hg (II) ion or its detection probe (Rhodamine B quenched by gold nanoparticle) was individually encapsulated in droplets, merged together and then well mixed. As these two kinds of droplets mixed together, the fluorescence intensity increased. The enhancement was investigated as an evidence to identify Hg (II) ions [25]. 2. Materials and methods 2.1. Chip fabrication
∗ Corresponding authors. E-mail addresses: [email protected] (W. Liu), [email protected] (S.-S. Guo), [email protected] (X.-Z. Zhao). 1 These two authors contribute equally to this work. 0924-4247/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.sna.2011.09.019
The microfluidic device was fabricated by two-layer polydimethylsiloxane (PDMS) chip production technical practices [18]. First of all, the silicon wafer with photoresist patterns for fluidic
Z.-X. Guo et al. / Sensors and Actuators A 172 (2011) 546–551
channels was prepared by conventional photolithography method. The photoresist (AZ50TX, Clariant Corporation, USA) pattern was spin-coated at 1200 rpm for 30 s, which produced a uniform photoresist film of about 37 m in thickness. In order to pattern microfluidic channels network, photoresist layer was exposed by UV light through a mask, and the exposed region was washed out using a commercial developer solution (Clariant Corporation, USA). In addition, to form a semicircle shape for the well sealing of the valves, this pattern was also posited in the 120 ◦ C hot plate for 10 min. For the fabrication of the pneumatic control layer, the silicon wafer was patterned with negative photoresist (SU8 2050, Electronic Materials) of 40 m in thickness using a similar way like the liquid layer. All silicon wafers with photoresist patterns were modified in trimethoxychlorosilane (TMCS, Aldrich) vapor for easy separation of PDMS molds from the photoresist patterned substrate [26,27]. PDMS (GE RTV615) processor A and crosslink B were well-mixed at ratio 5:1, degassed, and poured onto the fluid layer silicon wafer to form a thick fluidic layer. Another portion of PDMS with processor A and crosslink B, ratio 20:1, was mixed and then spin-coated on the control layer silicon wafer at 1400 rpm for 60 s. The fluidic and control layers PDMS mode were cured at 80 ◦ C for 20 min, aligned and then bonded by baking at 80 ◦ C over night. Holes were punched onto the designed pattern to form ports connected to reagents or oil inlets and outlets in the fluidic layer, and to create inlets of the pneumatic control channels in the control layer. Then the two layers PDMS mode was bonded to the glass substrate through oxygen plasma treatment. Then microfluidic device was baked at 80 ◦ C oven for one day to make PDMS surface of the liquid channel hydrophobic [28]. 2.2. Materials Soybean oil (Beiya Medical Oil Co. Ltd., China) or the mineral oil (Sigma, USA) with 0.5% Span 80 (China National Medicines Co. Ltd., China) was used as the continuous phase. Then the food dye or reagent solution was preloaded into the PTFE microbore tubing (Cole-parmer, USA) for on chip operation. Mercury bichloride (HgCl2 ) (China National Medicines Co. Ltd., China) was dissolved into the DI water to form a 100 mM solution for further experiment. Gold nanoparticle was synthesized by traditional Frens method [29]. Typically, 10 mL 38.8 mM Trisodium citrate (China National Medicines Co. Ltd., China) solution was added to 100 mL 1 mM boiling HAuCl4 solution (Shanghai Qiangshun Chemical Reagents Co. Ltd., China), stirred for 15 min. When the solution was cooled to room temperature, the as-prepared gold nanoparticles (AuNPs) could be used directly in further steps. Borate saline buffer with pH 9.0 was used to dilute AuNPs to proper concentration. 4 L Rhodamine B (RB) (1 mM) was added into the 10 mL 6 nM AuNPs solution and the mixture was stirred for 2 h, after that 0.5 mL 20 mM 2,6-pyridinedicarboxylic acid (PDCA) was mixed with this solution. This solution was used as the detection probe of Hg (II) ions in the next step on chip detection [28]. 2.3. On chip operation 2.3.1. Experiment The valves integrated into microfluidic device (Fig. 1) were filled with deionized water and actuated by 50 psi compressed air through corresponding control channel and regulated by electronic solenoid valves (Series S070, SMC, Japan) in our home-made pneumatic control system. Those valves were automatically controlled through a data acquisition module (USB-4750, Advantech, USA) driven by a custom software programmed in LabView (National Instruments, USA). Through these pneumatic valves, the fluidic channel was opened or closed as a desire loop for reagents handling. The oil flow was continuously injected into the
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Fig. 1. Schematic picture of valve-based microfluidic device design, including three parts: droplet generators, active fusion units and mixing channel. Under control of pneumatic valves (with green color), all the reagents were loaded and operated in the fluidic network of oil and reagents inlets in liquid layer (with blue color). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
main channel by syringe pumps (Harvard Apparatus, USA). The food dye solutions or the reagents were loaded into the liquid channel of the chip through PTFE microbore tubing, connected via short stainless steel tubes under precise pressure regulator (Model 83100, Porter Instrument Co. Ltd., USA). The whole droplet operation process was observed and recorded by an inverted fluorescence microscope (IX71, Olympus, Japan) coupled with a color CCD (DP72, Olympus, Japan) or using a CCD camera (BigC Dino-Lite). 2.3.2. Droplet generation The single droplet generation unit includes reagent loading channel, main chamber and three control microvalves V1, V2 and V3 in Fig. 2. “T” junction geometry under the control of three pneumatic microvalves in the aqueous flow channel and main oil flow channel was invented for droplet production and operation. The process of water-in-oil droplet generation in this automatic microfluidic device was shown in Fig. 2. In the whole operation, oil flow was injected into main channel with a certain flow rate. In the starting step (S1), initializing, with all valves sealed, the oil flow stopped in the main chamber and waited for reagent injection in time T1. In the second step (S2), reagent loading, the reagent was pushed into the main chamber under certain pressure within a controllable loading time T2, while V2 opened. In the third step (S3), droplet generating, droplet was produced and trapped in the main chamber for further observation or operation when all the valves were sealed in time T3. In the last step (S4), droplet driving, the droplet was driven away by the oil flow to next droplet generation unit or to the mixing channel in driving time T4. 2.3.3. Droplets fusion Once two droplet generators integrated as Fig. 3(a) shown, the second one could also serve as the droplet fusion unit, as the schematic picture described in Fig. 3(b). At the first step of the circle (S1), with all the valves closed, the oil flow stopped and the DI water flow and blue food dye flow were all sealed in the reagent channels.
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Fig. 2. (a) Droplet generation process, containing four main steps: step 1 (S1) initializing with V1 to V3 closed (C); step 2 (S2) reagent loading into the main chamber with V1 and V3 closed, V2 open (O); step 3 (S3) droplet generating with V1–V3 closed; step 4 (S4) droplet transporting with V1 and V3 open, V2 closed. (b) The droplet volume depended on the loading pressures at different carrying oil flow rates (40 l/h, 50 l/h and 60 l/h).
However, DI water droplet formed in the last circle was trapped in the region where the second droplet would be produced on the optimized condition as Fig. 3(b) shown. In the step 2 (S2), with V2 and V4 opened and other valves closed, the DI water flow and the blue food dye flow were individually loaded into the main chamber under certain loading pressures within a controllable loading time T2. In the step 3 (S3), when all the valves were sealed, DI water droplet was produced in the first droplet generation unit, and the blue food dye droplet was formed at the very location where the last DI droplet stayed so that it merged with DI water droplet directly. In the step 4 (S4), with V2 and V4 closed, V1, V3, and V5 opened, the merged droplet was driven by oil flow to mixing channel. At the same time, the DI water droplet, produced by the first generation unit, was driven a certain distance to the second droplet generation unit. When the next circle started, this DI water droplet was trapped in position as the Fig. 3(b) shown and ready for the fusion process. 3. Result and discussion
loading pressure and the flow rate of the oil phase were the main variables to manipulate droplet volume. For instance, droplet generation process was fixed with initialization step time 50 ms, loading step time, 50 ms, droplet generating step time 50 ms, and driving time 500 ms. On this condition, the droplet volume dependency to the loading pressure and the oil flow rate was investigated as Fig. 2(b) shown. Considering the geometrical parameters of the microchannel and the size of droplets, the droplets were confined to be disk-like or plug-like shape in the main channel. Therefore, the volume of droplets could be expressed and calculated by V = AH, where A was the area of the droplet and H was the height of the main channel. Obviously, the droplet volume increased when turning the loading pressure up from 14 psi to 24 psi, and the droplet volume was almost linear to the loading pressure, as Fig. 2(b) shown. Moreover, once using the same loading pressure, droplet volume decreased when the flow rate of the oil phase rising from 40 l/h to 60 l/h. This tendency would be caused by the pressure enhancement inside the microfluidic channels with increasing oil flow rate. However, the coefficient of the droplet volume to the loading pressure was kept around 0.160–0.169 with the oil flow rare variation.
3.1. Droplet volume 3.2. Droplet micromixer For further operation or assay, well controlling of the volume of the droplets is extremely important, relevant factors were considered and studied, such as the loading pressure, the flow rate of oil, and separate operation time of the steps. When all the droplets were produced in the same automatically controlled period, the
After obtaining the experimental data of the single component droplet generation, the two component droplet generation units were not only used for two individual droplet generation but also employed as active droplet micromixer. Two individual droplets
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Fig. 3. (a) Schematic of two component droplets micro-mixer. (b) The droplets mixing process in the second droplet generation unit: step 1 (S1) droplet trapping with V3–V4 closed (C); step 2 (S2) other reagent loading into the main chamber with V3 and V5 closed, V4 open (O); step 3 (S3) second droplet generating and merging with V3–V5 closed; step 4 (S4) merged droplet transporting and mixing by the oil flow with V3 and V5 open, V4 closed.
with different components could be merged together through the automatic microfluidic operation. The mechanism of the mixing requires accurate controlling of the movements of the droplets, typically, the fusion efficiency of the droplets was determined by both the position where the droplet generated by the first unit was trapped in last circle and the position where the other droplet was produced by the second unit in present circle. Therefore, the flow rate of the oil and the driving time which affected the action of the droplets would be the critical factors for the two component droplet fusion. With constant operation time of the initializing, reagent loading, droplet generating 100 ms, 100 ms and 100 ms, respectively and each droplet volume almost unchanged (1–1.3 nl), the droplet fusion condition was optimized by adjusting the flow
rate of the oil phase and the droplet driving time (T4) of each period as the curve shown in Fig. 4(d). Under oil flow rate of 30 l/h, those two component droplets were merged together when the driving time was 1550 ms. Then the droplets were well mixed through the long and structured channel as Fig. 5(a) described. The optical signal was calculated by subtracting the droplet light absorption intensity from the background light absorption intensity. Fig. 5(b) was used to demonstrate the good mixing efficiency of the droplet through the mixing channel. While the driving time got shorter or longer, 1350 ms or 1900 ms, respectively, the droplet with blue food dye could not catch up with the red one in the former case or would go further than the second droplet could active reach and mix in the latter case, as Fig. 4(a)–(c) shown.
Fig. 4. (a)–(c) Droplets behavior with separate driving time 1350 ms, 1550 ms and 1900 ms under the oil flow rate of 30 l/h, with the other three steps unchanged. (d) Droplet fusion condition about the oil flow rate and driving time, with confined droplet sizes (volume 1–1.3 nl).
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Fig. 5. (a) The merged droplet (D1–D5) were transporting in the mixing channel. (b) The optical intensity analysis of the merging droplet from D1 to D5 in the mixing channel with same mixing ratio.
Fig. 6. Droplet-based Hg (II) detection: (a) Fluorescence image of Hg (II) ion droplet in mineral oil flow with 0.5% Span 80. (b) Fluorescence image of probe (RB quenched by modified AuNPs) droplet in mineral oil flow with 0.5% Span 80. (c) Fluorescence image of mixed droplet of Hg (II) ion and RB quenched by AuNPs in the mineral oil flow with 0.5% Span 80 (scale bar 100 m).
3.3. Hg (II) ion detection
4. Conclusion
With the droplet generation and fusion condition optimized, this device with multiple component droplet generation and active mixing function was used to test mercury ions through the fluorescence enhancement which was induced by free fluorescence dye releasing from AuNPs-RB probes, when the probes were exposed to Hg (II) ions [28]. The Hg (II) solution and the detection probe solution was loaded into two loading channel separately under the automatic control of the pneumatic valves, and the channels was pretreated by 0.5% BSA solution for 30 min to inhibit reagent adsorption. On the beginning, reagents were encapsulated into droplets and driven by mineral oil with 0.5% Span 80. Then fluorescence images of the Hg (II) droplet and probe droplet were taken and shown as Fig. 6(a) and (b) with a very weak fluorescent intensity on the same exposure time. However, once the 100 mM Hg (II) ion droplet and the probe droplet (RB quenched by AuNPs) were mixed together, the fluorescence brightness in the droplet region was clearly enhanced although RB molecular could diffuse in the oil phase (Fig. 6(c)). Obviously, this kind of multi-component droplet generator and micromixer could be used to detect various chemical and biological samples in microfluidic systems.
In conclusion, we have developed a microfluidic device integrated precise liquid handling functional units for multiple components droplets generation and individual droplets automatic fusion, which could be used for Hg (II) ion detection. Using this droplet handling device, the tiny volume sample could be encapsulated into the nl or pl droplet and automatic operated to react with other reagents. This device also containing three or more reagents inlets could be used for assay of two or more individual samples and could be developed for further multiple sample detection for the chemistry, biology or bioengineering study. The reagent droplets could be mixed together with required concentration or specific ratio. The complicated biochemistry condition screen could be realized in this enlarged scale droplet micro-mixer. With the characters of sophisticated liquid handling, this platform would be valuable for clinical diagnosis or environmental test with tiny volume samples and extremely expensive probes. Acknowledgements This work was partially supported by China National Funds for Distinguished Young Scientists (Grant No. 50125309), National
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Biographies Zhi-Xiao Guo received her Bachelor’s degree in 2005 in College of Life Science at Wuhan University. Now she is pursuing her PhD degree in School of Physics and Technology at Wuhan University. Her interest focuses on droplet-based microfluidics. Qian Zeng received his MSc from Wuhan University in 2007. Currently, he is preparing his PhD in School of Physics and Technology at Wuhan University. His research interests involve the integration of Surface Acoustic Wave (SAW) elements in microfluidic device and its application. Meng Zhang finished her undergraduate study at School of Physics and Technology, Wuhan University in 2009, and now she is a graduate student at Wuhan University. Her research interest focuses on the design and fabrication of smart material and novel digital microfluidic device. Long-Ye Hong got his undergraduate study at School of Physics and Technology, Wuhan University in 2010, and now he is a graduate student at Wuhan University. His research interest is microfluidic device. Yang-Feng Zhao is an undergraduate student at School of Physics and Technology, Wuhan University. He focuses on droplet microfluidics. Wei Liu received his PhD degree in physics at Wuhan University in 2008 and presently is a lecturer in School of Physics and Technology at Wuhan University. His field of interest is nanomaterials and Lab on a Chip. Shi-Shang Guo received his PhD degree in physics at Wuhan University in 2004 and presently is an associate professor in School of Physics and Technology at Wuhan University. His field of interest is Acoustic microfluidics and sensors. Xing-Zhong Zhao received his PhD in physics at University of Science and Technology of Beijing in China in 1989 and presently is a professor in School of Physics and Technology at Wuhan University. His current fields of interest are Lab on a Chip.
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators A: Physical journal homepage: www.elsevier.com/locate/sna
Valve-based microfluidic droplet micromixer and mercury (II) ion detection Zhi-Xiao Guo 1 , Qian Zeng 1 , Meng Zhang, Long-Ye Hong, Yang-Feng Zhao, Wei Liu ∗ , Shi-Shang Guo ∗ , Xing-Zhong Zhao ∗ Key Laboratory of Artificial Micro- and Nano-structures of Ministry of Education and School of Physics and Technology, Wuhan University, Wuhan 430072, PR China
a r t i c l e
i n f o
Article history: Received 22 March 2011 Received in revised form 6 July 2011 Accepted 14 September 2011 Available online 19 September 2011 Keywords: Microfluidic Droplet Valve Micromixer
a b s t r a c t A valve-based microfluidic micromixer was developed for multiply component droplets generation, manipulation and active mixing. By integrating pneumatic valves in microfluidic device, droplets could be individually generated, merged and well mixed automatically. Moreover, droplet volume could be controlled precisely by tuning loading pressure or the flow rate of the oil phase, and certain droplets fusion conditions were also investigated by adjusting the droplet driving times and oil flow rates. In these optimized conditions, fluorescence enhancement of droplets was used to detect Hg (II) ions in droplet by mixing with probe droplets (Rhodamine B quenched by gold nanoparticle). This method would have powerful potential for tiny volume sample assay or real-time chemical reaction study. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The use of microfluidic device or microreactor to investigate chemical and biological reaction emerges great practical advantages to conventional methods [1–3]. Particularly, the droplet microfluidic technology, with characters such as economic sample consumption, high-throughput production, minimal cross-contamination, and adaption to different kinds of detections, arouses more and more interest for nanoliter or picoliter chemical or biological reagents handling and chemical reactions controlling [4–6]. Several studies about droplet microfluidics were developed to generate uniform droplet in regular sequences or manipulate them in passive or active ways [7–9]. For passive droplet manipulation, well designed microfluidic device with structured channels were developed to achieve various functions like droplet fusion, breaking, trapping and sorting [10–13]. Moreover, passive droplet actuators was applied widely for high throughput screening of gene assays, enzyme kinetic study, protein crystallization, cell assay and drug discovery according to certain capabilities of the droplets [14,15]. However, in order to control the individual droplet more accurately and enlarge the scale of parallelization of chemical or biological assay in microfluidic devices, active droplet manipulation technology was still of great need. So far, a variety of external actuators based on
optical, magnetic, acoustic, electric methods were integrated into microfluidic channel for effective and precise controlling of the movements of emulsion droplets [16–18]. Specially, mechanical droplet manipulation methods attract much attention for their robust operation and adaption to large scale integration [19–21]. For example, Abate et al. adopted mechanical methods to control emulsion flow or vary the size and the generation frequency of emulsion droplets by single side wall in single layer chips [22,23]. Zeng et al. demonstrated a mechanical valve actuated system for individual droplet generation and manipulation [24]. Here, we reported a valve-based microfluidic device with functions such as individual droplet generation, active mixing and trapping. The tiny volume reagents could be used to be encapsulated into the individual water-in-oil droplet with controllable volume. Then the droplets produced from different droplet generator could be actuated to merge together and with uniform mixing efficiency. Using this automatic microfluidic droplet micromixer, Hg (II) ion or its detection probe (Rhodamine B quenched by gold nanoparticle) was individually encapsulated in droplets, merged together and then well mixed. As these two kinds of droplets mixed together, the fluorescence intensity increased. The enhancement was investigated as an evidence to identify Hg (II) ions [25]. 2. Materials and methods 2.1. Chip fabrication
∗ Corresponding authors. E-mail addresses: [email protected] (W. Liu), [email protected] (S.-S. Guo), [email protected] (X.-Z. Zhao). 1 These two authors contribute equally to this work. 0924-4247/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.sna.2011.09.019
The microfluidic device was fabricated by two-layer polydimethylsiloxane (PDMS) chip production technical practices [18]. First of all, the silicon wafer with photoresist patterns for fluidic
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channels was prepared by conventional photolithography method. The photoresist (AZ50TX, Clariant Corporation, USA) pattern was spin-coated at 1200 rpm for 30 s, which produced a uniform photoresist film of about 37 m in thickness. In order to pattern microfluidic channels network, photoresist layer was exposed by UV light through a mask, and the exposed region was washed out using a commercial developer solution (Clariant Corporation, USA). In addition, to form a semicircle shape for the well sealing of the valves, this pattern was also posited in the 120 ◦ C hot plate for 10 min. For the fabrication of the pneumatic control layer, the silicon wafer was patterned with negative photoresist (SU8 2050, Electronic Materials) of 40 m in thickness using a similar way like the liquid layer. All silicon wafers with photoresist patterns were modified in trimethoxychlorosilane (TMCS, Aldrich) vapor for easy separation of PDMS molds from the photoresist patterned substrate [26,27]. PDMS (GE RTV615) processor A and crosslink B were well-mixed at ratio 5:1, degassed, and poured onto the fluid layer silicon wafer to form a thick fluidic layer. Another portion of PDMS with processor A and crosslink B, ratio 20:1, was mixed and then spin-coated on the control layer silicon wafer at 1400 rpm for 60 s. The fluidic and control layers PDMS mode were cured at 80 ◦ C for 20 min, aligned and then bonded by baking at 80 ◦ C over night. Holes were punched onto the designed pattern to form ports connected to reagents or oil inlets and outlets in the fluidic layer, and to create inlets of the pneumatic control channels in the control layer. Then the two layers PDMS mode was bonded to the glass substrate through oxygen plasma treatment. Then microfluidic device was baked at 80 ◦ C oven for one day to make PDMS surface of the liquid channel hydrophobic [28]. 2.2. Materials Soybean oil (Beiya Medical Oil Co. Ltd., China) or the mineral oil (Sigma, USA) with 0.5% Span 80 (China National Medicines Co. Ltd., China) was used as the continuous phase. Then the food dye or reagent solution was preloaded into the PTFE microbore tubing (Cole-parmer, USA) for on chip operation. Mercury bichloride (HgCl2 ) (China National Medicines Co. Ltd., China) was dissolved into the DI water to form a 100 mM solution for further experiment. Gold nanoparticle was synthesized by traditional Frens method [29]. Typically, 10 mL 38.8 mM Trisodium citrate (China National Medicines Co. Ltd., China) solution was added to 100 mL 1 mM boiling HAuCl4 solution (Shanghai Qiangshun Chemical Reagents Co. Ltd., China), stirred for 15 min. When the solution was cooled to room temperature, the as-prepared gold nanoparticles (AuNPs) could be used directly in further steps. Borate saline buffer with pH 9.0 was used to dilute AuNPs to proper concentration. 4 L Rhodamine B (RB) (1 mM) was added into the 10 mL 6 nM AuNPs solution and the mixture was stirred for 2 h, after that 0.5 mL 20 mM 2,6-pyridinedicarboxylic acid (PDCA) was mixed with this solution. This solution was used as the detection probe of Hg (II) ions in the next step on chip detection [28]. 2.3. On chip operation 2.3.1. Experiment The valves integrated into microfluidic device (Fig. 1) were filled with deionized water and actuated by 50 psi compressed air through corresponding control channel and regulated by electronic solenoid valves (Series S070, SMC, Japan) in our home-made pneumatic control system. Those valves were automatically controlled through a data acquisition module (USB-4750, Advantech, USA) driven by a custom software programmed in LabView (National Instruments, USA). Through these pneumatic valves, the fluidic channel was opened or closed as a desire loop for reagents handling. The oil flow was continuously injected into the
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Fig. 1. Schematic picture of valve-based microfluidic device design, including three parts: droplet generators, active fusion units and mixing channel. Under control of pneumatic valves (with green color), all the reagents were loaded and operated in the fluidic network of oil and reagents inlets in liquid layer (with blue color). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
main channel by syringe pumps (Harvard Apparatus, USA). The food dye solutions or the reagents were loaded into the liquid channel of the chip through PTFE microbore tubing, connected via short stainless steel tubes under precise pressure regulator (Model 83100, Porter Instrument Co. Ltd., USA). The whole droplet operation process was observed and recorded by an inverted fluorescence microscope (IX71, Olympus, Japan) coupled with a color CCD (DP72, Olympus, Japan) or using a CCD camera (BigC Dino-Lite). 2.3.2. Droplet generation The single droplet generation unit includes reagent loading channel, main chamber and three control microvalves V1, V2 and V3 in Fig. 2. “T” junction geometry under the control of three pneumatic microvalves in the aqueous flow channel and main oil flow channel was invented for droplet production and operation. The process of water-in-oil droplet generation in this automatic microfluidic device was shown in Fig. 2. In the whole operation, oil flow was injected into main channel with a certain flow rate. In the starting step (S1), initializing, with all valves sealed, the oil flow stopped in the main chamber and waited for reagent injection in time T1. In the second step (S2), reagent loading, the reagent was pushed into the main chamber under certain pressure within a controllable loading time T2, while V2 opened. In the third step (S3), droplet generating, droplet was produced and trapped in the main chamber for further observation or operation when all the valves were sealed in time T3. In the last step (S4), droplet driving, the droplet was driven away by the oil flow to next droplet generation unit or to the mixing channel in driving time T4. 2.3.3. Droplets fusion Once two droplet generators integrated as Fig. 3(a) shown, the second one could also serve as the droplet fusion unit, as the schematic picture described in Fig. 3(b). At the first step of the circle (S1), with all the valves closed, the oil flow stopped and the DI water flow and blue food dye flow were all sealed in the reagent channels.
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Fig. 2. (a) Droplet generation process, containing four main steps: step 1 (S1) initializing with V1 to V3 closed (C); step 2 (S2) reagent loading into the main chamber with V1 and V3 closed, V2 open (O); step 3 (S3) droplet generating with V1–V3 closed; step 4 (S4) droplet transporting with V1 and V3 open, V2 closed. (b) The droplet volume depended on the loading pressures at different carrying oil flow rates (40 l/h, 50 l/h and 60 l/h).
However, DI water droplet formed in the last circle was trapped in the region where the second droplet would be produced on the optimized condition as Fig. 3(b) shown. In the step 2 (S2), with V2 and V4 opened and other valves closed, the DI water flow and the blue food dye flow were individually loaded into the main chamber under certain loading pressures within a controllable loading time T2. In the step 3 (S3), when all the valves were sealed, DI water droplet was produced in the first droplet generation unit, and the blue food dye droplet was formed at the very location where the last DI droplet stayed so that it merged with DI water droplet directly. In the step 4 (S4), with V2 and V4 closed, V1, V3, and V5 opened, the merged droplet was driven by oil flow to mixing channel. At the same time, the DI water droplet, produced by the first generation unit, was driven a certain distance to the second droplet generation unit. When the next circle started, this DI water droplet was trapped in position as the Fig. 3(b) shown and ready for the fusion process. 3. Result and discussion
loading pressure and the flow rate of the oil phase were the main variables to manipulate droplet volume. For instance, droplet generation process was fixed with initialization step time 50 ms, loading step time, 50 ms, droplet generating step time 50 ms, and driving time 500 ms. On this condition, the droplet volume dependency to the loading pressure and the oil flow rate was investigated as Fig. 2(b) shown. Considering the geometrical parameters of the microchannel and the size of droplets, the droplets were confined to be disk-like or plug-like shape in the main channel. Therefore, the volume of droplets could be expressed and calculated by V = AH, where A was the area of the droplet and H was the height of the main channel. Obviously, the droplet volume increased when turning the loading pressure up from 14 psi to 24 psi, and the droplet volume was almost linear to the loading pressure, as Fig. 2(b) shown. Moreover, once using the same loading pressure, droplet volume decreased when the flow rate of the oil phase rising from 40 l/h to 60 l/h. This tendency would be caused by the pressure enhancement inside the microfluidic channels with increasing oil flow rate. However, the coefficient of the droplet volume to the loading pressure was kept around 0.160–0.169 with the oil flow rare variation.
3.1. Droplet volume 3.2. Droplet micromixer For further operation or assay, well controlling of the volume of the droplets is extremely important, relevant factors were considered and studied, such as the loading pressure, the flow rate of oil, and separate operation time of the steps. When all the droplets were produced in the same automatically controlled period, the
After obtaining the experimental data of the single component droplet generation, the two component droplet generation units were not only used for two individual droplet generation but also employed as active droplet micromixer. Two individual droplets
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Fig. 3. (a) Schematic of two component droplets micro-mixer. (b) The droplets mixing process in the second droplet generation unit: step 1 (S1) droplet trapping with V3–V4 closed (C); step 2 (S2) other reagent loading into the main chamber with V3 and V5 closed, V4 open (O); step 3 (S3) second droplet generating and merging with V3–V5 closed; step 4 (S4) merged droplet transporting and mixing by the oil flow with V3 and V5 open, V4 closed.
with different components could be merged together through the automatic microfluidic operation. The mechanism of the mixing requires accurate controlling of the movements of the droplets, typically, the fusion efficiency of the droplets was determined by both the position where the droplet generated by the first unit was trapped in last circle and the position where the other droplet was produced by the second unit in present circle. Therefore, the flow rate of the oil and the driving time which affected the action of the droplets would be the critical factors for the two component droplet fusion. With constant operation time of the initializing, reagent loading, droplet generating 100 ms, 100 ms and 100 ms, respectively and each droplet volume almost unchanged (1–1.3 nl), the droplet fusion condition was optimized by adjusting the flow
rate of the oil phase and the droplet driving time (T4) of each period as the curve shown in Fig. 4(d). Under oil flow rate of 30 l/h, those two component droplets were merged together when the driving time was 1550 ms. Then the droplets were well mixed through the long and structured channel as Fig. 5(a) described. The optical signal was calculated by subtracting the droplet light absorption intensity from the background light absorption intensity. Fig. 5(b) was used to demonstrate the good mixing efficiency of the droplet through the mixing channel. While the driving time got shorter or longer, 1350 ms or 1900 ms, respectively, the droplet with blue food dye could not catch up with the red one in the former case or would go further than the second droplet could active reach and mix in the latter case, as Fig. 4(a)–(c) shown.
Fig. 4. (a)–(c) Droplets behavior with separate driving time 1350 ms, 1550 ms and 1900 ms under the oil flow rate of 30 l/h, with the other three steps unchanged. (d) Droplet fusion condition about the oil flow rate and driving time, with confined droplet sizes (volume 1–1.3 nl).
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Fig. 5. (a) The merged droplet (D1–D5) were transporting in the mixing channel. (b) The optical intensity analysis of the merging droplet from D1 to D5 in the mixing channel with same mixing ratio.
Fig. 6. Droplet-based Hg (II) detection: (a) Fluorescence image of Hg (II) ion droplet in mineral oil flow with 0.5% Span 80. (b) Fluorescence image of probe (RB quenched by modified AuNPs) droplet in mineral oil flow with 0.5% Span 80. (c) Fluorescence image of mixed droplet of Hg (II) ion and RB quenched by AuNPs in the mineral oil flow with 0.5% Span 80 (scale bar 100 m).
3.3. Hg (II) ion detection
4. Conclusion
With the droplet generation and fusion condition optimized, this device with multiple component droplet generation and active mixing function was used to test mercury ions through the fluorescence enhancement which was induced by free fluorescence dye releasing from AuNPs-RB probes, when the probes were exposed to Hg (II) ions [28]. The Hg (II) solution and the detection probe solution was loaded into two loading channel separately under the automatic control of the pneumatic valves, and the channels was pretreated by 0.5% BSA solution for 30 min to inhibit reagent adsorption. On the beginning, reagents were encapsulated into droplets and driven by mineral oil with 0.5% Span 80. Then fluorescence images of the Hg (II) droplet and probe droplet were taken and shown as Fig. 6(a) and (b) with a very weak fluorescent intensity on the same exposure time. However, once the 100 mM Hg (II) ion droplet and the probe droplet (RB quenched by AuNPs) were mixed together, the fluorescence brightness in the droplet region was clearly enhanced although RB molecular could diffuse in the oil phase (Fig. 6(c)). Obviously, this kind of multi-component droplet generator and micromixer could be used to detect various chemical and biological samples in microfluidic systems.
In conclusion, we have developed a microfluidic device integrated precise liquid handling functional units for multiple components droplets generation and individual droplets automatic fusion, which could be used for Hg (II) ion detection. Using this droplet handling device, the tiny volume sample could be encapsulated into the nl or pl droplet and automatic operated to react with other reagents. This device also containing three or more reagents inlets could be used for assay of two or more individual samples and could be developed for further multiple sample detection for the chemistry, biology or bioengineering study. The reagent droplets could be mixed together with required concentration or specific ratio. The complicated biochemistry condition screen could be realized in this enlarged scale droplet micro-mixer. With the characters of sophisticated liquid handling, this platform would be valuable for clinical diagnosis or environmental test with tiny volume samples and extremely expensive probes. Acknowledgements This work was partially supported by China National Funds for Distinguished Young Scientists (Grant No. 50125309), National
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Biographies Zhi-Xiao Guo received her Bachelor’s degree in 2005 in College of Life Science at Wuhan University. Now she is pursuing her PhD degree in School of Physics and Technology at Wuhan University. Her interest focuses on droplet-based microfluidics. Qian Zeng received his MSc from Wuhan University in 2007. Currently, he is preparing his PhD in School of Physics and Technology at Wuhan University. His research interests involve the integration of Surface Acoustic Wave (SAW) elements in microfluidic device and its application. Meng Zhang finished her undergraduate study at School of Physics and Technology, Wuhan University in 2009, and now she is a graduate student at Wuhan University. Her research interest focuses on the design and fabrication of smart material and novel digital microfluidic device. Long-Ye Hong got his undergraduate study at School of Physics and Technology, Wuhan University in 2010, and now he is a graduate student at Wuhan University. His research interest is microfluidic device. Yang-Feng Zhao is an undergraduate student at School of Physics and Technology, Wuhan University. He focuses on droplet microfluidics. Wei Liu received his PhD degree in physics at Wuhan University in 2008 and presently is a lecturer in School of Physics and Technology at Wuhan University. His field of interest is nanomaterials and Lab on a Chip. Shi-Shang Guo received his PhD degree in physics at Wuhan University in 2004 and presently is an associate professor in School of Physics and Technology at Wuhan University. His field of interest is Acoustic microfluidics and sensors. Xing-Zhong Zhao received his PhD in physics at University of Science and Technology of Beijing in China in 1989 and presently is a professor in School of Physics and Technology at Wuhan University. His current fields of interest are Lab on a Chip.