Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Summary

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Abstract

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 µl) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Introduction

W/O microdroplets^1-5 have many important applications for the study of biochemistry and bioengineering, including protein synthesis⁶, protein crystallization⁷, emulsion PCR^8,9, cell encapsulation¹⁰, and construction of artificial cell-like systems^5,6. To produce W/O microdroplets for these applications, important criteria are control of size and monodispersibility of the W/O microdroplets. Microfluidic devices for making monodisperse, size-controllable W/O microdroplets¹¹ are based on the co-flowing method^12,13, flow-focusing method^14,15, and the T-junction method¹⁶ in microchannels. Although these methods produce highly monodisperse W/O microdroplets, the microfabrication process requires complicated handling and specialized techniques for the preparation of microchannels, and also requires a large amount of sample solution (at least several hundred µl) because of the inevitable dead volume in the syringe pumps and tubes that conduct the sample solution to the microchannels. Thus, an easy-to-use and low-dead-volume method to generate monodisperse W/O microdroplets is needed.

This paper, along with videos of experimental procedures, describes a centrifugal capillary-based axisymmetric co-flowing microfluidic device¹⁷ for generating cell-sized, monodisperse W/O microdroplets (Figure 1). This simple method achieves size monodispersity and size controllability. It requires just a tabletop mini-centrifuge and a capillary-based axisymmetric co-flowing microfluidic device fixed in a sampling microtube. Our method needs only a very small volume (0.1 µl), and does not waste any significant volume of the sample.

Protocol

1. Fabrication of a Capillary-based Microfluidic Device

1. Set up of the holders
   Note: The holder design is presented in Figure 2A.
   1. Cut out each of the four discs of the holders (Figure 2A(i)-(iv)) from 2-mm-thick polyacetal plastic plate using a milling machine. Use the following dimensions for each of the four discs of the holder: (i) disc 1 diameter 8.5 mm, capillary hole (CH) diameter 1.3 mm, screw hole (SH) diameter 1.8 mm; (ii) disc 2 diameter 8.7 mm, CH diameter 2.0 mm, SH diameter 1.8 mm; (iii) disc 3 diameter 8.7 mm, CH diameter 0.5 mm, SH diameter 1.8 mm; and (iv) disc 4 diameter 9.1 mm, CH diameter 1.0 mm, SH diameter 1.8 mm.
   2. Assemble the holders using M2 × 40 screws (Figure 2B). A bottom part of the holder (Figure 2B) consists of the disc 1 and the disc 2 in Figure 2A(i), (ii) and an upper part (Figure 2B) of the holder consists of the disc 3 and the disc 4 in Figure 2A(iii), (iv).
      1. To construct the bottom part of the holder, insert the screw in three SH of each disc 1 and 2. Shorten the screws by nipping off a piece of the thread portion. Keep the length of screw at 0.9 cm (the same length as the bottom part of the holder).
      2. To construct the upper part of the holder, insert screws into the two SH of each disc 3 and 4. Shorten the screws by nipping off a piece of the thread portion. Keep the length of screw at 0.7 cm (the same length as the upper part of the holder).
      3. To assemble the holder, join the bottom and upper parts of the holder using a long screw.
         Note: Keep the length of the each part of the holder exact: the bottom part is 0.9 cm; the upper part is 0.7 cm (Figure 2B).
2. Fabrication of the glass capillaries
   1. Use two types of glass capillaries: an inner glass capillary (Outer diameter (OD)/ Inner diameter (ID): 1.0/0.6 mm), and an outer glass capillary (OD/ID: 2.0/1.12 mm).
   2. Use a glass cutter to divide the outer glass capillary into three equal parts, and then use the glass cutter to divide the inner glass capillary into two equal pieces.
   3. Sharpen each divided inner and outer glass capillaries using a glass capillary puller (Figure 3A). Set the weight of the puller at max. Set the heat level of the puller at 60 degrees for the outer glass capillary and 70 degrees for the inner capillary. Carefully sharpen the glass capillary.
      1. Keep the length of the tip within the constricted part of the glass capillary: the inner capillary is 1.5-1.8 cm; the outer capillary is 0.8-1.0 cm (Figure 3C). If this length is shorter or longer than the described length, please adjust the heat level of the puller.
   4. Fix the inner or outer glass capillaries to the microforge stand using tape (Figure 3B).
   5. Cut off the tip of the glass capillary using the microforge in three steps (Figure 3B): (i) touch the tip of the glass capillary to the glass beads on a platinum wire, (ii) heat the platinum wire by stepping on a foot switch for 1-2 sec, and (iii) after 1-2 sec, cut off the tip of the glass capillary by cooling the platinum wire.
      1. Adjust the diameters of the inner (d_i) and outer (d_o) capillary orifices, respectively. The orifice diameter of the inner glass capillary is 5, 10, and 20 µm (d_i= 5,10, 20 µm) and the outer glass capillary (d_o) is 60 µm (d_o= 60 µm) in this experiment.
         Note: The glass capillary is disposable. Repeat the fabrication of the glass capillaries.

2. Procedure for Generating W/O Microdroplets

1. Fill an outer glass capillary with oil containing surfactant. The mixture of oil and surfactant is hexadecane containing 2% (w/w) sorbitan monooleate in this experiment (Figure 4A).
   Note: There are many combinations of oils and surfactants (e.g., oils may be fluorinated or carbonated; surfactants may be ionic, nonionic, or fluorochemical).
   1. Introduce 10 µl of hexadecane containing sorbitan monooleate into an outer glass capillary. In Figure 4A, the orifice diameter of the outer glass capillary (d_o) is 60 µm (d_o= 60 µm). To adjust the orifice of glass capillary, return to steps 1.2.4-1.2.5.
2. Set the outer capillary in the bottom part of the holder (Figure 4B).
3. Draw about 0.1 µl of an aqueous solution into an inner glass capillary (Figure 4C) by capillary action. In Figure 4C, the orifice diameter of inner glass capillary (d_i) is 10 µm (d_i= 10 µm). To adjust the orifice of the glass capillary, return to steps 1.2.4-1.2.5.
4. Set the inner capillary in the upper part of the holder (Figure 4D-a). Insert the inner capillary into the outer capillary (Figure 4D-a). Looking at the white dot circle as in Figure 4D-a, observe the position of the inner capillary inside the outer capillary (internal diameter of the outer capillary (w) = 130 µm) (Figure 4D-b,c) using a digital microscope. The position of the inner capillary in the outer capillary must be set to w = 100-150 µm.
   Note: To change the position of the inner capillary in the outer capillary, please turn the screw in the upper part of the holder. Thereby, distance w can be controlled precisely.
5. Introduce 100 µl of hexadecane containing sorbitan monooleate (2% w/w) into the bottom of a 1.5 ml sample microtube. Install the holder, with the inner and outer capillaries, in the sample microtube (Figure 4E-a). Be sure to check the outer capillary to keep it away from the air-oil interface (Figure 4E-b).
6. Centrifuge the sample microtube using a tabletop swinging-out-type mini-centrifuge at a gravity of 1,600 x g for 1-2 sec to generate microdroplets (Figure 4F). Carry out all experiments at RT.
   Note: Use a swinging-out-type centrifuge. A droplet may collide with a sidewall of the sample microtube and disintegrate when a fixed-angle-type centrifuge is used.
7. Slowly draw up the W/O droplets by pipette, and then, put them on a glass slide.
8. Capture images of the microdroplets generated using a digital microscope (magnification, 200X).

Results

In this study, we present a simple method for the generation of cell-sized W/O microdroplets by using a capillary-based centrifugal microfluidic device (Figure 1). The microfluidic device was composed of a capillary holder (Figure 2B), two glass capillaries (inner and outer glass capillaries in Figure 3C), and a microtube containing an oil including surfactant. We injected 0.1 µl of sample solution into the inner glass capillary and place...

Discussion

Using this device, the monodisperse W/O microdroplets were generated by Plateau-Rayleigh instability of a jet-flow¹⁷. Microscopic examination did not reveal the presence of satellite droplets. In the fabrication of the device, three critical steps are essential to successfully generate monodisperse W/O microdroplets. First, to supply a straight flow of oil containing surfactant and aqueous solution, the capillary holes of four discs must be arranged in a concentric pattern. Second, the inner capillary was care...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-mm-thick polyacetal plastic plate	Tool	Nikkyo Technos, Co., Ltd. (Japan)	244-6432-08
Milling machine	Tool	Roland DG Co., Ltd. (Japan)	MDX-40A
End Mill RSE230-0.5*2.5	Tool	NS Tool Co., Ltd. (Japan)	01-00644-00501
M2*40 screws	Tool	Jujo Synthetic Chemistry Labo. (Japan)	0001-024
Glass Capillry Puller	Tool	Narishige (Japan)	PC-10
Microforge	Tool	Narishige (Japan)	MF-900
Inner Glass Capillary	Tool	Narishige (Japan)	G-1
Outer Glass Capillary	Tool	World Precision Instruments Inc. (USA)	1B200-6
1.5 ml Sample tube	Tool	INA OPTIKA CO.,LTD (Japan)	ST-0150F
Hexadecane	Reagent	Wako Pure Chemical Industries Ltd. (Japan)	080-03685
Sorbitan monooleate (Span 80)	Reagent	Tokyo Chemical Industry Co., Ltd. (Japan)	S0060
Milli Q system	Reagent	Merck Millipore Corporation (Germany)	ZRQSVP030
Swinging-out-type Mini-centrifuge	Tool	Hitech Co., Ltd. (Japan)	ATT101
Digital Microscope	Tool	KEYENCE Corporation (Japan)	VHX-2001