Archive for October, 2015

Highly precise alignment for the rapid fabrication of Plexiglas® microfluidic devices

G. Simone

University of Naples Federico II, Piazzale Tecchio 80, 80125 Napoli, Italy.


Why is this useful?

Most of microfluidic devices use channels with rectangular cross-sections. The microfabrication of rectangular shaped channels is straightforward with standard tools such as photolithography.

Fluid dynamics, rheology, soft matter and, recently, biology-based investigations need circular cross-section microchannels; indeed, pre-fabricated capillaries are normally used to carry out the studies. However, capillaries are impractical for some investigations requiring complicated designs.

For Plexiglas® (or other plastic) devices, microfabrication by micromilling is a low cost procedure, which, in the last decades, has gained popularity in the field of microfluidic applications.1-2 The fabrication and the sealing of Plexiglas® microchannels with circular cross-section can be challenging.

Here, a method of fabrication of plastic microfluidic devices with circular cross-section is presented. The method is low cost and it can be performed by minimally trained users. The alignment step builds on a procedure first introduced by Lu et al. that used circular magnets to align layers of polydimethylsiloxane (PDMS).3 The protocol is validated for circular cross-section channels, but it can be used for fabricating rectangular channels or special inlets as well.


What do I need?

  • Plexiglas® sheets (thickness 1mm) Rohm Italy
  • CNC MicroMill (Minitech, US)
  • Ball nose end-mills (0.001 inch, PMT Endmill)
  • Magnets 4 mm × 4 mm× 4 mm, DX
  • Microscope Bresser 58-02520
  • Clamps RS Italy
  • Ethanol
  • Microscope (or stereomicroscope)
  • Glass slides for microscopy (50 mm × 75 mm)


What do I do?

1. Computer aided design of the device (CAD).

  • Design the microfluidic channels with CAD software. Fig. 1a displays the top and bottom layer of the main channel and, in particular, aligned square through-holes in each layer.
  • Translate the CAD file to Computer Numerical Control (CNC) code for micromilling.

2. Micromilling

  • The endmill should be aligned to set the point z=0 by using a microscope.
  • Then, the microchannels (top and bottom) must be milled. Channel with depends on the endmill diameter.4 It is worth emphasizing that setting the correct work parameters (such as tool speed and depth) is crucial. Indeed, if the latter are not appropriate, the plastic workpiece develops internal stresses that during the bonding result in cracks and chip breakdown.
  • Finally, the through holes and the frame have to be milled. The number of through holes for locating the magnets depends on the dimension of the microfluidic device. It is good practice for the rectangular through-holes to have a pitch of roughly 10 mm (this dimension depends on the size of the microchannels and of the magnets).

3. Alignment

  • Align the top and bottom layers with a stereomicroscope (Fig. 1b). The bottom layer should be set on a solid surface and the magnets inserted into the through-holes. Then, the second layer should be placed on the first and a second set of magnets inserted in the square through-holes of the second layer.
  • The magnets located in the first and second layer naturally provide a good “first alignment” of the microchannels, while leaving freedom to slightly adjust the layers (i.e., this is a reversible sealing). Once the magnets are fixed, the quality of the alignment should be checked with a microscope.

    Fig. 1. The Fabrication. a. CAD design of a microfluidic channel with circular cross section. The length of the microfluidic device is 40mm, the width is 15mm. The depth of grooves is 50μm, width 25μm. b. Magnets employed for the bonding. C. Clamps used for sealing the microchannel.

4. Bonding

  • Clamp the Plexiglas® layers together and submerge assembly in ethanol for 15 minutes (Fig. 1c). For optimal bonding, the clamping should be done with clamps positioned at the edges of the Plexiglas®.5
  • After 15 minutes, the sealing of the microfluidic channels should be checked. If the Plexiglas® is sufficiently bonded, the magnets can be removed and glass slides placed on either side of the Plexiglas® layers. Clamp the glass slides and allow the assembly to rest for another 5-15 minutes.
  • The device is ready to be used for different applications as shown in Fig. 2a. Capillary tubing can be easily connected to the channel for modular design (Fig. 2b).

    Fig. 2. Examples of microfluidic devices. a. Perfusion of the samples through hole at the inlet. b. Perfusion through capillaries.

In conclusion, analyzing the protocol, the following advantages can be emphasized:

  1. The process is designed for different materials, but it fits perfectly with Plexiglas®
  2. The equipment necessary for the fabrication and assembly includes simply a micromilling machine and a (stereo)microscope
  3. The use of square magnets (instead of circular ones) allows for more precise alignment due to further restriction to the sliding of the top and bottom layers.


References

  1. G. Simone, G. Perozziello, J. Nanosc. Nanotech., 2010, 11, 2057.
  2. G. Simone, RSC Advances, 2015, 5, 56848.
  3. J-C Lu, W-H Liao, Y-C Tung, J. Micromech. Microeng. 2012, 22, 075006-075014.
  4. G. Perozziello, G. Simone, P. Candeloro, F. Gentile, et al. Micro and Nanosystems, 2010, 2, 227-238.
  5. G. Medoro, G. Perozziello, A. Calanca, G. Simone, N. Manaresi, 2010, US Patent App. 13/257,545.

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Direct Delivery of Reagents from a Pipette Tip to a PDMS Microfluidic Device

Hoon Suk Rho1, Yoonsun Yang2, Henk-Willem Veltkamp1, and Han Gardeniers1

1Mesoscale Chemical Systems Group, MESA+ Institute for Nanotechnology, University of Twente, The Netherlands.

2Medical Cell BioPhysics Group, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, The Netherlands.


Why is this useful?

Most common way to handle and transport reagents in chemical or biological labs is by using a pipette. However, tubing connection is generally used for the delivery of reagents into a microfluidic device. Even though the connection with commercial tubing and connectors allows various choices on the sizes and materials of the tubing and easy connection, difficult sampling from stock solutions, dead volume in tubing and connectors, and extra sterilization on tubing and connectors for biomaterials, still remain challenges. Here we demonstrate a direct connection of pipette tips to a PDMS device and loading reagents by pressure driven flow.


What do I need?

  • Stainless still puncher (Syneo LLC)
  • Precision stainless steel tip (23 gauge, #7018302, Nordson Corporation)
  • Tygon tubing (0.020″ x 0.060″OD, #EW-06418-02, Cole-Parmer Instrument Company)
  • 3D printed plug
  • Pliers

What do I do?

Punching inlet and outlet on a PDMS device

1. Select the size of a puncher based on the size of a pipette tip will be connected. We achieved tight connections of pipette tips on a PDMS substrate when we punched holes by using punchers with outer diameter of 2.4 mm, 1.8 mm, 1.3 mm, and 1.0 mm for 50 – 1000 µl, 2 – 200 µl, 0.5 – 20 µl, and 2 – 200 µl (capillary) pipette tips, respectively.

Fig. 1 Direct connection of pipette tips on a PDMS device.

Plug connection preparation

1. Separate a pin from a precision stainless steel tip. The pin can be easily removed by twisting the plastic part while the pin is held by pliers.

2. 3D-print a plug. The outer diameter of the plug depends on the size of the pipette tip that will be connected. The detailed dimensions of the plug are shown in Fig. 2.

3. Connect a precision stainless steel tip, tubing, a pin, and a plug. Because the plastic part of the tip is luer tapped, it can be connected to male luer connectors and commercial plastic syringes.

Fig. 2 Tubing connection with a 3D-printed plug.

Solution loading

1. Pipette the sample, connect the pipette tip into the inlet of a PDMS device, and take off the pipette. When an empty pipette tip is connected into the outlet of the device, the sample from the outlet can be collected in the tip.

2. Insert the plug into the tip and connect the tip to a pressure source. When pressure is applied into the pipette tip, the solution in the tip is pushed into a microchannel (Fig. 3A). The luer tip can be connected to a syringe and a syringe pump can be used as a pressure source (Fig. 3B). The flow rate of the sample solution can be controlled by the syringe pump. The luer tip also can be connected to a pressure regulator with a luer fitting. Fig. 3C shows the flow rate of sample loading at various applied pressures controlled by a pressure regulator. In this case an external gas source is required. However, this system is cheaper than commercial microfluidic flow control systems. Also a digital pressure regulator can be used for accurate flow rate control at the low flow rate regime less than 1 µl/min.

Fig. 3 A. Loading blue food dye solution into a microchannel, B. Solution loading by a syringe pump, and C. Solution loading by using a pressure regulator.


What else should I know?

No leakage of the solution was observed in the connection of a pipette tip onto a 1mm thick PDMS substrate. However at least a thickness of 3 mm is highly recommended to achieve tight fitting and stable support for the pipette tip. The pin obtained from a precision stainless tip is very useful for tubing connections. For example two separated tubes can be connected by the pin and also the pin can be inserted into an inlet or outlet of a PDMS device punched by a puncher with an outer diameter of 1mm. Also the pin can be easily bent by using pliers for compact connection to a PDMS device.

Fig. 4 Tubing connection by using the pin from a precision stainless tip.

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