Chips and Tips

Christopher Y. Wong*, Georgian Ionut Ciobanu*, Mohammad A. Qasaimeh, and David Juncker
Micro and NanoBioEngineering Laboratory, Biomedical Engineering Department, Faculty of Medicine, McGill University, Montreal, Canada.
* Authors contributed equally

Why is this useful?

Microfluidic and lab-on-a-chip (LOC) devices are typically connected to external sample reservoirs. A significant issue for LOC devices is interfacing multiple sample reservoirs and connecting them both to external pressure controllers or syringe pumps and to the microfluidic chip [1,2]. Researchers typically use home-made reservoirs and interfacing them to the chip and the controllers requires specialized skills and training and/or additional fabrication steps.[3,4] Often the connections are unreliable and leaky, and as a consequence much time is lost in setting them up and it is difficult to add or remove connections from the reservoirs.

Here we introduce a “plug-and-play” reservoir that allows for multiple connections to be simply plugged in. The reservoirs are made using off-the-shelf threaded microcentrifuge tubes and screw-on septum caps. Needles, capillaries and steel tubing can be inserted one at a time – or in combination – through the cap’s septum and used to connect the reservoir to a pressure source and to one or several inlets of a microfluidic chip. The connectivity between reservoir and pressure source(s) and chip(s) can be adapted to specific needs, for example to form a reservoir with a single input and a single output, or with multiple inputs and outputs. Reservoirs with single or multiple input/output lines can be switched easily by by unscrewing the microcentrifuge tube and screwing the cap with the capillaries on another microcentrifuge tube containing a different solution.

The reservoirs are cheap, disposable, easy to assemble, and robust enough to support pressures up to 345 kPa (and even higher pressures with the variant described in the supplementary material) while having almost no dead volume. The connection of reservoirs to microfluidic chips – or to other reservoirs so as to form complex circuits – can be performed in a matter of seconds. Arrays of off-chip reservoirs can be assembled within racks and many lines can be connected at high density to a microfluidic chip. These plug-and-play reservoirs are a versatile solution to the world-to-chip interface and should find widespread application for microfluidic experimentation.

What do I need?

For the reservoir:

• threaded screw cap with membrane/septum [5]
• threaded microcentrifuge tube (any volume, as long as it has the same thread type as the cap) [5]

For the connections:

• sharp hypodermic needle with Luer-Lock (any size from 23 to 16 gauge) [6]
• 1/16″ ID Tygon tubing or other preferred tubing [7]
• male Luer-lock to 1/16″ ID hose barbed tubing adapters [7]
• glass capillary (Note: OD of glass capillary needs to be smaller than ID of needle) [8]

Optional for other connection types (shown in supplementary materials):

• 0.0250″ OD hollow steel tubing [9]
• 0.0200″ ID microbore Tygon Tubing [10]

Fig. 1 shows the required parts. Fig. S1 in the supplementary materials shows the alternative options in our laboratory of microcentrifuge tube sizes and input/output connection types. (A) Threaded screw cap with membrane/septum [5]. (B) Threaded microcentrifuge tube [5]. (C) Sharp 20G1½ hypodermic needle with Luer-Lock [6]. (D) 1/16″ ID Tygon tubing [7]. (E) Male Luer-lock to 1/16″ ID hose barbed tubing adapter [7]. (F) Glass capillary of 350 µm OD [8].

What do I do?

1. In order to insert a glass capillary to serve as an output, use a sharp hypodermic needle with an inner diameter (ID) that is just larger than the capillary’s outer diameter (OD). Insert the needle into the membrane screw cap and then slide the capillary through the needle, as shown in Fig. 2.

2. Screw the cap onto a reservoir of the desired size and then remove the needle while holding the capillary in place, as shown in Fig.3. Repeat steps 1 & 2 for every additional glass capillary connection required.

3. Insert the input pressure needle to appropriate depth (see next section for details) and connect to the Tygon tubing to the needle using the Luer-lock to tubing adapters, as shown in Fig.4. Repeat this step for every additional Tygon tubing connection required.

4. The reservoir is completed. Unscrew the cap from the container and fill it with your reagent and screw the cap back on.

5. (Optional step) Instead of using a glass capillary, a steel tube connected to Tygon microbore tubing can be used. We used 0.0250″ OD steel tubing, inserted into 0.0200″ ID Tygon tubing and then forced the pin through the septum, as seen in Fig. 5. Multiple steel-Tygon tubing connections can be connected to one septum as shown in Fig. S2.

6. Connect the 1/16″ Tygon tubing to the pressure source and the capillary (or the microbore Tygon tubing) to the chip. Make sure to create slightly smaller sized holes for the input connections on the chip to create a tight seal. When using multiple reservoirs, they can be placed into microcentrifuge tube racks for easy handling (Microtube Storage Racks), as shown in Fig. 6.

7. Everything is now ready to use!

What else should I know?

This setup is simple, easy to build and can be done in less than a minute. Depending on the cap, the membrane/septum provides an airtight seal, but after inserting and removing needles more than a few times, especially with larger needles, leakage becomes apparent. Since the system is pressure controlled, a small leakage rate relative to the supply flow rate can be tolerated. Only a limited number of connections, up to 10 in our experience, can be connected to a single cap or else leakage becomes excessive. The reservoirs work well up to 345 kPa, and for higher pressure applications solid caps can be used as described in the supplementary material.

It is important to maintain the needle connected to the pressure source above the liquid surface in order to prevent the formation of bubbles. Be careful of the sharp points and remember to dispose of the needles properly.

Supplementary materials

Figure S1. The different available options (excluding glass capillaries) in our laboratory of microcentrifuge tubes sizes, membrane caps, steel tubing, and input/output connections.

The presented setup above was not tested at pressures higher than 345 kPa, and at pressures above 140 kPa, there was minimal gas leakage. However, we have developed another setup that works without any leakage, even at 345 kPa, but requires several minutes to make and a precision micro-drill. This setup does not use a membrane cap, but a conventional screw cap without a membrane. Using suitable micro-sized drill bits, different sized holes can be made in the micro tube or the cap. After inserting the output capillaries or steel tubing and the input syringe needle, Instant Krazy glue [10] is applied to affix the inputs and outputs at the precise place. Dual melt glue [11] is then applied to seal the terminals from air/liquid leaks. The final setup is shown in Fig. S3.

We mention here that this setup is permanent and should be robust at high pressure applications. At the same time, it requires a precision micro drill, more time to make, and it is less flexible in the sense that it is harder to modify the interface.

References

[1] S. Li and S. Chen, IEEE Trans. Adv. Packag., 2003, 26, 242-247.
[2] R. Lo and E. Meng, Sens. Actuators, B, 2008, 132, 531-539.
[3] T. Liu, E. V. Moiseeva and C. K. Harnett, Integrated reservoirs for PDMS microfluidic chips, Chips & Tips (Lab on a Chip), 22 April 2008.
[4] T. Das et al., Interfacing of microfluidic devices, Chips & Tips (Lab on a Chip), 27 February 2009.
[5] Sarstedt AG & Co.: http://www.sarstedt.com/
[6] BD: http://www.bd.com/
[7] McMaster-Carr Supply Company: http://www.mcmaster.com/
[8] Polymicro Technologies: http://www.polymicro.com/
[9] New England Small Tube: http://www.nesmalltube.com/
[10] Small Parts, Inc.: http://www.smallparts.com/
[11] Krazy Glue: http://www.krazyglue.com/
[12] The Stanley Works: http://www.stanleyworks.com/

Jean-Christophe Baret
Institut de Science et d’Ingénierie Supramoléculaires (ISIS),
Université de Strasbourg, CNRS UMR 7006, Strasbourg, France
jc.baret[at]unistra.fr

Why is this useful?

In suspension, cells, beads and particles tend to sediment at a speed given by the balance of viscous drag and gravity force. In classical biological experiments, the most straight-forward solution to keep cells in suspension is to shake large volumes. This type of system is relevant at high Reynolds number but is not very compatible with the classical systems used to inject small amounts of liquid in confined geometries, such as microfluidic systems.

Another common way to prevent sedimentation is to match the densities of the particles and the carrier fluid, or to increase the viscosity of the carrier fluid to increase sedimentation times. However, these solutions are not always suitable due to the biological or physical constraints of a microfluidic experiment. For example, increasing viscosities increases the pressure drop in microchannels, and additives for density matching are not always biocompatible.

Previously in the Chips and Tips section a system has been described for cell injection [1]. With a similar system, we observed that sedimentation along the tubing binding the syringe reservoir to the chip hinders good mixing of the cells. This tubing should therefore be as short as possible, which is sometimes incompatible with the large footprint of the syringe pumps or other equipment around the microfluidic chip. Interestingly, in another Chips and Tips section, a system for temperature control of syringe pumps has been described [2]. The idea there was to use a syringe pump to pump liquid in order to move the piston of another syringe placed in a temperature-controlled bath. This system still showed problems related to the length of the connection tubing to the chip.

Here we combine both systems [1,2] to inject cells, beads and particles efficiently into microfluidic devices using small amounts of liquids (typically ~1 mL). The cells are initially placed in a syringe close to the chip and are automatically shaken inside the syringe using a small motor and a magnet. This syringe is then remotely actuated by another syringe mounted on a syringe pump [2] which reduces the footprint of the injection system in the vicinity of the chip. The tubing length from the syringe containing the cells to the chip can then be as small as 5 cm, which reduces the residence time of cells in the tubing and therefore reduces sedimentation.

What do I need?

• Syringes: BBraun Injekt (2 mL or 5 mL)
• Syringe needles: Terumo NEOPLUS 23G
• Tubing: Fisher Scientific PTFE tubing (i.d. 0.56 mm, o.d. 1.07 mm)
• Small magnets: Supermagnete, e.g. S-03-01-N
• Teflon magnets: Fisher Scientific, length 6 mm, diameter 3 mm
• Motor: Radiospares, e.g. 20GN steel 50:1 gear DC motor, 175rpm 12V
• Power supply: Radiospares, e.g. LASCAR – PSU 130
• Small equipment: stands and support, steel wire

What do I do?

1) The first part of the procedure deals with preparing the syringes. This system is very close to the one presented in [1]. Cut the plungers of 2 syringes, and insert a magnet in one (Fig 1).

2) Bind them together with the metal wire (Fig 2).

3) Fill the syringe that does not contain the magnet with water and hold it vertical (Fig 3).

4) Insert the needles in the tubing, fill a third syringe with water, place the needle in position, flush the tubing with water and connect the syringe with the free needle (Fig 4). Make sure no air bubbles are present in the water phase: they will increase the response time of the system to flow rate modifications.

5) Invert the syringe to have the magnet in the upper part and fill this part with the liquid containing the cells, beads or particles (Fig 5) and connect a short needle with tubing (Fig 6).

6) Rotate the syringe and mount the motor with the magnets. Turn on the power supply (Fig 7). The speed of the motor is simply controlled by the voltage to guarantee gentle agitation of cells.

7) The system is now ready to be connected to a chip on one hand and to a syringe pump on the other (see video below). More sophisticated systems can probably be adapted to allow a temperature control of the syringe.

References

[1] R. Cooper and L. Lee, Preventing suspension settling during injection, Chips & Tips (Lab on a Chip), 21 August 2007.
[2] L. Capretto, S. Mazzitelli and C. Nastruzzi, An easy temperature control system for syringe pumps, Chips & Tips (Lab on a Chip), 22 April 2008.

Tamal Das
Department of Biotechnology, IIT Kharagpur-721302, India

Debapriya Chakraborty, Suman Chakraborty*
Department of Mechanical Engineering, IIT Kharagpur-721302, India
* e-mail: suman[at]mech.iitkgp.ernet.in

Why is this useful?

The integration of PDMS chips, with pumps or connections to reservoirs, has been found to pose serious difficulties because of its associated problems in leaking, interfacing with tubing, etc. Here, we present a simple approach for making reservoirs and also connecting devices with tubing for interfacing with pumps, without the aid of commercially available connectors and using commonly available consumables in the laboratory.

The PDMS mould is bonded with glass in two ways – using hydrophilic and hydrophobic means. Hydrophilic bonding involves using a plasma cleaner for oxidation. Hydrophobic bonding is done using a PDMS mixture. Here, we report easy and cost effective hydrophilic and hydrophobic bonding protocols. For example, the hydrophilic bonding can also be done without the use of conventional plasma oxidation.

What do I need?

• PDMS base and curing agent (SYLGARD 184 silicone elastomer kit) in 10:1 wt/wt ratio
• micropipette tip (2-200 µl pipette tips)
• 20 gauge (outer diameter) needles
• tubing (20 gauge inner diameter)
• Piranha solution (H₂O₂ and H₂SO₄ solution in 1:1 vol/vol ratio)

What do I do?

1. Cut the syringe needles to the desired length (approximately 1.5 cm), preferably using wire electrical discharge machining. Any other needle cutter can be used if it can cut sharply the ends of the needles.

2. Punch holes in the PDMS mould using these needles at the inlet/outlet.

3. Bonding the PDMS mould to the substrate (Figure 2):

• Hydrophilic bond:

1. Oxidize the glass slide and the PDMS mould in freshly prepared Piranha solution.
2. Dip the glass slide in the solution for 5 mins, and the PDMS mould for 10 secs. Dipping the PDMS mould for larger times may lead to damage to the mould because of strong oxidation
3. Rinse them in deionised water at least three times. This step is required to remove any residual acid as well as to prevent hydrophobic recovery.
4. Dry both the glass slide and the mould using a compressed stream of dry nitrogen gas.
5. Place the mould over the glass slide and heat it at 70°C for 30 minutes.

• Hydrophobic bond:

1. Coat the glass slide with a thin layer (~ 10 µm thickness) of PDMS mixture (preferably by spin coater).
2. Heat it at 70°C for 7 mins (this makes the PDMS hardened but still sticky)
3. Place the PDMS mould on the sticky PDMS surface. In order to avoid wrinkles and air gaps, lay the mould down from one end to the other.
4. Heat it for at least 30 mins at 70°C.

4. After bonding the PDMS mould to the glass, place the needles in the holes. Apply a small amount of PDMS mixture to the cylindrical surface of the needles. The PDMS mixture slowly settles down near the junction. Applying larger quantities of PDMS mixture may lead to seepage into the channel through the junction. Heat it immediately for 10-15 mins at 70°C. (Figure 3)

5. Cut a small piece of micropipette tip with at least one planar surface. Put a bit of PDMS mixture around the periphery of the planar surface. Place it centering the needles and heat it for 20 mins at 70°C. This makes the reservoirs. (Figure 4)

6. Pour PDMS mixture into the reservoirs around the syringe needle tips and heat it at 70°C for 25-30 mins until the supports become rigid. This provides the support for the needles. (Figure 5)

The tubing can be connected to these cut out needles, and the other ends connected to a syringe or peristaltic pump (Figure 6). If the connection to the pump is not desired and only the reservoirs are required then steps (1, 4, 6) are not required.

Alexander Iles
Dept. Chemistry, University of Hull, Cottingham Rd, Hull, HU6 7RX, UK

Problem

In microfluidics it is often necessary to interface chips using capillaries or plastic tubing. This can introduce a number of problems. For example, with glass chips it is rarely the case that the tubing or capillary is a perfect match for the reservoir hole that has been drilled into the device. Hence when using adhesives to attach tubing, the glue can seep down into the microdevice and clog its channels. Alternatively, with PDMS it is difficult to find anything that will stick to it and also securely hold a connecting tube in place. Nanoports can be used to interface devices, but they are expensive [1]. Small pieces of PDMS can be plasma bonded to a chip to create ports [2], but this involves extra fabrication steps and a plasma oven.
The solution:

To use fumed silica to create thixotropic adhesives. Adding fumed silica to epoxy resins or to PDMS creates adhesives that are extremely viscous and will not flow. Hence it can be added to epoxy resins to prevent seepage between access holes and tubing with glass chips and also it can be added to PDMS to make highly effective ports for PDMS chips.

Materials

• A small quantity of premixed epoxy resin or PDMS
• Fumed silica. Fumed silica is manufactured by Degussa and can be obtained as a free sample by visiting their website[3]
• A container and a spatula for mixing

Procedure

1. Take a small quantity of your resin of choice (epoxy or PDMS). Place this in your mixing container.

2. To the resin, add a small amount of fumed silica, and using a spatula, mix it into the resin. A good starting point is 1 part fumed silica to 11 parts pre-mixed PDMS (by weight). Warning! Always wear a dust mask when handling fumed silica, it is like very fine dust. One sneeze and it will go everywhere! Bear in mind the working life of your epoxy resin, some resins set in 5 minutes or less, which will not give much time for mixing in fumed silica.

3. Once the fumed silica has been incorporated into the resin, add more as necessary to obtain the required thixotropy. Then it is ready for application.

4. For glass chips, always wipe down the surface of the device with acetone and allow it to evaporate before applying epoxy, to ensure a good bond.

5. Place the tube or capillary into the hole. Use a pipette tip or small applicator to apply a small blob of adhesive around the interface. The resin mixture will not flow, so it will be necessary to smooth it around the tube or capillary with the applicator. The procedure is similar for PDMS devices, except that a hole must first be bored into the chip, the tube inserted and then the thixotropic PDMS applied. In both cases, cure times can be reduced and adhesion strengths improved if an oven at 60-70 ºC is used for curing. Refer to Figures 1 and 2 for step by step photographic explanations and further details.

Figure 1. Procedure for making thixotropic epoxy for interfacing glass chips: (a) Mix the epoxy in a suitable container and have some fumed silica ready for use. (b) Add the fumed silica and mix in. (c) Insert a capillary or tube into a hole on the glass chip, after wiping it with acetone. (d) Apply thixotropic epoxy to the interface between the chip and the connector and leave to cure.

Figure 2. Procedure for making thixotropic PDMS for interfacing PDMS chips: (a) Mix the PDMS in a suitable container and have some fumed silica ready for use. (b) Add the fumed silica and mix in. (c) The PDMS should now look like this. (d) Cut a hole using a boring tool in your PDMS chip. (e) Insert a tube (e.g. PTFE) of a suitable size into the hole on the PDMS chip. (f) Apply thixotropic PDMS to the interface between the chip and the connector tube. (g) Leave it to cure, either at room temperature or at 60-70 ºC. (h) The completed interface. A silica capillary from, for example, a syringe pump, can now be securely inserted into the PTFE tubing and removed again as often as required without leaking. The PTFE tubing has an I.D. of 300 µm, which makes a good interference fit with standard 375 µm O.D. silica capillaries.

Conclusion

Adding fumed silica to PDMS or epoxy resins allows the researcher to fine tune the viscosity of their system as desired. Thixotropic PDMS is probably one of the best materials for interfacing PDMS devices, since few things will stick better to PDMS than PDMS itself!

References

[1] C. Koch, J. Ingle, and V. Remcho,  Bonding Upchurch® NanoPorts to PDMS, Chips & Tips (Lab on a Chip), 12 February 2008.

[2]  S. Mohanty, D. J. Beebe and G. Mensing, PDMS connectors for macro to microfluidic interfacing, Chips & Tips (Lab on a Chip), 23 October 2006.

[3] http://www.aerosil.com/aerosil/en/default