APPLICATION NOTEAUTOMATED PNEUMATIC VALVEACTUATION TO PERFORMMICROFLUIDIC PROTOCOLSINTRODUCTION
Microfluidic technology has been utilized in multiple research and industrial fields, providing
advantages in fluid handling capabilities, scalability, robustness, and automation. In thepast 10 years, effort has been devoted to the development of microfluidic platforms capableof performing several assays using programmable fluidic operations within an array ofmicrovalves. Similar to logic circuits where multiple computing routines are executed on asingle device, programmable microfluidic platforms have been implemented3. This allows one
to perform fluidic operations such as mixing, sampling, washing, and reacting automaticallyon a microfluidic chip by modifying the sequence/order of fluidic operations with software. Thisis useful in biological and chemical applications, such as quantitative metabolic biomarker
and genetic analysis4,5, protein-based biomarker detection6, or small molecule chemical andenvironmental analysis7. These platforms usually consist of a 2D-array of microvalves that permit
flow regulation, on/off switching and sealing of liquids, gases or vacuums8. Several microvalves
have been developed using pneumatic, electrokinetic and electrochemical actuators. Amongthese mechanisms, pneumatic actuation is the most reliable due to the simplicity of fabrication,ease-of-use, scalability, and high degree of accuracy, precision, and reliability. Pneumaticallyactuated microvalves utilize the deflection of an elastomer (typically PDMS) membrane tocontrol fluid flow1. Today, most platforms in the academic field are customized: solenoid valves
are connected to a specially designed optocoupler, all contained in a custom casing. This ismonitored using a microcontroller connected to PC, and a customized software is implemented
for controlling the valves and sequence/order of fluidic operations. An additional software isrequired for controlling pressure (figure 1 on next page). As a result, setting up the infrastructure
to control such platforms is often time-consuming, and requires specific engineering expertiseand extensive research facilities. As a consequence, a major barrier to entry for using microfluidic
devices with integrated valves can be implementing control software and electronics9.
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APPLICATION NOTE

ComputerFor device control

DAQ module
for data acquisition

MFCSTM-EZ
Pressure controller

Solenoïd valves
3-port/2-position

Chip support
with pneumatic inlet/outlet
Figure 1: Schematic of a typical microfluidic platform with integrated valves10

The LineUpTM P-SWITCH is designed to address these concerns: When combined withpressure controllers, it is a ready-to-use microfluidic electrovalve controller (positiveand negative pressures) that can connect to any microfluidic system with pneumaticchannels. Using Fluigent MAT software, it is possible to implement complex sequencingof fluidic operations with accurate pressure steps, without requiring additional materials
or software. Elementary fluidic functions such as fluid transfer, volume calibration,
mixing, aliquoting and linear dilutions can be parallelized, automatically controllingcomplex operations.
We demonstrate the use of the LineUpTM system with a P-SWITCH and using MAT software
for the sequential injection of fluids within a microfluidic chip to perform pumping,mixing, sampling, and cleaning.

1For additional information, contact us by email : contact@fluigent.com or consult our website : www.fluigent.com

APPLICATION NOTE

MATERIALS
Microfluidic pressure based control

LineUpTM P-SWITCHThis module contains 8 3-port/2-position solenoid valves. It allows one toswitch between 8 pressure outlets and 2 different supplied pressures(P1 and P2). Those pressures are common to all the valves and can becontrolled within the range of - 800 mbar to + 2000 mbar. It can be usedto actuate quake or other pneumatic valves.

LineUpTM Flow EZ
The most advanced flow controller for pressure-based fluid control. The
Flow EZTM is available in a variety of pressure ranges from -800 to +7 000mbar. It can be combined with a FLOW UNIT to control pressure or flowrate. Two Flow EZTM 1 000 mbar and one Flow EZTM -800 mbar are used
here.

LineUpTM LINKThe LINK provides connection of LineUp™ series modules to a PC forsoftware control. Fluigent software solutions allow one to control andmonitor the devices in real time as well as record data (All-in-One), orto automate pressure/flow-rate steps and microfluidic valve actuation(MAT).

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APPLICATION NOTE
Microfluidic chip
ABFigure 2: (A) Chip holder + Flowstretch microfluidic chip with valve chambers to actuate liquids
directly from syringes, (B) Focus on a chamber
The microfluidic system used was designed by the CEA-Leti. The device a is a hybrid cardconsisting of three polymer cards and a stretchable membrane, illustrated in figure 3.A below.
The first card contains reagents storage and network channels to connect the fluidic chambers(1). The second card contains pneumatic valves and chambers at the top layer and pneumatic
network at the bottom layer (2). The third card allows for the pneumatic channels to close (3).
The stretchable membrane is positioned between the fluidic card (1) and the pneumatic card (2).
The elastic membrane is made of Ecoflex00 50, a bi-component silicone material that withstands
larger deformations than PDMS, allowing to totally empty and fill the chambers at low pressures(few hundreds of mbar), which simplifies the pneumatic instrumentation required to actuate
the chip.

A

B
Figure 3: (A) Overview of the chip layers, (B) Schematic
representation of the chip chambers and connections.

3

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APPLICATION NOTE
The chambers and valves shift between two states. The fluidic system is closed when a positive
pressure is applied to the chamber or the valve and open when negative pressure is applied.
The fluidic network channel consists in 13 chambers of 5 µL and 1 chamber of 200 µL, allowing
for pumping, mixing and sampling operations.

Figure 4: Working principle of the chip quake valves

Sotware control and automation
The Fluigent Microfluidic Automation Tool (MAT) is a software package
for developing and running time based microfluidic protocols using
Fluigent instruments. It allows one to easily create protocols for automatedexperiments, including a wide range of operations and loops as well as TTL
input/output.

Figure 5: Overview of Fluigent Microfluidics Automation Tool interface(protocol shown unlinked to the current application note)
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4APPLICATION NOTEMETHODS & RESULTS
System set-upCaption

Computer with MAT software
Automation tool for the protocols

Pneumatic tubing 4mm OD
Pneumatic tubing 3mm OD

Microfluidic system
Chip holder with inlets+ Flowstretch chip

Power supply
Pressure supply
VacuumsupplyLineUp controllers chain
LINK, 2 Flow EZ 1 bar,
2 P-SWITCH, 1 Flow EZ -800
mbar
Figure 6: Complete system set-up schematic withdevices, supplies and pneumatic tubing connections

The system used is shown in figure 6 above. An external pressure source is connected to theLineUpTM system consisting in a LINK module, two P-SWITCHTM, two Flow EZTM +1000 mbar andone Flow EZTM -800 mbar. Each Flow EZTM supplied with positive pressure is connected to one
distinct P-SWITCHTM while the Flow EZTM with negative input is connected to both P-SWITCHTM.
This way each quake valve controller (P-SWITCH) is able to provide positive pressure or use
a vacuum on the chip chambers. The 8 outlets lines from one P-SWITCHTM are connected tothe pneumatic chambers of the microfluidic system, while the outlet tubing from the second
P-SWITCHTM is connected to the pneumatic valves. The chambers are pressurized at +100 mbar,
the valves are pressurized at +450 mbar, and the negative pressure applied is - 100 mbar
on the whole pneumatic system. Syringes containing red dye and distilled water are directly
connected to the two inlets of the microfluidic chip, and a syringe is connected to the outlet to5

collect waste.
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APPLICATION NOTE

Automation of sequenced fluid operations
In order to automate the fluidic operation steps, the Fluigent Microfluidic Automation Tool
(MAT) software is used. A new sequence block is added to the protocol section. To help building
the protocol, a picture of the microfluidic chip is uploaded, and each chamber and valves areannotated. Here, A and B represent the two P-SWITCH and the numbers represent the valves
buttons of each P-SWITCH (figure 7).

Each P-SWITCH module actuates 8 valves on the microfluidic chip. On
the MAT software, those valves are indicated with a letter (A or B) forthe module, and a number from 1 to 8 for the corresponding outlet.

AB

Possibility to rename the sequence
Uploadable pîcture
Number of

Add a stepcorresponding

Delete a stepvalve, Letter for

Duplicate a step

the module

Load a picture
Possibility to rename P1 and P2
Sequence (rename, set duration, set positions)Figure 7: MAT function bloc to automate pressure steps using P-SWITCH

The step sequences are next edited by modifying the valve position. When the valves are
displayed in green, it corresponds to a positive pressure (closed fluidic system: the liquid
cannot flow in the chamber/valve), when the valves are displayed in orange, it corresponds
to a negative pressure (open fluidic system: the liquid is allowed to flow in the chamber/valve).
“Pumping”, “mixing”, “sampling,” and “cleaning” steps are edited. The steps are implemented
sequentially, with specific duration time. Note that it is also possible do it manually using theLineUpTM local control feature.
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APPLICATION NOTE

Pumping
In order to ensure precise fluid delivery, a pumping step is often performed within a microfluidic
chip, provide accurate liquid control and compensate for swept or dead volumes. The liquid is
introduced through the microfluidic chip for filling these volumes. To illustrate this process, a red
dye is injected, and flows sequentially through the chambers 6B, 5B, and 4B, before exiting thechip through the outlet. This is performed by applying vacuum to the selected fluidic chambers,
as shown in figure 8 below.

Step 1

Step 2

Step 3

Step 4

Step 5Figure 8: Valves steps to perform the automated pumping protocol
Figure 8 shows the pumping within the microfluidic chip. We can observe that fluid is precisely
injected on the targeted chambers for each steps, and that it fully exits the chip at the end of thesequence. A 6th step is added to close all valves. The protocol is performed in less than 5 seconds.

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APPLICATION NOTE

Mixing (calibration)
Mixing, is often required for sample dilution, reagent homogenization, and chemical orbiological reactions. Mixing can be challenging under microfluidic conditions due to laminar
flow. Turbulent flow (improving mixing) is prevented. By using pneumatic actuation with amembrane deflection, fluid motion between the chambers turns out to be very fast11. This canbe used to create fluidic recirculation between two solutions within a chamber, and enhancemixing. To improve the mixing performance, liquids can also move back and forth by actuating
the chambers again. Here, an automated protocol was developed to demonstrate fluid mixing
between two solutions using pneumatic actuation. A red dye solution of 5 µL is moved to the 5Bchamber of the chip, and 200 µL of distilled water is moved to 1A chamber. Next, the red dye is
moved to the 7B chamber, and finally moves to 1A for mixing. Liquid is moved back and forthby actuating the chamber 7B 3 times to ensure complete mixing.

Figure 9: Valves steps to perform calibration of 5µL and 200µL volumes

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8APPLICATION NOTEMixing
Figure 10 shows the solutions positions after calibration. The red dye solution is correctly moved
to 5B position, and distilled water is moved to 1A.

Figure 10: Microfluidic chip state after calibration protocol, ready to mix
Figures 11 (A) and (B) show the injection of the red dye solution to the 200 µL chamber containing
distilled water (1A position), and figures 11 (C) and (D) show the consecutive re-injections forimproved mixing. Fluidic recirculation can be observed and the solution seems homogeneousafter three fluid transfers, in less than 6 s.

Figure 11: Valves steps to perform mixing protocol

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APPLICATION NOTE
Sampling
The process of sampling is also typically performed in fully integrated micro total analysis
system sor Lab-on-a-Chip devices. To demonstrate this process using our system, the solution
previously mixed in 1A is sampled into 3 chambers, each connected to 2 consecutive chambers.
The complete protocol is described in figure 12. Briefly, the 2B and 2A valves are open, allowing
to move to solution into the 2A chambers, and subsequently to the consecutive chambers (3Aand 4A), before exiting the chip through the outlet.

Figure 12: Valves steps to perform calibration of 5µL and 200µL volumes
Figure 12 shows the sampling process. The red solution is equally dispensed into the threechambers (2A) in less than 2 s. The solution is next moved to the consecutive chambers, asimplemented on the protocol, and no solution is remaining within the previous chambers (2Aand 3A). The sampling protocol functioned well, and took less than 4 s.
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10APPLICATION NOTECleaning
At the end of a microfluidic protocol, cleaning a reusable chip is needed to ensure proper
function for the next experiments. Here, an automated protocol is developed to perform acleaning step. The complete protocol is described in figure 13. Briefly, distilled water is moved
to the 200 µL chamber. Next, the neighboring chambers (5B, 7B,2B, 2A, 3A and 4A) are open,
allowing for the liquid to flow into them. The chambers are consecutively closed and the liquid
is pushed through the outlet of the microfluidic system, to the waste syringe.

Figure 13: Valves steps to perform cleaning protocol

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APPLICATION NOTE

Figure 14 shows the cleaning process. This step is implemented right after the sampling protocol
We observe in figure 14 (A) that distilled water is injected within the 200 µL chamber, removingthe red solution from the chamber. Figure 14 (B) shows the microfluidic chip at the last step of thecleaning process. We observe that there is no red dye remaining in the microfluidic chambers,
confirming the functioning of the cleaning step.

Figure 14: Microfluidic chip state during cleaning protocol, after sampling

CONCLUSION
We have demonstrated the use of the LineUpTM P-SWITCH and Fluigent MAT software for
implementing sequential fluid operations, including pumping, mixing, sampling andcleaning, with a custom microfluidic chip. Using the P-SWITCH brings many advantages suchas quick changing of liquids, no liquid contact with the instrument, less cross contamination(compared to fluidic valves). Moreover, the system is expandable as several P-SWITCHes can bestacked. This microfluidic system is useful in many applications such as:-

Quake/Mathies valve control-

Fast medium switching-

Sequential sample injection-

On-demand droplets-

Medium perfusion switch for cell biology-

Microfluidics in flow chemistry, cell culture, …

This application note, protocols and experiments havebeen performed in conjunction with the CEA-LetiFor additional information, contact us by email : contact@fluigent.com or consult our website : www.fluigent.com

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APPLICATION NOTEREFERENCES[1] Kim, J., Stockton, A. M., Jensen, E. C. & Mathies, R. A. Pneumatically actuated microvalve circuits
for programmable automation of chemical and biochemical analysis. Lab Chip 16, 812–819 (2016).
[2] Han, K. N., Li, C. A. & Seong, G. H. Microfluidic Chips for Immunoassays. Annu. Rev. Anal. Chem.
6, 119–141 (2013).
[3] Thorsen, T., Maerkl, S. J. & Quake, S. R. Microfluidic large-scale integration. Science (80-. ). 298,580–584 (2002).
[4] Jensen, E. C., Bhat, B. P. & Mathies, R. A. A digital microfluidic platform for the automation ofquantitative biomolecular assays. Lab Chip 10, 685–691 (2010).
[5] Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Rep.
5, 795–800 (2004).
[6] Erik C. Jensen, Yong Zeng, Jungkyu Kim, and R. A. M. Microvalve Enabled Digital Microfluidic
Systems for High Performance Biochemical and Genetic Analysis. 15, 455–463 (2011).
[7] Kim, J., Jensen, E. C., Stockton, A. M. & Mathies, R. A. Universal microfluidic automaton forautonomous sample processing: Application to the mars organic analyzer. Anal. Chem. 85, 7682–7688 (2013).
[8] Oh, K. W. & Ahn, C. H. A review of microvalves. J. Micromechanics Microengineering 16, (2006).
[9] White, J. A. & Streets, A. M. Controller for microfluidic large-scale integration. HardwareX 3,135–145 (2018).
[10] Parent, C. Développement d’une plateforme autonome et portable et pour des applicationssanté. (2019).
[11] Parent, C. et al. Quantitative biological assays with on-chip calibration using versatilearchitecture and collapsible chambers. Sensors Actuators, B Chem. 261, 106–114 (2018).

13For additional information, contact us by email : contact@fluigent.com or consult our website : www.fluigent.com

APPLICATION NOTE

LIST OF FIGURES
Figure 1: Schematic representic a typical microfluidic platform withintegrated valves10

1

Figure 2: (A) Chip holder + Flowstretch microfluidic chip with pnematic
valve chambers to actuate liquids directly from syringes, (B) Focus on achamber

3

Figure 3: (A) Overview of the chip layers, (B) Schematic representation of
the chip chambers and connections.

3

Figure 4: Working principle of the chip pneumatic valves

4

Figure 5: Overview of Fluigent Microfluidics Automation Tool interface(protocol shown unlinked to the current application note)

4

Figure 6: Complete system set-up schematic with devices, supplies andpneumatic tubing connections

5

Figure 7: MAT function bloc to automate pressure steps using P-SWITCH

6

Figure 8: Valves steps to perform the automated pumping protocol

7

Figure 9: Valves steps to perform calibration of 5µL and 200µL volumes

8

Figure 10: Microfluidic chip state after calibration protocol, ready to mix

9

Figure 11: Valves steps to perform mixing protocol

9

Figure 12: Valves steps to perform calibration of 5µL and 200µL volumes

10

Figure 13: Valves steps to perform cleaning protocol

11

Figure 14: Microfluidic chip state during cleaning protocol, after sampling

12

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