Theo T. Veenstra
6.3 MICROFLUIDIC COMPONENTS IN DINAMICS
From the previous section, it was seen that the DNA-isolation function, the PCR function and the detection were designed with a microfluidic mindset.
Most of DINAMICS building blocks need additional in-/out-puts apart from the ones for the sample-liquid itself. Depending on the function of the building block, this calls for either the capability of switching fluid-streams or for combining two fluid-streams into one single fluid-stream. These basic fluidic functions raise the need for the design/development of valves and mixers, which then can be applied within the functional building blocks.
So the total list of components to be developed consists of the following items:
• Valve (On/Off, combination of valves to create e.g. three-way valves)
• Mixer
• DNA isolation chamber
• PCR chamber
• Detection chamber
These components were developed separately, but as they are to be integrated into a single chip, the fabrication technologies of all separate chips/components have to be compatible with each other. The way this is done is shown in the next paragraph.
6.3.1 The valve component
For the DINAMICS project a valve was developed with minimal dead and displaced volume. As the valve will be used for example in the PCR chip it has to be able to withstand at least 1 Bar of pressure (as the temperature within the PCR chip can be as high as 99°C). Pressures higher than this are not expected in the system at any given time.
The valve design is shown in Figure 6.2. A channel in the chip is covered with a flexible membrane (either Viton or PDMS are ok for this). The channel ends with a through hole to the other side of the chip. Around the entrance of the through hole, a ridge is placed which blocks the flow path when the membrane is forced down. This ridge is the valve-seat. Without any actuation force on the membrane, build-up of pressure will lift the membrane opening a flow path.
A typical test of the performance of the valve is shown in Figure 6.3. During this test run a fixed flow rate was pushed towards the valve. The pressure just in front of the valve is measured with a pressure sensor. The connecting tube to the pressure sensor contains an air bubble, which gives some compliance to the system.
At some point during the pressure build-up in time, the valve starts to leak (Figure 6.3). It can be seen that the pressure at which the valve starts to leak is about 8 Bars, after which the valve is able to hold 7 Bars, which is sufficient for the DINAMICS system.
6.3.2 The mixer component
The mixing component of the DINAMICS system is the next basic fluidic component that needs to be developed.
In microfluidics all fluidic flow is laminar. Mixing effects due to turbulence can not be expected, which leaves only diffusion as the driving force for mixing. This means that a trick has to be employed which brings down the diffusion distance. In the literature, several approaches to this end are described (Hessel, 2005) The solution we have chosen is the“lamination mixer,”the two fluid streams A and B are brought
Pressure over valve
0 1000 2000 3000 4000 5000 6000 7000 8000 9000
0 100 200 300 400 500 600 700 800
Time [sec]
Pressure [mBar]
Start flow
Actuate valveValve leakage Figure 6.3 Measurement on the valve-performance.
F
Force on the membrane blocks the flowpath With no applied force on the membrane, a flowpath is available
Top-view of the channel.
Photograph of the valve-chip.
Figure 6.2 Schematics and photograph of the“DINAMICS”valve.
Detection of Pathogens in Water Using Micro and Nano-Technology 84
together such that a distribution ABABA…ABA is constructed over the width of the channel (Figure 6.4).
This is done in a wide portion of the channel. After narrowing the channel, the different sections of A and B are only in the order of 1 µm wide, ensuring fast diffusion-based mixing of the compounds in A and B.
The way this is realized is by creating an array of 39 shielded inlets for fluid B in the stream of fluid A. See also the SEM picture in Figure 6.5. The shielding of the individual inlets is necessary as otherwise the incoming fluid would stay at the bottom of the channel, resulting in a distribution of fluid B at the bottom of the channel with fluid A above that. This would result in a diffusion-distance of over 50 microns which would take a relatively long time–especially for large molecules as DNA.
In the mixing section, 39 streams of fluid B are intertwined in 40 streams of fluid A. When this combined stream has arrived at the channel section of 100 microns wide, the average laminate-width is only 1,25 microns, ensuring almost instantaneous mixing. From simulations it was learnt that the distribution of the flow is nearly homogeneous over the inlets (see Figure 6.6). The center of the channel shows a slightly higher flow-rate, locally resulting in a slightly wider section. This does not significantly interfere with the mixers’performance.
With the two most basic fluidic functions of valving and mixing established, the advanced functions will be implemented. First the DNA isolation will be discussed.
In1: A In2: B
Out: A+B
Figure 6.4 Left: Top view of Lamination Mixing principle: fluid B is injected at multiple inlets into a stream of fluid A. Right: crossectional view of chip.
Figure 6.5 SEM picture of the mixing section.
6.3.3 The DNA isolation component
For the extraction of DNA from a sample liquid, commercial kits are available. These kits require a number of pipetting, agitation and flushing steps to obtain a sample with the desired DNA extracted from the cells in the original sample solution. The working principle of these kits is that the DNA from the disrupted cells binds to a glass surface (mostly in the form of small glass beads). A flushing step makes sure that all other cell-material is disposed of. Using an appropriate buffer solution, the bound DNA is re-eluted from the glass beads into the buffer solution.
The procedure from a standard commercial kit (Roche MagnaPure LC total nucleic acid-kit) is taken as example upon which our chip-system will be based (Figure 6.7).
Figure 6.6 Simulation on the mixer’s performance.
Sample material
Cell disruption and protein digestion by the addition of Lysis Buffer and
Proteinase K
Nucleic acid binding to the surface of Magnetic
Glass Particles
Magnetic separation of
the nucleic acid-bead complex
Removal of cellular debris
by extensive washing steps
Magnetic separation of
the nucleic acid-bead complex
Nucleic acid elution at high
temperatures during the removal
of the Magnetic Glass Particles
Figure 6.7 Steps in the commercial kit from which the chip based procedure derives.
Detection of Pathogens in Water Using Micro and Nano-Technology 86
To translate the“open fluidics”commercial kit into a“closed fluidics”system, some means of trapping the glass material within the chip has to be realized. To be able to do this, a switch is made from glass beads to a glass fibre material. The glass fibre material is placed within a cavity in the chip before closing the chip (see also Figure 6.8). The dimensioning of the cavity with respect to the glass fibre pad is critical: if a flow-path is available around the glass fibre pad, the liquid containing the DNA will go around the envisioned binding sites, resulting in a very low DNA-extraction efficiency of the chip. A donut-shaped cavity is proposed. With the inlet on the outside of the donut and the outlet in the centre, no short-circuiting flow-path exists around the perimeter of the fibre-pad, as long as the height of the chamber is controlled correctly during the fabrication: all sample liquid must pass through the fibre pad.
The chip has two fluidic inlets; one is used for supply of sample liquid, the other is used for the re-elution buffer. The flow of the re-elution buffer is controlled through a syringe pump with a small syringe, such that a high degree of control over the disposed/displaced volume is available.
As seen in Figure 6.8, the channel is closed with a Viton (or PDMS) seal, ensuring a leak-tight assembly.
This way it is also possible to exchange the fibre pad for example the investigation of different binding materials. Figure 6.9 shows how the chip in its holder looks before and after assembly.
Preliminary tests have been performed with the DNA-isolation chip. It was shown that the DNA-capturing efficiency was in the same range as for the commercial kit (see Figure 6.10).
Figure 6.8 Left: schematic of the proposed geometry of the DNA isolation chip, showing how the fiber-pad is placed in the chip. Right: Top view of the chip. Two inlet channels connect to a glass-fiber pad (hatched area).
The outlet of the chip is in the center of the glass-fiber pad.
Figure 6.9 Photographs of the chip and its holder. The white patch seen on the right is the glass fiber pad.
After the isolation of the DNA-material, the sample is processed further in a PCR-chip. This chip is described in the next section.
6.3.4 The PCR reaction component
The chip described in this section has as task the multiplication of the amount of (targeted) DNA present in the sample-solution. This multiplication is done by the classic Polymerase Chain Reaction (PCR) procedure (see vast amounts of literature for details on“PCR”). For this reaction, the sample needs to be mixed with a
“PCR-mix”, after which a number of temperature steps need to be performed. The temperature steps are indicated in Figure 6.11. The temperature is cycled a number of times between 95, 55 and 75°C, effectively doubling the amount of targeted DNA with each cycle.
The chip that has to perform a PCR reaction needs to fulfil quite a number of essential requirements. First, the sample liquid needs to be mixed with the‘PCR buffer’. For this, the mixer described earlier can be applied. Next the sample liquid has to be run through a number of the temperature cycles of the PCR-cycle. As the temperature will get as high as 95°C, the pressure within the chip will reach
Comparison LioniX vs Commercial kit
10 11 12 13 14 15 16 17 18 19 20
1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07
Concentration (CFU/ml)
CT [cycle]
Lionix Commercial kit
Figure 6.10 Comparison of the efficiency of the reference kit to the implemented chip-procedure.
95
55 75
RT
Time [a.u.]
Temp [C]
1 PCR cycle
Figure 6.11 The temperatures of the PCR cycle in time.
Detection of Pathogens in Water Using Micro and Nano-Technology 88
approximately 1 Bar. Without any counter measures, the PCR chip would boil empty. To prevent this, all the inlets and outlets of the chip must be closed tightly to prevent any liquid leaving the chip during the temperature cycling. For the first chip-series, it was chosen to use external commercial zero-dead-volume valves to fulfil this function (Upchurch rotary valves). Finally, some form of temperature management has to be implemented. To this end, a temperature dependant resistor was implemented on the chip with which it is possible to measure the temperature directly on the chip. Heating of the chip was achieved with an integrated heater-resistor, whereas the cooling-function was implemented with a Peltier cooling element in the chip-holder. See also Figure 6.12 and Figure 6.13.
Last but not least, the inner surface had to be coated in order to be compatible with the compounds within the PCR-mix. This coating is realized by flushing the chip (when mounted in its holder) with SigmaCote™. Following the prescribed procedure for the sigma-cote, the chips interior is fully covered with a monolayer of water-repellent protective molecules.
Testing the PCR-chips, it was found that the performance is just below the theoretical level; the multiplication factor of 2 per cycle was not fully met. Adding a few extra multiplication cycles solved this issue easily.
The sample coming from the PCR-chip should contain enough DNA-material for detection. This sensing-part is covered in the following section.
Figure 6.12 Schematic drawings of the geometry of the PCR-chip. Left: cross-sectional view. Right: Top view.
The PCR-chamber/channel is strongly elongated to avoid no-flow regions.
Photograph of chip (Front) Photograph of chip (Backside) Photograph of (chip in) holder Figure 6.13 Photographs of the realized PCR chips and its holder.
6.3.5 The hybridization chamber component
After the DNA has been multiplied in the PCR-unit, it should be possible to detect the DNA using an appropriate sensor. The sensor shown here is employed by the University of Bologna. The working principle of the sensor is a change in the capacitance between two electrodes as DNA selectively binds to the electrodes’surface.
The change in electric capacitance or of other label-free or label-dependent electrochemical properties of the electrode interface then can be translated to a DNA-concentration in the sample liquid. Details on the working principle can be found in the chapter by Gazzola and co-workers in this book (Chapter 7). The event of DNA binding to the surface is called“hybridization”, hence the chip is named the hybridization chip.
The pictures and photographs in Figures 6.14 and 6.15 show the realized hybridization chip. Four parallel channels with each a private electrode-set are employed on the chip.
6.3.6 Fabrication–Technology platform
All fluidic components from the previous sections were fabricated in silicon and glass technology (MEMS) using different fabrication schemes. These fabrication schemes are summarized in Figure 6.16 and Table 6.1. Some of the dimensions are dictated by the function of the component, whereas others could freely be chosen.
As the ultimate goal for the DINAMICS project was the integration of all microfluidic functions into one single chip, all fabrication schemes of the standalone components have to be aligned to fit within one fabrication technology.
Figure 6.14 Left: schematic cross-section of the Hybridization chip. Right: top view of design of the chip. The oval outlines of the channels are shown with the fluidic in- and out-lets at each end. The measurement electrodes are connected to the two arrays of connector pads.
Figure 6.15 Photographs of the chip-half with the electrodes for the Hybridization-chip (left) and the hybridization chip mounted in its holder (right).
Valve Mixer DNA Isolation PCR Hybridization
Figure 6.16 Different fabrication schemes of the different components.
Detection of Pathogens in Water Using Micro and Nano-Technology 90
The used fabrication schemes for the single components were mostly selected for their convenience at the single component level. Now some compromises have to be made in order to be able to fit all components into the single production scheme. For example this might mean that the depth for a certain section of the chip will be leading, forcing another component to a non-optimal depth. This reconfiguration process is schematically shown in Figure 6.17.
Table 6.1 Used fabrication techniques for the different parts of the microfluidic chips.
Valve Mixer DNA isolation PCR Hybridization
Viton Viton/PDMS Viton PDMS Clamped glass
Through holes Powder
Valve DNA Isolation Mixer PCR Hybridization
Figure 6.17 Overview of the technology shifts in the different componenents in order to align the fabrication technologies into the fully integrated chip.
From the last schematic in Figure 6.17 it can be seen that the depth of the PCR chamber is dictated by the depth of the Isolation chamber, due to the thickness of the fiber-glass pad. All other etched channels are used only for transportation purposes, for which purpose the exact depth is not too important. Here a trade-of between internal volume [low volume gives lower sample broadening (dispersion)] and pressure-drop over the channel is made. A depth of around 100 microns works out fine for these channels.