Microfluidics: Liquid handling
The aim of this document is to scratch the surface of microfluidics, trying to describe the most significant phenomena at this scale.
The first goal of microfluidics is to take advantage of the benefits of scaling down: better control, lower cost, faster results and more. These benefits are especially relevant for biological reactions.
The effects that become dominant in microfluidics include laminar flow, diffusion, fluidic resistance, surface area to volume ratio, and surface tension. As the magnitude of these physical effects is different to the ones experienced at the macroscopic scale, fluid integrated microdevices must be designed from first principles and not simply by miniaturizing macroscopic devices.
Liquid flow in the microdomain belongs to the regime of viscous dominated flow. There is a fundamental change in hydrodynamics that occurs here, which significantly affects microfluidic operations like mixing. This barrier occurs when the Reynolds number is of the order of unity. At these scales, viscous forces dominate over inertial forces, turbulence is nonexistent, surface tension can be a powerful force, diffusion becomes the basic method for mixing, and evaporation acts quickly on exposed liquid surfaces. At low Reynolds numbers, fluid dynamics is dominated by viscous drag rather than by inertia and this is why devices that rely on inertial effects for their operation will no longer work.
One consequence of laminar flow is that two or more streams flowing in contact with each other will not mix except by diffusion. Diffusion is the process, by which a concentrated group of particles in a volume will, by Brownian motion, spread out over time so that the average concentration of particles throughout the volume is constant.
As diffusion times can be short at the microscale, microchannels can be used to create concentration gradients having complex profiles. Mixing schemes at the microscale must find ways to maximize the interfaces between solutions to allow diffusion to act quickly.
Surface area to volume ratio is another factor that becomes important at the microscale. The inverse characteristic length scaling of the surface-area-to-volume ratio implies that heat and mass transfer into or out of a chip can be enhanced as the dimensions of the device are reduced.
When working at the micro scale, another element as the surface tension forces become significant. Surface tension is the result of cohesion between liquid molecules at the interfaces. The surface free energy of a liquid is a measure of how much tension its surface contains. The path a fluid will travel through a capillary is directly related to the water’s surface free energy and inversely related to the radius of the capillary.
When microchannels with dimensions on the order of microns are used, the lengths liquids travel based only on capillary forces are significant. Surface energies have been widely exploited in microfluidics as pumping systems.
Microfluidics deals with the behaviour, precise control and manipulation of fluids that are geometrically constrained to a small (typically sub-millimetre) scale. This kind of research and work involves the usage of different technologies, components and materials, witch are key factors in microfluidic area.
Usually, micro means one of the following features:
Microfluidics is a multidisciplinary field intersecting engineering, physics, chemistry, microtechnology and biotechnology, with practical applications to the design of systems in which such small volumes of fluids will be used. Microfluidic area emerged in the beginning of the 1980s and is used in the development of inkjet printheads, DNA chips,lab on a chip technology, micro-propulsion, and micro-thermal technologies.
In this field microLIQUID develops and produces from the simplest microfluidic chip to complex microfluidic devices.
Our manufacture process allows us to integrate different designs and devices in a wafer, reducing time and cost of manufacturing.
microLIQUID offers standard microfluidic products ( microfluidic chips and encapsulate) and develop customized microfluidic structures and chip holders (connectors).
Microfluidic Prototyping: Molds for PDMS Microfluidic Chips
In microLIQUID, we manufacture SU8 molds. The moulds are used as tools for soft casting polymers such as PDMS or polymers Hot Embossing.
Microfluidic Prototyping systems
In microLIQUID, we fabricate the microfluidic structures according to the customer specific requirements.
With no minimum quantities requirements, we start the fabrication from 1 unit at affordable prices.
Our in-house facilities allows us to use different fabrication methods and/or materials.
Techniques: Photolithography, Hot Embossing, Etching (Wet and dry), stereolithography, inject moulding, micromachining, sputtering and vaporization
Materials: Plastics, glass and silicon
The microfluidic channel geometries may vary with high aspect ratio, curved channels, high precision height or length and no depth-width constraints.
Heaters and sensors: We integrate the electrodes inside the microfluidic structures. Made in: Gold, Platinum. Titanium, Chromium or Aluminium or other materials.
Experience:We’ve been developing our microfluidic expertise in the last years through several projects as: Microprobe flexible-semiflexible, Combining delivery and electrical signalling, qPCR Lab on a Chip, Electrolysis pump, Embedded microcantilevers, SU8 free standing structures embedded in microchannels for microfluidic control
Microfluidics Manufacturing: Photolitography technique
Photolitography is manufacturing process commonly used in microfluidics.
When it comes to challenging microfluidic arquitectures such as interdigitated electrodes(<10µ), high aspect ratio channels and small features this is a much more precise technique for the fabrication. It uses UV light to transfer a geometric pattern from a Cromium (<10µ) or acetate(>10µ) photomask with very high dimensional resolution
This manufacturing process can transfer these patterned structures to a wafer made of Silicon, Quartz, Glass, Polymers or even Metals.
The alignment of the photomask is a crucial process and is done with a mask aligner for accurate results.
The photolitography, when using the light, only allows to create rectangular or square channels, as the “attack” comes from the upper side and is straight(90º).
Also is used for manufacturing of molds for PDMS casting or hot embossing.
Microfluidic Chips: Injection Molding
Injection Molding in microfluidics is the last process of the development and launch of a microfluidic product.
Once the whole process of a microfluidic product workflow is done, and the product has interest for the market, the prototyping system has to be abandoned and the manufacturing process changes.
Due to more need of microfluidic chips manufacturing and once the material of the microfluidic architecture is decided, the technique to obtain fast and cost effective pieces is the INJECTION MOLDING.
Injection molding is basically the process to melt a thermoplastic under certain conditions and then, pushed within the cavities of a mold(injected), where it is cooled to a temperature. Once this temperature is reached, the pieces can be removed without deformation.
Once the mold is designed and done(is the main investment at this moment), the manufacturing process is replicable and the production batches can be enlarged and costs reduced. Time and cost shavings make this technique the best one to manufacture large series of microfluidic cartridges.
The molds can have only one cavity or several cavities(which allows to have more production per injection slot but costs are higher) and the polish process is a key element to obtain higher quality microfluidic cartridges.
Once you have the mold totally refined, the company is prepared for mass production of the microfluidic pieces.
Microfluidic Chips: Thermoplastic Material Cyclic olefin polymer COP
Cyclic olefin polymer COP is an amorphous polyolefin with a cyclic structure in the main chain.
The following figure shows the polymerization scheme and polymer structures:
According to it, COP is polymerized by Ring Opening Metathesis Polymerization of norbornene derivatives, followed by hydrogenation of double bonds that provides more stability in terms of heat and weather resistance.
Compared to other thermoplastics such as PP, PC, PS and LDPE, COP provides significantly improved moisture and vapor barrier properties
COP is a glass-like & UV transparent polymer, which exhibits excellent optical properties and performances which enable to get a higher optical signal quality for small complex parts, increase resolution and lower detection limits in fluorescence spectroscopy, as well as to get a high dimensional stability under a harsh and humid environment. This material exhibits high transparency (92% light transmittance).
COP offers extremely low fluorescence across the excitation/emission spectrum and has been proven to increase resolution and lower detection limits in fluorescence spectroscopy for in vitro and in vivo imaging.
COP absorbs virtually no moisture and shows no dimensional changes even under conditions of high temperature and humidity. Thanks to its low water absorption, the refractive index of COP remains constant after exposure to humid environments.
Microfluidics Application: Real time PCR (Polymerase Chain Reaction)
Real time quantification during simultaneous amplification(PCR – Polymerase Chain Reaction) is enabled by gene-specific probes, that is, relatively short DNA strands binding specifically to the target sequence during the annealing step.
When the polymerase elongates the target sequence, it comes across the probe and digests it. Digestion of the probe disassociates two additional molecules that are bound to the probe. These molecules are the reporter dye and a quencher molecule. The reporter dye generates a fluorescence signal when it is stimulated by a light of a certain wavelength.
However, as long as it is bound to the primer together with the quencher molecule, the fluorescence signal is suppressed due to fluorescence energy transfer (FRET).
When the enzyme disassociates the reporter dye and quencher from the probe, the reporter dye is free to generate fluorescence signals that can be measured by photomultiplier diode. The digestion effect is irreversible thus leading to an increasing fluorescence signal the more probes are digested and amplicons are generated. The reaction mechanism for the real time PCR is shown in the following scheme:
The reporter dye is equipped with a fluorescence label that has a specific absorption and emission wavelength. By combination of labels with different emission spectra, real time multiplex PCR is enabled.
Fluorescence signals are measured as relative fluorescence units that can be normalized to a standard so that the relative change of fluorescence can be determined. This is how deviations due to variations in reporter dye concentration in the mixture or bleaching effects can be equilibrated.
Signal detection in a thermocycling device starts with a constant low fluorescent noise during the first thermocycles. At a certain amplification level, the fluorescence signal starts to increase exponentially. After a few further PCR cycles, the signal increases linearly and finally comes into saturation because the probes that are contained in the reaction mix are used up.
A high concentration of target DNA leads to an early signal amplification.
In contrast, little concentrations require more thermocycles to reach a certain threshold until their fluorescence signal is sufficiently detectable. Therefore, the sample concentrations can be discriminated by comparing the time points at which a defined fluorescence level is reached.
This threshold is plotted as a horizontal line in real-time graphs and this way, the respective threshold cycles (cT) can be easily compared and related to the initial concentrations at the sample.
Microfluidics Application: Polymerase Chain Reaction PCR
The polymerase chain reaction (PCR) is a biochemical method designed to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
The technique was developed in 1984 by the American biochemist Kary Mullis who received the Nobel Prize for developing it in 1993. The technique amplifies specific DNA fragments from minute quantities of source DNA material, even when that source DNA is of relatively poor quality.
PCR has developed into the standard method for the identification and replication of DNA strands and is nowadays a key element in modern techniques of genetic analysis which finds its application ranging from pathogen detection to genetic fingerprinting in forensic laboratories.
Due to its high specificity, PCR is playing now a major role in future individualized medicine, namely in point-of-care diagnostics (POC).
The principle of PCR is the multiplication of defined strands of DNA with the use of enzymatic polymerase molecules. The duplication of a DNA strand is carried out in three principle steps:
– Denaturation: In the first step, the mix is heated to 95 ºC so that the hydrogen bonds that stabilize the original double stranded DNA break and it splits in two single-stranded DNA-molecules.
– Annealing: This step takes place at lower temperatures, between 50 and 65°C, and once the temperature is reduced, the small complementary DNA-molecules (so-called primers) attach themselves to defined sites of the single-strand DNA molecules (“prime annealing”).
– Elongation: The temperature is raised up to 72 ºC, specific to the thermostable DNA polymerase. The polymerase molecules attach themselves to the single-stranded DNA and form the complementary DNA strand (“primer elongation”), leading to a final identical copy of the original double-stranded DNA.
These two molecules then undergo the same cycle again and again, which leads to an exponential growth of the number of identical DNA strands which at the end of the process exists in a sufficient amount to be used for further analysis or other purposes.
Microfluidic Chips Manufacturing: Product workflow
From microfluidic design and prototype fabrication to final validation and mass production
In microLIQUID, these are the main steps we work with our customers. The design of the microfluidic architecture, based on the needs of the partner, is the spark of the project management process.
Once the microfluidic device design is accepted and developed, the second phase is the prototyping where the cost and the flexibility are key elements. We have the range of technologies to reach both objectifs
After the microfluidic prototypes are defined, we have to check that the design and the prototype meets the requirements, test the microfluidics and the biology/detection components to see they are working as expected. If so, we end with a final design of the microfluidic architecture.
This final design is the starting point for medium and mass production processes, where the customer has the concept tested, and the proximity to the market allows higher investments.
The final material(thermoplastics) , the architecture and the biological tests need to be decided, and these elements define the production process of medium batches, like hot embossing or photolitography.
If all the elements are clear, the injection moulding solution is the cheapest option for mass production, where, once the first investment is done, the replication of the microfluidic structures is cost effective and replication can be obtained.
To obtain the replication, the validation of the microfluidics, biology, design, detection and functionality are the final steps.