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.
Microfluidics Manufacturing: Clean Room Facilities
microLIQUID has in-house the means to work in microfluidics and medical devices. Our main facilities are:
INJECTION MOLDING CLEAN ROOM – ISO 7&8 for silicone and thermoplastic components.
EXTRUSION CLEAN ROOM – ISO 7&8 for silicone tubing and silicone and thermoplastic catheters.
LABORATORY AND TESTING CLEAN ROOM – for project development & testing activities.
These means allow the company to develop:
Integration of the microfluidic manufacturing with biotechnological process treatments in the same manufacturing line ( surface treatmentes, Ag/Ab immobilization, freeze-drying etc) , Point of Care and Point of use Microfluidic Cartridges
Also, the company has the means to characterise and validate the microfluidic functionality required for final applications, with a Lab dedicated
microLIQUID has developed products for Biotech companies where the microfluidic development is done at the same time that the packaging and the equipment respecting the automation and the miniaturization (E.g. qPCR, test ELISA etc)
In our 400 m2 Clean Room Facility , the company does:
Design and simulation
Some Activities & Processes:
Hot Embossing etc…..
Thermoplastics for Microfluidic Chips
Polymers for Microfluidic Applications offer a broad range of attractive properties and have thus been considered for Lab-on-a-Chip systems since the late 90’s.
Common thermoplastics such as polyethylene (PE), polystyrene (PS), polyethylene terephtalate (PET), polypropylene (PP), polycarbonate (PC) or cyclic olefin (co)polymers (COC/COP) are widely available as monolayer foils.
Polymers consist of single macromolecules that are iteratively linked to each other forming constituting long chains. These chains are arranged in linear or branched fashion in the polymer matrix.
Knowledge on the temperature behaviour is important for polymer processing. Heating a polymer matrix induces energy that leads to breakage of secondary valences between adjacent polymer chains. Above certain temperature, these chains are free to slide along each other resulting in higher chain mobility and thus elasticity. The temperature required to soften the material is called glass transition temperature Tg.
Thermoplastics are a highly attractive substrate material for microfluidics systems, with important benefits in the development of low cost disposable devices for a host of bioanalytical applications.
Thermoplastics are a class of synthetic polymers which exhibit softening behavior above a characteristic glass transition temperature (Tg) resulting from a long-range motion of the polymer backbone, while returning to their original chemical state upon cooling. Thermoplastic polymers differ from elastomer or thermoset plastics by their ability to be softened or fully melted and reshaped upon heating, while remaining chemically and dimensionally stable over a wide range of operational temperatures and pressures.
More recently cyclic olefins (COC and COP) have emerged as highly attractive microfluidic materials, with high optical clarity into the deep-UV range (~250 nm), low water absorption, and exceptionally good resistance to solvents including organics such as acetonitrile commonly used in liquid chromatography.
Microfluidic Chip using Bonding Technique
Bonding process : The sealing of the open microchannels is necessary to produce the final enclosed fluid paths, and thus a critical step in the fabrication process invariably involves bonding a caping layer to the microchannel substrate. Bond strength is a critical consideration and bond interfaces must provide suitable chemical or solvent compatibility to prevent degradation during use, without compromising dimensional control of the microchannels due to deformation during the bonding process.
Other important considerations for the bond interface include surface chemistry, optical properties, and material compatibility and homogeneity of the channel sidewalls.
Additional issues such as manufacturability and compatibility with off- microfluidic chip interconnects can limit the selection of bonding methods.
Microfluidics bonding techniques may be categorized as either indirect or direct. Indirect bonding involves the use of an adhesive layer to seal two substrates and encapsulate microchannels fabricated in one or both of the substrates. In contrast, direct bonding methods fix the two substrates without any additional materials added to the interface.
While in direct bonding the bulk polymer itself comprises the adhesive giving a as a result microchannels with homogeneous sidewalls, indirect bonding methods require an intermediate adhesive that results in channel sidewalls with different chemical, optical and mechanical properties than the bulk polymer.
In general, bonding forces between mating surfaces arise from either molecular entanglement or charge interactions. Entanglement can occur by mechanical interlocking of diffusion between surfaces, while bonding due to charge interactions can result from electrostatic or chemical (covalent) bonding, acid-base interactions, or van der Waals forces.
Thermoplastic bonding methods like thermal fusion bonding, solvent bonding, localized welding and surface treatment and modification bonding are mainly achieved by molecular entanglement.
Adhesive bonding is achieved from charge interactions. In most cases bonding at high temperatures can greatly enhance polymer entanglement and interaction at the bonding interface resulting in high bong strength. However, bonding methods for microfluidic chips must be adapted and optimized for the task of enclosing micron-scale fluidic channels without excessive deformation of the channel cross sections.
Sealing injected pieces, both flat and structured pieces, is one of our well-established fabrication procedures. Alignment accuracy is around units of microns and the bonding yield reaches the 97% of the total area.
We are sealing polymers such as COC, COP, PMMA, PET and also PS, PC, but we are able to create a bonding setup for any polymer needed, doing short series or mass production.
This setup enables:
1. Accurate Flatness (Deformation ± 1 micron)
2. Ideal products for optical applications
3. Impurities-free product, done in clean room
Microfluidic Manufacturing Technique: Hot Embossing for Microfluidics
Hot Embossing technique used in microfluidics is a prototype and production method for creating microfluidic structures.
The first step is to create the SU8 mold of the structures you want to stamp in the polymer chip. The resolution you want to get is based on the mould, so the mask to choose is a key element. Resolutions of structures under 10 micron, a chromium mask is needed.
Once the mold is defined and manufacture, the hot embossing process starts. The temperature to reach is very important here. The objective is to get a certain temperature in the process, where the material (a polymer) reaches the glass transition temperature(called Tg and is different for each polymer)
At this temperature the polymer is very “malleable”, so you can set and replicate the SU8 structures of the mould in the polymer.
Once you cool the material above the Tg Temperature it becomes hard and is alike the glass.
With this technique you are able to replicate complicated microfluidic structures in the different materials(polymers) we use, and create different designs/ microfluidic chip from one mold.
Microfluidic Manufacturing Technique: Xurography
Microfluidic Lab on a chip (LOC) technology opened the possibility of handling very small volumes, bringing about the opportunity to analyze samples that were previously beyond our reach.
In addition, it has proven to have the capacity to increase both speed and sensitivity, which combined with the fact that it is a tool on the same scale as the single cell and many fundamental biological processes makes LOC a well suited tool for investigation and manipulation of these processes.
During the last decade, there has been enormous amount of research towards finding the best material, simplifying fabrication techniques, improving biocompatibility and miniaturizing the device scale in order to develop devices which are more efficient, cheaper, faster, and have a higher throughput. In this framework, the need for a fabrication tool that speeds up the research work becomes clear.
Xurography is a prototyping technique that employs a knife plotter to structure thin foils.
This technique uses a cutting plotter traditionally used in the sign industry for cutting graphics in adhesive vinyl films
Manufacturers specify the resolution of the cutting plotters in terms of mechanical and addressable resolution. The mechanical resolution specifies the resolution of the motors, while the addressable resolution is the programmable step size.
There are three types of cutting methods, and the specific one is chosen as a function of the application.
– Drag knife: Drag knife cutting uses a swivel blade that follows the cutting path of the feature as it moves relative to the material. This introduces lateral force from the blade at sharp feature corners, which can break the tip when cutting harder or thicker substrates.
– True tangential: Controls blade position with an addressable motor. When cutting corners, the blade lifts completely out of the material and rotates to the new direction. Line segments can be over-cut to ensure the material is completely cut from top to bottom at feature corners. This is useful when cutting thick materials.
– Emulated tangential: Uses a swivel blade but lifts the blade just to the surface of the material before pivoting on the tip at a feature corner. This reduces lateral force on the blade. Over-cuts in emulated tangential plotters bring the blade into position before initiating a cut and ensure feature corners are completely severed from the rest of the material.
Microfluidic Chip: Lab on a chip solution
Microfluidic Lab on a Chip (LOC) is a Device which integrates one or several laboratory functions on a single chip of only milimeters to few centimeters in size and that are capable of handling extremely small fluid volumes down to less than pico liters.
The main ADVANTAGES are:
– Low fluid volume consumption
– Faster analysis and response times
– Better controlled reactions
– Optimized heat and mass transfer
– Low fabrication costs
There is an increasing interest in MEDICINE , BIOLOGY and ANALYTICAL CHEMISTRY to develop these devices due to the flexibility, cost and speed.
Microfluidic Chips Manufacturing Technologies
microLIQUID uses several technologies for the fabrication of microfluidic structures in prototypes and serial production of microfluidic flexible chips.
For Microfluidic structures
Etching (Wet and dry).
Breaking and Dicing.
Bonding (thermal and anodic), we bond any polymer or substrate.
For Serial production
For Chip holders: microfluidic connectors