Flow Cytometry : Advantages of using Microfluidics
Positive aspects about Flow Cytometer based on Microfluidics:
1. To have a high purity and recovery for the sorted cell population.
2. To sort based on an intracellular characteristic in which magnetic beads would not have access.
3. To sort cell labelled with fluorescent probes for nuclear or other intracellular targets.
4. To have information about cell surface molecules.
5. To sort different receptors even if they are low in density.
6. To sort cells according to absence, density or presence of the receptors.
Flow Cytometry using Microfluidics
Flow cytometry is a laser-based technology used in cell counting, cell sorting, biomarker detection or protein engineering.
By suspending cells and passing these suspending cells by passing them using an electronic detection machine, flow cytometry is done.
This technique allows making simultaneous analysis of the different characteristics of the more than a thousand particles per second.
This technique is usually used in the diagnosis of health disorders, in most of the cases blood cancers, but it is used in many medical trials, such as to sort particles according to their properties or purify populations of interest.
Microfluidic Manufacturing Technique: CNC Micromachining
CNC Micromachining is defined as the removal of material at micro level.
During the last year the interest over the micro machining technology has increased.
Due to this, every manufacturing and industry segment has started to work, segments such as aerospace, automotive world or medical appliances.
Even , nowadays, there are still several technical challenges the potential for product miniaturization continues to grow.
The micromachining technologies involves to work with features smaller than 0,001”, that is why, it is necessary to work with accuracy in the 0,0001” or less range, using always cutters smaller than 1/8 or about 3mm. It takes significant speed to effectively use such small-diameter tools, and the machines have to be, as said before, very accurate.
Taking into account the microfluidic field of technology, we can say that micromachining can be used in this field, because recent developments in microfabrication enables the integration of hard and soft structures, making possible to control the microfluidic systems structures. This structures can be applied to drug delivery.
Moreover, there is a manufacturing method which involves laser micromachining for the structure of microfluidic channels in a thin metallic sheet.
It is important to say that some polymers are better to use over silicone when building microfluidic devices because they have biocompatible properties as well as cheapness. Also, using micromachining it is possible to make fewer processing steps than using the conventional way.
Microfluidic Event in Spain
Also the international approach with 34% of delegates attending from outside of Spain and fully business oriented. Is one of the largest Forum in the world with more than 850 companies coming and in one to one meetings, over 3.000 every edition.
The meeting will be in Bilbao, in the Bilbao Exhibition Center from the 28th to the 30th of September and here you can find the schedule/program: http://www.biospain2016.org/Program
microLIQUID will be there with one stand, within the Biobasque pavilion where we will show our latest developments and services. It is a great opportunity to meet and explain our capabilities for the market.
Options for collaboration/partnering:
For more information: [email protected]
Microfluidics Application: Point of Care Devices
Our aim is to provide portable diagnostic tools to ensure rapid, affordable and simple analysis in many scenarios of our society (hospitals, airports, doctor’s practice, roadside police controls, natural environment etc).
However, conventional analytical methods often require a large volume of sample and complicated time-consuming protocols.
The more portable ones are based on slow immunochromatographic strips or low-sensitivity electrochemical detection systems, whereas desktop systems are sensitive and semi-automatic but bulky and heavy.
microLIQUID tries to improve quality of life and medical services through the development of quick diagnostic devices that will carry out sample preparation and detection reducing the incidence of current society threats.
Our idea is to create intelligent and portable systems across many sectors for efficient treatment(environment monitoring, health, food , veterinary), by integrating cost-efficiently manufactured Lab-on-a-Chips.
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).
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.
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.
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.