From micro-total-analysis-system (MTAS) to neurons-on-chip – the application range is broad and has given major new insights into the spread of the disease on a patient level as well as on a molecular scale. Most important findings were, that the size of the protein aggregates found in patients and in in-vitro assays is related to their toxicity. Especially the larger aggregated protein assemblies (oligomers) are considered as the damage causing species as described in an interview with the biotech company Fluidic Analytics¹.

This fact is the main driver in the research field for the development of protein sizing technologies of higher precision and specificity. In particular – since a single misfolded protein can corrupt others and spread the disease – we, therefore, seek methods to detect these specimens on a single-molecule level. Microfluidics has provided powerful tools for protein misfolding disease research: Microdroplet devices, diffusional sizing devices and electrophoresis-on-chip have proven invaluable for the study of protein aggregates (See Fig.1 A-C). However, to truly reach MTAS and analyze protein solutions on the single-molecule level, the nanofluidic regime opens up a totally new set of applications which are challenging to achieve or not possible at all with conventional microfluidics.

Fig. 1: Illustration of micro-, and nanofluidic chip designs used in protein misfolding disease research (A) Microdroplet generators are used to separate single cells (FACS) or confine protein into independent experiments in micron-sized droplets; (B) Diffusional-sizing devices use the spread of a sample injected in the centre of a microfluidics channel and its developing diffusion profile to estimate its hydrodynamic radius; (C) Electrophoresis on chip allows to measure the charge of molecules flowing through a perpendicular applied electric field; (A*) Smaller channel cross-sections allow the generation of nanodroplets; (B*) Diffusional sizing can be facilitated by observing single molecules diffusing in nanochannels between two reservoirs. If channels widths reach the size of proteins also filtration becomes possible; (C*) Electrophoretic trapping in nanofunnels can be used to concentrate and capture charged molecules in solution (e.g. DNA);

The new chip designs that arise from smaller channel widths are similar to microfluidic chip layouts but allow to significantly decrease the needed sample concentrations to the femtomolar range and the usage of new physics by reaching the nanoscale (See Fig.1 (A*)-(C*)). Nanodroplet maker chips (A*) produce even smaller independent nanoreactors for massive parallelization of drug testing or drug encapsulation. Nanochannels (B*) in between two microfluidic reservoirs can be used to measure single proteins in solution as they propagate from one reservoir to the other. This allows to measure the sizes of protein monomers (approx. 0.5 nm) up to macromolecular assemblies such as oligomers (2-10 nm), exosomes (40-100 nm) or viruses (80-120 nm) in solution, without permanent surface immobilization and a relatively simple chip design. Nanofunnels (C*) concentrate charged proteins and short stranded DNA (e.g. µDNA) in solution by application of an electric field, which facilitates an easier detection of these with optical setups. Especially for the analysis of protein solutions, nanochannels have the major advantage of eased experimental setup and conduction of experiments. Microfluidic diffusional sizing devices need external machinery such as multiple-precision syringe pumps, optical microscopes, high-voltage power supplies and standardized flow measurement protocols to allow for reliable protein sizing measurements. With a nanochannel device, a single 50 µl injection manually pipetted into a chip mounted on top of a commercial microscope can be used to evaluate e.g. antibody binding events with the highest precision at femtomolar concentrations. This decreases the total sample consumption by orders of magnitude, increases throughput and eases the training of staff for conduction of single-molecule experiments drastically. However, the availability of nanofluidic devices is currently limited to research laboratories and companies² with expensive clean-room facilities or researchers with expertise in nanofabrication³– where high prototyping costs put additional constraints.

A way to circumvent this matter is soft lithography⁴, which allows a cost-effective fabrication of disposable nanofluidic devices from a single master wafer. Recently, we demonstrated the scalable integration of nanofluidic functionalities into existing microfluidic designs using two-photon lithography as an effective alternative fabrication method in comparison to conventional electron beam lithography⁵. Commercial 2-photon lithography systems (e.g. Nanoscribe’s Quantum X and Photonic Professional GT2) are available and provide a sophisticated way to produce nanofluidic master wafers, but open-source systems can also be found in the community, and provide an even more cost-effective nanolithography solution for master fabrication and soft lithographic chip imprinting without cleanroom facilities.

Commercialization potential in nanofluidics, therefore, lies within three sectors: Firstly, in the application of nanofluidic devices for biotechnological diagnostics and antibody development e.g. antibody testing to specific targets such as viruses, exosomes or protein complexes in solution at minimal sample consumption. Secondly, in the cost-effective fabrication and distribution of nanofluidic devices tailored to customer’s needs using fast and flexible fabrication techniques and thirdly in the key-knowledge transfer of chip architecture and physical effects happening on the nanoscale (similar to PCB design parameters known from the electronics industry) which can be exchanged by consulting services. However, for all of this to be economically relevant, the fabrication and prototyping need to be realized in a cheap and scalable manner without expensive cleanroom facility maintenance costs and at faster design-to-device delivery times than conventional chip industry allows. The combination of two-photon lithography with soft lithography, therefore, provides an effective integration of nanofluidics into existing microfluidic designs and paves the way for a broad implementation of nanofluidic chips for various applications related to neurodegenerative disease research and cancer diagnostics.

Neuron image photocredit: Oliver Vanderpoorten and Colin Hockings

References

<sup>1. Ruairi J MacKenzie, „Size Matters: Diffusion Technique Sorts Out Pathological Proteins”, NNR, https://www.technologynetworks.com/neuroscience/blog/size-matters-diffusion-technique-sorts-out-pathological-proteins-322669

2. Wunsch, B., Smith, J., Gifford, S. et al. Nanoscale lateral displacement arrays for the separation of exosomes and colloids down to 20 nm. Nature Nanotech 11, 936–940 (2016). https://doi.org/10.1038/nnano.2016.134

3. Levin, S., Fritzsche, J., Nilsson, S. et al. A nanofluidic device for parallel single nanoparticle catalysis in solution. Nat Commun 10, 4426 (2019). https://doi.org/10.1038/s41467-019-12458-1

4.Qin, D., Xia, Y. & Whitesides, G. Soft lithography for micro- and nanoscale patterning. Nat Protoc 5, 491–502 (2010). https://doi.org/10.1038/nprot.2009.234.

5. Vanderpoorten, O., Peter, Q., Challa, P.K. et al. Scalable integration of nano-, and microfluidics with hybrid two-photon lithography. Microsyst Nanoeng 5, 40 (2019). https://doi.org/10.1038/s41378-019-0080-3.</sup>

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Oliver Vanderpoorten

Oliver Vanderpoorten is part of the Centre for Misfolding Disease at the University of Cambridge where he conducts research on nanolithography and nanoscopy methods. He holds two master degrees in engineering subjects which give him an applied attitude towards biotechnological applications: He holds a Master in Electrical and Microsystems Engineering from OTH Regensburg (2015) and a second Master’s degree in Sensor Technologies from the University of Cambridge (2016). During his PhD in the EPSRC funded Sensor CDT he explored the landscape of nanofluidic chips for various applications related to life sciences and protein misfolding diseases and developed 2-photon lithography as fabrication method for nanofluidic devices. In his free time he’s interested in entrepreneurship, sailing, painting and spends his time with outreach to explain science to the public in an informal way (https://youtu.be/TPJPYvumErM?t=100).

The post Bridging the micro to the nanoscale – how to facilitate the transition to nanofluidic devices as new standard in life sciences appeared first on The MicroFluidic Circle.

In general, PDMS belongs to the silicone family with some unique features which have turned PDMS to the most desirable material in microfluidics. Transparency, biocompatibility, flexibility (Young’s elastic modulus of ~ 1 – 3 MPa), high gas-permeability, low dielectric constant, low surface tension, and low solubility are some of PDMS characteristics.

The PDMS strength stems from its capability in soft lithography. First, PDMS base monomer is thoroughly mixed by its curing agent, then this prepolymer is degassed to remove all air bubbles, and finally, it is ready to be poured on the mold. The mold can be made by conventional methods such as photolithography or newer technologies such as 3D printing. Regardless of the mold type, PDMS can replicate features on the mold from macroscale to nanoscale. After curing and peeling off the PDMS replica from the mold, the PDMS part should be sealed by a flat surface. Here, another advantage of PDMS fabrication comes to play. There are a variety of techniques to seal a PDMS replica such as conformal contact, physical bonding, vacuum bonding, oxygen plasma bonding, corona surface activation, flame bonding, wet-bonding, adhesive bonding, and to name a few. Some of these methods are reversible, and some are irreversible which can be chosen based on applications. In most of these techniques, there is no need to use a chemical or solvent to achieve the sealing. The chemical- or solvent-free bonding of PDMS to PDMS substrates or other substrates eliminates any chance of chemical contamination to tested samples.

PDMS soft lithography allows researchers to fabricate devices with multilayers of PDMS. This process is called “sandwiching,” meaning that several layers of PDMS replica can be stacked on top of each other to build a more complex geometry. In sandwiching, other components such as membranes (porous or non-porous) can be added between layers to create the desired device. There are different ways to bond these membranes to PDMS: (1) membranes first can be treated by a silane molecule (such as 3-aminopropyltriethoxysilane), and then both treated membrane and PDMS are exposed to oxygen plasma and be bonded, (2) some adhesive (double-sided tapes or adhesives) may be used to attach membranes to PDMS if no high pressure is sought, and (3) silicon dioxide (SiO₂) can be coated on a membrane by sputtering and be bonded to PDMS using oxygen plasma.

There are a lot of strategies to tailor the bulk or surface properties of PDMS. This allows researchers to modify their PDMS devices based on their needs. For tailoring bulk properties of PDMS, these methods can be used: (1) changing the ratio of base and curing agent, (2) playing with curing conditions (temperature and time), (3) adding other molecules to PDMS, and (4) adding fillers such as SiO₂ to PDMS. Playing with these parameters can impact the bulk properties of PDMS such as elasticity, transparency, photothermal effect, refractive index, electrical conductivity, and to name a few. The review paper which was written by P. Wolf et al., comprehensively reviews all different techniques for PDMS bulk or surface modification. In most scenarios, surface modification of PDMS is the area of interest in microfluidics. The most common way is to use oxygen or air plasma, corona discharge, and ultraviolet light or ozone exposure to introduce silanol (Si-OH) groups to the surface of PDMS at the cost of methyl groups (Si–CH₃). This results in forming a thin layer of few nanometers on the surface (it may contain some cracks as well) and decreasing water contact angle to lower than 5°. When a long-lasting surface modification strategy is desired, the surface of PDMS can be tailored by polydopamine (PDA), various polyethylene glycol (PEG) derivatives, or silane-based molecules. Hydrophobicity or hydrophilicity of PDMS surfaces can be easily rendered using one of these methods. There is a broad range of strategies and techniques to tailor PDMS properties in the literature which can be considered as one of the advantages of PDMS over other materials for microfabrication.

Tailoring the bulk properties of PDMS to improve its conductivity has become a topic of research in flexible and stretchable electronics. Different fillers such as carbon nanotubes, graphite, silver particles, nanowires, and gold nanotubes have been added to PDMS to reach higher conductivity. As PDA is conductive, it has been coated on PDMS to integrate electrochemical sensors for various applications.

With the origin of organ-on-a-chips and microfluidic cell culture platforms, PDMS has gained more attractions from researchers from other fields. As a result, the surface of PDMS has been coated with PDA, gelatine, collagen, or fibronectin to enhance the adhesion, proliferation, and the growth of cells inside a chip. Besides, the surface of PDMS first can be modified by PEG or a silane molecule or PDAas linker, and then a bioactive molecule can be attached to the linker for achieving specific applications such as differentiation or detection of a secreted biomolecule by cells.

PDMS is permeable to gases such as oxygen and carbon dioxide. This led to the origin of microfluidics blood oxygenators in which gas exchange between blood and air is needed.PDMS membranes can be easily fabricated by spin-coating wet PDMS on a flat substrate such as a wafer. The properties of the PDMS membrane can be tuned by changing the speed of the spinner, curing agent ratio, and curing temperature. PDMS membranes as thin as ~ 1 µm can be produced without observing pinhole defects. When the permeability should be avoided, the thickness of PDMS can be increased over 1 mm.

In a nutshell, PDMS has shown its potential in the realization of so many applications in microfluidics. Microfabrication with PDMS enables us to fabricate devices with simple designs to devices with very complex features. PDMS has been extensively studied, and this provides fantastic resources for everyone to optimize PDMS properties based on their needs.

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Mohammadhossein Dabaghi

Mohammadhossein Dabaghi is currently a postdoctoral fellow at the department of medicine at McMaster University. He completed his Ph.D. at McMaster University majoring in Biomedical Engineering and worked on new microfabrication technologies to develop an Artificial Placenta device for preterm neonates with respiratory failure. He earned a master’s degree in Biomedical/Chemical Engineering (2014), and a BS in Chemical Engineering from Sharif University of Technology, Iran (2012). His research interests focus on lung-on-a-chips, biosensors for organ-on-a-chips, and bioprinting.

• https://www.researchgate.net/profile/Mohammadhossein_Dabaghi2

The post PDMS and Its Role in The Realm of Microfluidics appeared first on The MicroFluidic Circle.

Preterm (premature) babies with RDS (respiratory distress syndrome) require additional respiratory supports to let their lungs develop. Usually, mechanical ventilation is the first option to deal with RDS. If mechanical ventilation is not sufficient, another life support device, named extra-corporeal membrane oxygenation (ECMO) machine, will be used. In ECMO, the blood is pumped outside of the body and oxygenated by a gas exchange device called the blood oxygenator. Both methods are invasive and can cause severe long-term side-effect and complications. In mechanical ventilation, positive pressure applied in the lungs can seriously damage the tissue of the lungs. ECMO may be less destructive to the lungs but it has own issues, especially for those babies with extremely low birth-weight. Generally, ECMO devices need a high volume of blood to be primed (for instance around 40 mL for the device such as Quadrox-i Neonatal & Pediatric), however, a one-kilogram preterm baby has only about 100 mL of blood. As a result, donor blood from adults needs to be transfused to keep the whole system operational. Nonetheless, baby blood and adult blood have different physiology in which red blood cells (RBCs) of newborn babies have a higher affinity to oxygen compared to RBCs in adults. Additionally, surgery is required to get access to the large vessels and a pump is needed to keep blood flowing in the circuit which may damage RBCs and lead to hemolysis and thrombus formation.

Artificial Placenta as an Alternative Solution

The artificial placenta (AP) concept refers to a postnatal lung assist device (LAD) which is connected to a premature baby via the umbilical cord and can provide additional gas exchange besides the baby’s lung. The AP concept has been around for a long time back to 1961 when the first attempts were made by Callaghan et. al. However, there is still no commercial AP device in clinics due to inefficiency and incompatibility of available LADs for this purpose. An ideal AP device should be operable by a baby’s heart instead of an external pump while provides sufficient gas exchange in room air. Also, such a device should have very low priming volume to eliminate any need for donor blood transfusion and be filled only with saline solution. In addition, the volume of needed saline should not surpass 10 percent of the total blood of the baby’s body to avoid blood dilution. The first and main job of an AP device is to compensate for the loss in gas exchange caused by under-developed lungs. Also, some effort was made to expand AP application more and provide nutrition to premature babies and remove the metabolic waste at the same time.

Conventional blood oxygenators (used for ECMO) which are usually made from hollow fiber membranes are not suitable for artificial placenta application as they have high priming volume and need a pump for operation. A hollow fiber membrane (HFM) device is made by twisting bundles of hollow porous tubes (fibers) in a plastic-made shell. Usually, oxygen is introduced to these fibers and blood flows around these porous fibers where oxygen transfers to blood and carbon dioxide is released from the blood. Because of the nature of the hollow fiber bundle, the blood experience a non-physiological travel which may damage RBCs and initiate clotting. Also, there is a great potential of blood leakage through pores or contamination of the blood. An ECMO device is connected to the body in venous-artery or venous-venous configuration where a pump is needed to remove blood from the body and send it back to the ECMO device and vice versa.

It should be noted that an AP device is not going to operate in the same way that an ECMO machine works. In fact, an AP device is connected to a baby’s body in parallel to the blood circulatory system wherein the inlet of the device is attached to an artery and the outlet to a vein. This particular type of arterio-venous connection causes the blood entering the device has already been traveled through the baby’s lungs and be partially oxygenated. Under-developed lungs of a premature baby cannot fully saturate the blood with oxygen, whereby the oxygen saturation level may only be 10%–30% below full saturation. Therefore, an AP device only needs to raise the oxygen saturation level from ~ 70 % to 100 % which is higher than the amount of oxygen saturation level entering an ECMO device.

The Solution

Over the last decade, researchers have been using microfabrication technologies to overcome the shortcoming of HFMs and trying to introduce the next generation of blood oxygenators. Microfluidic blood oxygenators have shown promising improvement in gas exchange compared to convention ECMO devices over the last decade due to mimicking nature lungs by reducing the size of blood channels as small as capillaries in the lungs. Additionally, microfluidic blood oxygenators provide a more physiological flow path to the blood which is critical in designing an AP device for the long-term application.

Recently microfluidic blood oxygenators have been specifically designed to address the needs of the neonates and have been developed with low priming volume, low-pressure drop to operate pumpless, and capable of working in the ambient air. Rochow et. al.used microfluidic blood oxygenators to construct such a LAD for premature babies suffering from RDS. Their device had a low priming volume of 4.8 mL and could be successfully connected to the umbilical vessels of newborn piglets and perfused solely by the pressure differential provided by the animals’ heart. Although the gas exchange of this LAD was not sufficient enough but they showed that a pumpless LAD with low priming volume was not a fantasy anymore!

Challenges

Truly, the future belongs to blood oxygenators advancing microtechnologies owing to their unique design so much similar to nature lungs. However, they need to overcome some challenges to be able to compete with HFMs and replace them in the markets. The main challenges are listed below:

1. Scaling up: the main and most important limitation of all microfluidic blood oxygenators is their small size which constrains their operating blood flow rate below 10 mL/min. However, an AP device may need to support a higher blood flow rate up to 60 mL/min. Different strategies have been used to address this issue such as increasing the size of the device or connecting several devices together to increase the total capacity. It seems like a combination of both should be used to reach the desired gas exchange.

2. Hemocompatibility: although different approaches such as coating the surface of PDMS by antithrombin-heparin (ATH) or polyethylene glycol (PEG)have been used to improve the hemocompatibility of the blood-contact surfaces, more investigation is required to evaluate this coating reliability for a long-term condition as needed for an AP device.

3. Access to large bore vessels: an AP device is supposed to be connected to a premature baby via the umbilical cord. The main challenge is the constriction of the umbilical vessels right away after birth. As a result, a catheter is required to be inserted into the vessels and keep the vessels open and let blood flow with minimum resistance to blood flow. As Peng al. explained, a special type of catheter should be used for getting access through the umbilical cord to be able to minimize the resistance to blood flow and provide the maximum possible blood flow rate for an AP device.

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Mohammadhossein Dabaghi

Mohammadhossein Dabaghi is currently a postdoctoral fellow at the department of medicine at McMaster University. He completed his Ph.D. at McMaster University majoring in Biomedical Engineering and worked on new microfabrication technologies to develop an Artificial Placenta device for preterm neonates with respiratory failure. He earned a master’s degree in Biomedical/Chemical Engineering (2014), and a BS in Chemical Engineering from Sharif University of Technology, Iran (2012). His research interests focus on lung-on-a-chips, biosensors for organ-on-a-chips, and bioprinting.

• https://www.researchgate.net/profile/Mohammadhossein_Dabaghi2

The post Breathing Like in the Mother’s Womb appeared first on The MicroFluidic Circle.