Scientists at EPFL have developed a technique that can be a game-changer for genetics by making the characterization of DNA-binding proteins much faster, more accurate, and efficient.
Genes hold the DNA code for producing all the proteins of the cell. To begin this process, genes require a huge family of DNA-binding proteins called transcription factors, which are of enormous interest to biologists today. However, we still know very little about many transcription factors because of their large number, their ability to combine into different pairs, and the difficulty of studying their DNA-binding properties in the lab. Now, EPFL scientists have developed a microfluidics-based technique that can greatly speed up the process, with only a minimum of materials. Publishing in Nature Methods, the researchers have already used it to determine the DNA-binding properties of over 60 transcription factors, including nine new ones.
Mammals — including humans — have between 1300-2000 transcription factors, many of which combine with others into “heterodimers” in order to bind genes and induce their transcription into RNA. Heterodimers are estimated to range between 3000 and 25,000.
Consequently, the number of possible combinations can be very high. But we also need to understand their DNA-binding properties, e.g. their affinity and specificity for DNA. This is key if we are to ever exploit transcription factors for biotechnological or pharmaceutical purposes in the future. But profiling transcription factors is a daunting and very complex task, since it requires relatively large amounts of hard-to-make transcription factors.
There are several databases available, but in total they cover only around 500 single transcription factors, and only a fraction of heterodimers. Progress in this field is slow, and the available profiles in these databases are not very good in predicting which genes these transcription factors work on.
A microfluidics approach
The lab of Bart Deplancke at EPFL’s Institute of Bioengineering has now invented a new technique called SMiLE-seq, which can greatly speed up the process with only tiny amounts of transcription factors. The technique uses microfluidics, which control tiny amounts of liquids in equally tiny spaces. Microfluidics is fast-becoming an area of excellence at EPFL, bringing together a number of different fields and disciplines.
SMiLE-seq works by attaching small amounts of the transcription factor (or factors when studying heterodimers) in a microfluidic device — this is a chip with micrometer-size channels that allow liquid to flow through. Once the transcription factors attach to the chip’s surface, a large library of random DNA is pumped into the chip and flows over them. This allows the transcription factors to recognize their corresponding DNA sequences. The transcription factor-DNA complex is then physically trapped, while the DNA that is not bound is washed away.
Next, the bound DNA is taken off the device and prepared for sequencing to identify which part of it got caught by the transcription factors. This information is fed into specialized software that works out the DNA-binding properties of the transcription factors or heterodimers. In turn, this helps to better predict their DNA-binding profiles in living cells.
SMiLE-seq offers three advantages: First, it cuts down on the amount of transcription factors, as it only needs picograms of them. Second, it speeds up the process from days to less than an hour. And finally, SMiLE-seq is not limited by neither the length of the DNA target sequence, nor is it biased toward stronger affinity protein-DNA interactions.
Deplancke’s team used SMiLE-seq on 67 full-length human, mouse, and Drosophila transcription factors, successfully analyzing several that have never been studied before. He next plans to exploit the technique’s versatility for other molecules, e.g. RNA. His team has filed a patent, and a startup will take the concept of SMiLE-seq into the commercial world.
Learn more: Using microfluidics to improve genetics research
Method for moving fluids on a surface may find uses in condensers, microfluidics, and de-icing.
Researchers at MIT and elsewhere have developed a new way of driving fluid droplets across surfaces in a precisely controlled way. The method could open up new possibilities for highly adaptable microfluidic devices, as well as for de-icing technologies, self-cleaning surfaces, and highly efficient condensers.
The new system uses differences in temperature to push droplets of water or other fluids across a smooth surface, allowing precise control by simply turning heaters and coolers on and off. The finding is described this week in the journal Physical Review Fluids, in a paper by MIT associate professor of mechanical engineering Kripa Varanasi, professor David Quere at ESPCI in Paris, MIT postdoc Nada Bjelobrk, graduate student Henri-Louis Girard, Srinivas Subramanyam PhD ’16, and Hyuk-Min Kwon PhD ’13.
The differences in temperature on a surface, the researchers report, cause a change in the amount of surface tension across the droplet. That causes the droplet to move toward the direction that lowers its energy — the direction of higher surface tension. But this only works if the surface has been treated in a way that prevents droplets from getting pinned to it.
The surface treatment is one that Varanasi and his collaborators have been developing for years. It forms the basis of a startup company called LiquiGlide that is commercializing the technology for use in containers, such as ketchup bottles that can easily pour out all their contents.
This video shows the response of droplets on silicon surfaces, showing that untreated or textured surfaces do not allow the droplets to move in response to temperature differences, but that on a surface with a lubricant on a textured surface, the droplets move easily. (Video courtesy of Henri-Louis Girard/Varanasi Research Group. Video has been sped up.)
The treatment consists of texturing a surface at microscale and then impregnating it with a layer of oil, which fills the spaces between the posts and becomes trapped there by capillary forces. This trapped lubricant makes the surface slippery for the droplets. Furthermore, the droplets have a relatively large contact area with the surface, allowing for a rather large temperature difference across the droplet and a higher propulsion force. In contrast, droplets did not move on superhydrophobic surfaces inspired by lotus leaves, as their contact area is too small for the temperature gradient to be sufficient to move the droplet.
The basic effect this team is exploiting, called thermocapillary motion, has been demonstrated before by other researchers, but in those cases the process required very large temperature differences, and even then produced only very slow movements, making it unsuitable for most practical applications. The new system, with its slippery surface, requires much smaller temperature changes and significantly speeds up the movement of the droplets, propelling them up to 10 times faster.
“There have long been attempts to use thermocapillarity to propel water droplets on surfaces,” Varanasi says, but only now “can water droplets be moved at appreciable speeds,” which would be especially useful for many applications.
The underlying physics is similar to that of “tears” seen in wine glasses, where differences in surface tension caused by evaporation of alcohol can cause droplets of wine to travel upward along the side of the glass. In this case as well, the thermocapillary movement is caused by differences in surface tension across parts of the droplet.
The finding might be used to produce new kinds of microfluidic devices, for example for biomedical or chemical testing. Instead of using fixed, physical barriers to direct the flow of liquid, these devices could use arrays of heating and cooling elements to change the configuration of flows rapidly, at will, by simply adjusting the regional temperature differences on the surface.
“You could move drops around, mix them, move them to reaction sites,” Girard says, and thus create a highly flexible and adjustable “lab on a chip.” The system also allows precise control over the speed of the moving droplets. “You could pattern heaters in two dimensions and make the droplets follow a maze,” he says.
The process might also find applications, the researchers say, in areas such as de-icing airplane wings and other surfaces, or developing powerful condensers. In power plants, for example, the faster droplets can be shed from condensing surfaces, the more efficiently the plant can run.
The concept might also find applications for research in space, in a microgravity environment where normal laboratory devices that depend on gravity to move liquids around wouldn’t work.
Initially the research was basically “curiosity driven,” Varanasi says, and it began with a discussion at a conference, where he and Quere sketched the concept on a napkin. Now, he says, by allowing for a series of precisely controlled experiments, this system “also allows us to really understand the physics of thermocapillarity” better than ever before.
“Moving droplets on superhydrophobic surfaces has been considered for a while,” says Neelesh Patankar, a professor of mechanical engineering at Northwestern University who was not associated with this research. “However, pinning of the contact line has indeed restricted droplet movement achieved on superhydrophobic surfaces,” he says. “This work demonstrates a remarkable five-fold increase in droplet speed if liquid-impregnated surfaces are used instead. I will look forward to translation of this approach to microfluidic devices.”
UC Santa Cruz engineers use flexible silicone material to build an integrated optofluidic platform for biological sample processing and optical analysis
For well over a decade, electrical engineer Holger Schmidt has been developing devices for optical analysis of samples on integrated chip-based platforms, with applications in areas such as biological sensors, virus detection, and chemical analysis. The latest device from his lab is based on novel technology that combines high-performance microfluidics for sample processing with dynamic optical tuning and switching, all on a low-cost “chip” made of a flexible silicone material.
In previous devices from Schmidt’s lab, optical functions were built into silicon chips using the same fabrication technology used to make computer chips. The new device is made entirely of polydimethylsiloxane (PDMS), a soft, flexible material used in microfluidics as well as in products such as contact lenses and medical devices.
“We can use this fabrication method now to build an all-in-one device that allows us to do biological sample processing and optical detection on one chip,” said Schmidt, the Kapany Professor of Optoelectronics and director of the W. M. Keck Center for Nanoscale Optofluidics at UC Santa Cruz.
The flexibility of PDMS allows for novel ways of controlling both light and fluids on the chip. Using multilayer soft lithography techniques, senior graduate student Joshua Parks built chips containing both solid-core and hollow-core waveguides for guiding light signals, as well as fluidic microvalves to control the movement of liquid samples. Schmidt and Parks also developed a special microvalve that functions as a “lightvalve,” controlling the flow of both light and fluids.
“That opens up a whole new set of functions that we couldn’t do on a silicon chip,” Schmidt said. “The lightvalve is the most exciting element. In additional to a simple on-off switch, we built a moveable optical trap for analysis of biological particles such as viruses or bacteria.”
Parks and Schmidt reported the results of initial experiments with the new device in a paper published September 6 in Nature Scientific Reports.
In a previous study, Schmidt, Parks, and colleagues at BYU and UC Berkeley demonstrated a hybrid device in which a PDMS microfluidic chip for sample preparation was integrated with a silicon-based optofluidic chip for optical detection of viral pathogens. The new device combines both functions on the same chip. In addition, Schmidt said, the materials are relatively inexpensive, allowing rapid prototyping of devices.
“We can do the full chain of fabrication here in our lab, and we can make new devices very quickly,” he said.
Schmidt said the potential applications for this technology include a wide range of biological sensors and analytical devices. For viral diagnostic assays, for example, fluorescently labeled antibodies can be used to tag specific viral strains for optical detection. In a recent paper, Schmidt and colleagues demonstrated detection and identification of different flu strains using fluorescence detection in a multi-mode interference (MMI) waveguide. With the new device, they showed that they can actively tune an MMI waveguide on the chip.
The dynamic tuning of the optofluidic device is achieved by applying pressure to the optofluidic channel, changing its dimensions and thereby altering its photonic properties. “We can actually tune the spot pattern made in the channel by the interference waveguide, which we couldn’t do with the silicon chip,” Schmidt said.
Powered by a chemical reaction controlled by microfluidics, 3D-printed ‘octobot’ has no electronics
A team of Harvard University researchers with expertise in 3D printing, mechanical engineering, and microfluidics has demonstrated the first autonomous, untethered, entirely soft robot. This small, 3D-printed robot — nicknamed the octobot — could pave the way for a new generation of completely soft, autonomous machines.
Soft robotics could revolutionize how humans interact with machines. But researchers have struggled to build entirely compliant robots. Electric power and control systems — such as batteries and circuit boards — are rigid and until now soft-bodied robots have been either tethered to an off-board system or rigged with hard components.
Robert Wood, the Charles River Professor of Engineering and Applied Sciences and Jennifer A. Lewis, the Hansjorg Wyss Professor of Biologically Inspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS) led the research. Lewis and Wood are also core faculty members of the Wyss Institute for Biologically Inspired Engineering at Harvard University.
“One long-standing vision for the field of soft robotics has been to create robots that are entirely soft, but the struggle has always been in replacing rigid components like batteries and electronic controls with analogous soft systems and then putting it all together,” said Wood. “This research demonstrates that we can easily manufacture the key components of a simple, entirely soft robot, which lays the foundation for more complex designs.”
The research is described in the journal Nature.
“Through our hybrid assembly approach, we were able to 3D print each of the functional components required within the soft robot body, including the fuel storage, power and actuation, in a rapid manner,” said Lewis. “The octobot is a simple embodiment designed to demonstrate our integrated design and additive fabrication strategy for embedding autonomous functionality.”
Octopuses have long been a source of inspiration in soft robotics. These curious creatures can perform incredible feats of strength and dexterity with no internal skeleton.
Harvard’s octobot is pneumatic-based — powered by gas under pressure. A reaction inside the bot transforms a small amount of liquid fuel (hydrogen peroxide) into a large amount of gas, which flows into the octobot’s arms and inflates them like a balloon.
“Fuel sources for soft robots have always relied on some type of rigid components,” said Michael Wehner, a postdoctoral fellow in the Wood lab and co-first author of the paper. “The wonderful thing about hydrogen peroxide is that a simple reaction between the chemical and a catalyst — in this case platinum — allows us to replace rigid power sources.”
To control the reaction, the team used a microfluidic logic circuit based on pioneering work by co-author and chemist George Whitesides, the Woodford L. and Ann A. Flowers University Professor and core faculty member of the Wyss. The circuit, a soft analog of a simple electronic oscillator, controls when hydrogen peroxide decomposes to gas in the octobot.
“The entire system is simple to fabricate, by combining three fabrication methods — soft lithography, molding and 3D printing — we can quickly manufacture these devices,” said Ryan Truby, a graduate student in the Lewis lab and co-first author of the paper.
The simplicity of the assembly process paves the way for more complex designs. Next, the Harvard team hopes to design an octobot that can crawl, swim and interact with its environment.
“This research is a proof of concept,” Truby said. “We hope that our approach for creating autonomous soft robots inspires roboticists, material scientists and researchers focused on advanced manufacturing,”
Learn more: The first autonomous, entirely soft robot
New chip could help test drugs for ALS, other neuromuscular disorders.
MIT engineers have developed a microfluidic device that replicates the neuromuscular junction — the vital connection where nerve meets muscle. The device, about the size of a U.S. quarter, contains a single muscle strip and a small set of motor neurons. Researchers can influence and observe the interactions between the two, within a realistic, three-dimensional matrix.
The researchers genetically modified the neurons in the device to respond to light. By shining light directly on the neurons, they can precisely stimulate these cells, which in turn send signals to excite the muscle fiber. The researchers also measured the force the muscle exerts within the device as it twitches or contracts in response.
The team’s results, published online today in Science Advances, may help scientists understand and identify drugs to treat amyotrophic lateral sclerosis (ALS), more commonly known as Lou Gehrig’s disease, as well as other neuromuscular-related conditions.
“The neuromuscular junction is involved in a lot of very incapacitating, sometimes brutal and fatal disorders, for which a lot has yet to be discovered,” says Sebastien Uzel, who led the work as a graduate student in MIT’s Department of Mechanical Engineering. “The hope is, being able to form neuromuscular junctions in vitro will help us understand how certain diseases function.”
Uzel’s coauthors include Roger Kamm, the Cecil and Ida Green Distinguished Professor of Mechanical and Biological Engineering at MIT, along with former graduate student and now postdoc Randall Platt, research scientist Vidya Subramanian, former undergraduate researcher Taylor Pearl, senior postdoc Christopher Rowlands, former postdoc Vincent Chan, associate professor of biology Laurie Boyer, and professor of mechanical engineering and biological engineering Peter So.
Closing in on a counterpart
Since the 1970s, researchers have come up with numerous ways to simulate the neuromuscular junction in the lab. Most of these experiments involve growing muscle and nerve cells in shallow Petri dishes or on small glass substrates. But such environments are a far cry from the body, where muscles and neurons live in complex, three-dimensional environments, often separated over long distances.
“Think of a giraffe,” says Uzel, who is now a postdoc at the Wyss Institute at Harvard University. “Neurons that live in the spinal cord send axons across very large distances to connect with muscles in the leg.”
To recreate more realistic in vitro neuromuscular junctions, Uzel and his colleagues fabricated a microfluidic device with two important features: a three-dimensional environment, and compartments that separate muscles from nerves to mimic their natural separation in the human body. The researchers suspended muscle and neuron cells in the millimeter-sized compartments, which they then filled with gel to mimic a three-dimensional environment.
A flash and a twitch
To grow a muscle fiber, the team used muscle precursor cells obtained from mice, which they then differentiated into muscle cells. They injected the cells into the microfluidic compartment, where the cells grew and fused to form a single muscle strip. Similarly, they differentiated motor neurons from a cluster of stem cells, and placed the resulting aggregate of neural cells in the second compartment. Before differentiating both cell types, the researchers genetically modified the neural cells to respond to light, using a now-common technique known as optogenetics.
Kamm says light “gives you pinpoint control of what cells you want to activate,” as opposed to using electrodes, which, in such a confined space, can inadvertently stimulate cells other than the targeted neural cells.
Finally, the researchers added one more feature to the device: force sensing. To measure muscle contraction, they fabricated two tiny, flexible pillars within the muscle cells’ compartment, around which the growing muscle fiber could wrap. As the muscle contracts, the pillars squeeze together, creating a displacement that researchers can measure and convert to mechanical force.
In experiments to test the device, Uzel and his colleagues first observed neurons extending axons toward the muscle fiber within the three-dimensional region. Once they observed that an axon had made a connection, they stimulated the neuron with a tiny burst of blue light and instantly observed a muscle contraction.
“You flash a light, you get a twitch,” Kamm says.
Judging from these experiments, Kamm says the microfluidic device may serve as a fruitful testing ground for drugs to treat neuromuscular disorders, and could even be tailored to individual patients.
“You could potentially take pluripotent cells from an ALS patient, differentiate them into muscle and nerve cells, and make the whole system for that particular patient,” Kamm says. “Then you could replicate it as many times as you want, and try different drugs or combinations of therapies to see which is most effective in improving the connection between nerves and muscles.”
On the flip side, he says the device may be useful in “modeling exercise protocols.” For instance, by stimulating muscle fibers at varying frequencies, scientists can study how repeated stress affects muscle performance.
“Now with all these new microfluidic approaches people are developing, you can start to model more complex systems with neurons and muscles,” Kamm says. “The neuromuscular junction is another unit people can now incorporate into those testing modalities.”
A new technique, developed at EPFL, combines microfluidics and lasers to guide cells in 3D space, overcoming major limitations to tissue engineering.
Future medicine is bound to include extensive tissue-engineering technologies such as organs-on-chips and organoids – miniature organs grown from stem cells. But all this is predicated on a simple yet challenging task: controlling cellular behavior in three dimensions. So far, most cell culture approaches are limited to two-dimensional environments (e.g. a Petri dish or a chip), but that neither matches real biology nor helps us sculpt tissues and organs. Two EPFL scientists have now developed a new method that uses lasers to carve out paths inside biocompatible gels to locally influence cell function and promote tissue formation. The work is published in Advanced Materials.
In the body, cells grow in 3D microspaces that are specific to each type of tissue – liver, kidney, lung, heart, brain etc. These microenvironments are important because they control the behavior of the cells, e.g. how they interact with other parts of the tissue to help it develop, function, and repair. In addition, the microenvironments themselves are very dynamic and adaptable, sending the cells various biochemical signals to adapt their behavior to physiological changes.
This means that any successful merging of biology and engineering must first be able to grow cells in custom-built yet biologically active 3D spaces. Working at EPFL’s Institute of Bioengineering, Matthias Lütolf and his PhD student Nathalie Brandenberg have developed a method that uses a laser to cut three-dimensional pathways and networks for cells inside a hydrogel scaffold that matches their natural environment.
The method combines lasers with microfluidics – the science of controlling fluids in micrometer-sized spaces. Here, the scientists used focalized short-pulsed lasers, which can generate enough power to create tiny tunnels in different biocompatible gels already used in cell biology and tissue engineering. The laser can be applied before or even during 3D cell culture, meaning that the cells can be controlled in real time to match their natural growth.