“The whole point of having these inexpensive, easy-to-use devices is to make the information-gathering step — the intel — really inexpensive, almost negligible, so that the full health care dollar or rupee or whatever can be spent on the treatment,” she says.Droplet designer
For David Weitz, a professor of physics and of engineering and applied sciences at Harvard University, microfluidics isn't just about moving fluids through channels, but rather what those fluids carry.
Weitz's lab develops lab-on-a-chip systems using controlled creation of picoliter aqueous droplets in a hydrophobic carrier fluid, like a tightly controlled molecular vinaigrette. Here each droplet can act as an individual reaction vessel, which in turn can be sequestered into yet other vessels — a double emulsion in which an aqueous droplet is encapsulated within an oil droplet, which is itself suspended in aqueous solution.
To create these emulsions, Weitz uses a process akin to what happens with a leaky faucet. In that case, the water slowly collects in a drop until its weight exceeds the surface tension holding the droplet to the faucet. Similarly, Weitz's microfluidic analog extrudes an aqueous solution in a flowing stream of hydrophobic fluid until the shear force exerted by the flowing liquid exceeds the growing droplet's surface tension, creating a uniform structure. “Once we figured out how to do the fluidics, we could control them in a way that we could actually use them as reaction vessels,” Weitz says.
Weitz's lab has used this technology to address a wide range of questions. In one study, his team built a microscale fluorescence-activated cell sorter (FACS), one that enables experiments that aren't possible using conventional FACS machines. Researchers can look for cells that excrete specific antibodies or cytotoxins, for instance, or lyse cells inside the droplets and probe their contents directly. “You can't do that sort of stuff in a FACS machine, but you can do that in these drops,” he says.
The system also provided the infrastructure to drive a directed evolution experiment. In this case, the goal was to identify yeast expressing hyperactive forms of horseradish peroxidase. A yeast cell library expressing some 10 million HRP sequence variants was injected into a custom PDMS-based device and inserted into picoliter droplets such that each droplet contained a single cell along with fluorescent HRP substrate. As each droplet passed through the chip's plumbing, they were interrogated by a laser. Droplets that fluoresced most intensely were directed into a collection bin by an on-chip electrode while the rest were shunted to waste.
Using this strategy, Weitz's team was able to identify variants that were up to seven times more catalytically active than the wild type enzyme in only two rounds, all for a cost of about $2.50 — compared to an estimated $15.81 million for a comparable plate-based screen.Droplets of As, Gs, Cs, and Ts
Weitz's picoliter droplet technology forms the foundation of several commercial enterprises, including RainDance, which uses the approach to amplify selected genomic regions for DNA sequencing.
Another Weitz lab spin-off, called GnuBIO, uses the picodrop technology for the rapidly growing field of diagnostic sequencing. Upfront amplification and library preparation work is automated on the chip, so all a user need do is insert genomic DNA and press “Go.” Even PCR primers are “automated”; those come preloaded on the system's cartridges (which include the microfluidics) according to customer specifications.
The company's sequencing strategy uses a “picoinjector” to dispense the amplified DNA into a flowing “sequencing probe library,” a stream of droplets, each containing one of the 4096 possible hexanucleotide sequences. The sequence is read by monitoring hybridization of each hexanucleotide primer with the template, an event that is detected based on the ability of that primer-template pair to prime DNA polymerization.
Company CEO John Boyce likens the approach's plug-and-play simplicity to the now popular “K-Cup,” alluding to the ubiquitous pre-packaged coffee format.
And like brewing a K-Cup of coffee, the workflow is essentially non-existent, says Boyce — genomic DNA in, processed data out — making the platform especially useful in clinical labs. Clinicians, he explains, have neither the time nor the budget for the “sample prep gymastics” required with most sequencing platforms. “Being able to simplify their workflow and condense the steps of candidate gene capture, library preparation, and amplification onto a chip is absolutely key,” he says, “That will really open up the door.”