Also using microfluidics to power next-generation sequencing is Stephen Quake, a Howard Hughes Medical Institute Investigator at Stanford University.
As described in Nature Biotechnology in 2010, Quake's team built a device capable of lysing a single cell, separating the chromosomes, amplifying each one, and then shunting the resulting libraries back into collection ports, where they can be removed and sequenced.
The strategy actually solves a longstanding problem in human genetics, Quake says: how to properly haplotype a genome. Suppose an individual has two distinct mutations in a given gene. Are they both in the same copy of the gene, or is there one mutation each on the maternal and paternal chromosomes? In the former case, the individual can make a functional protein; in the latter case, he cannot. “So, it really matters to know that,” he says.
Sequencing data cannot resolve this issue, or as researchers put it, “phase” a chromosome. Quake's team was able to accomplish the feat by physically separating each chromosome and amplifying them independently. Perhaps best of all from a user's point-of-view, the chip automates a good deal of the sample preparation, a process that is, Quake admits, “a pain in the butt.” And costly; library preparation costs almost as much as sequencing these days.
Microfluidics company Caliper Life Sciences, part of PerkinElmer, has tackled one aspect of next-gen sequencing with its LabChip XT chip, which automates the process of sample fractionation, for instance, to enrich for sheared DNA fragments of a given size range. Fluidigm, a firm Quake co-founded, has also addressed next-gen sequencing. Using the company's PDMS-based Access Array, researchers can set up 2304 independent PCR reactions for targeted resequencing: 48 samples × 48 primer sets, each of which may contain as many as 10 distinct primer pairs.
Company CSO Marc Unger likens this kind of microfluidic device to DRAM — “taking a simple unit cell and making a lot more of them.” That's not to downplay their complexity — according to Unger, the company's 96.96 Dynamic Array chips contain nearly 30,000 valves and 100,000 pieces of pipe, the equivalent of a 1000-room hotel. But the company now is ramping up the complexity with integrated fluidic chips that are more like multifunctional computer CPUs. For instance, Fluidigm is working on a system that can capture a single cell, lyse it, and prepare a sequencing library from either RNA or DNA.
Micro-DIYThe applications of microfluidics technology are literally boundless. Case in point: Dutch researcher Albert van den Berg of the University of Twente in the Netherlands, with Ph.D. student Loes Segerlink, figured out how to use microfluidics to count and measure the motility of sperm, key indicators of fertility. (The chip gives sperm two alternate routes to travel, the default route of the flowing liquid, plus a second path that can be used only if the sperm are swimming. In both cases, motility is assessed via the electrical impedence changes caused as the cells pass one of two pairs of electrodes.)
But a researcher doesn't need to be an expert in fluid dynamics or even purchase systems from Fluidigm or Caliper to experiment with microfluidics. In fact, it's relatively easy for neophytes to do some preliminary groundwork and see if the approach makes sense.
Mark Burns, chair of the department of chemical engineering at the University of Michigan, has developed a set of 16 microfluidic assembly blocks — think PDMS LEGO pieces — for those who might want to experiment with microfluidics or just do some quick prototyping work.
The basic building blocks measure 4 mm × 4mm, though some are multiples of that size, such as an extended channel component measuring 1 block × 3. Each piece contains an individual fluidic circuit element, such as a reaction chamber, T-junction, or a zigzag channel.
Burns’ team uses these assembly blocks to prototype designs before switching to the more expensive task of synthesizing photolithography masks, a process that is at least an order of magnitude more expensive than using his assembly blocks.
Another option, developed by Boston-area high school physics teacher Joe Childs and perfected by Anas Chalah, director of instructional technology at the Harvard School of Engineering and Applied Sciences, uses a photocopier to create the raised molds used in PDMS-based microfluidic devices. In this case, a channel schematic is first designed and printed to a transparency, for instance in PowerPoint. This process is repeated over and over again, producing a raised relief image of the channel network. Finally, that design is imprinted into PDMS by pouring the liquid polymer over the transparency and letting it set.
The approach isn't perfect; photocopying is an imprecise process and microfluidic channels are just microns in size. Yet students in Chalah's engineering labs have used this approach, as well as other more standard techniques, to learn the fundamentals of chip design. “We are teaching fluid mechanics at the micro level,” he says; before that, the class used a massive apparatus that monitors fluid flow by the bucketful.
The new approach is so inexpensive researchers at other less-well-funded schools can use it to teach their students too, or dabble in the microfluidic space themselves. And that could pay perhaps the biggest dividends to the future of microfluidics: With so many young researchers dabbling, the number of microfluidics applications should only increase.
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