More often than not, you can’t put a price tag on the rewards of scientific research — satisfaction at solving a tough problem, the respect of peers, knowing your work will have a larger impact on the world. But sometimes you can: just ask Kamlesh (Ken) Patel (8621). He recently won the Society for Laboratory Automation and Screening (SLAS) $10,000 Innovation Award for his outstanding podium presentation, “Preparation of Nucleic Acid Libraries for Ultra-High-Throughput Sequencing with a Digital Microfluidic Hub.”
“The SLAS Innovation Award was created specifically to recognize cutting-edge research and the individual behind the work, and Kamlesh’s exploration into nucleic acid libraries for ultra-high-throughput sequencing with a digital microfluidic hub will impact the scientific community for years to come,” says SLAS Innovation Award Committee Chair Jörg Kutter.
While Ken’s name is on the award, he’s quick to point out that his work is part of a much larger effort with contributions from a multidisciplinary team. Led by principal investigator Todd Lane (8623), the RapTOR (Rapid Threat Organism Recognition) Grand Challenge, part of the International, Homeland and Nuclear Security strategic management unit, has the ambitious goal of rapidly identifying and characterizing unknown pathogens. (Lab News, Aug. 26, 2010). In an outbreak scenario, whether the result of bioterrorism or a fast-moving, deadly virus like Ebola, RapTOR could greatly accelerate the response. Until you know what’s making people sick, treatment is like throwing darts.
Leveraging DNA sequencing technology
Using the latest in DNA sequencing technology, RapTOR aims to transform slow, labor-intensive benchtop sample preparation methods to an automated microfluidic platform to create a fast, efficient, and flexible tool. “We’re taking advantage of DNA sequencing technology,” Ken says. “Reading the genetic code, the original building blocks, allows you to begin characterizing a pathogen at the most basic level.”
But getting at those building blocks is not easy — clinical samples are packed with information, most of which is not of use in characterizing an unknown pathogen. For example, more than 99 percent of the DNA in a blood sample is the human genome. DNA in a nasal swab is 90 percent human-derived and much of the rest is garden-variety bacteria. Suppressing all that background DNA is essential to get at the unknown pathogen.
DNA sequencing technology has evolved at a tremendous pace, even surpassing Moore’s Law, the 45-year-old prediction that computer processing power would double every two years. The pre-sequencing steps, however, have hardly changed since the bacteriophage genome was first sequenced in the mid-1970s.
Ken leads the Automated Molecular Biology (AMB) research to both scale down and automate traditional sample preparation methods such as normalization, ligation, digestion, and size-based separation — methods that traditionally require a skilled scientist and take days or even weeks. A critical component of RapTOR is bringing together the different sample prep steps to create a “one-stop shop” that connects to a DNA sequencer. Key to this is the digital microfluidic hub.
The hub is a Grand Central Station for samples, routing them from one step to the next with the flexibility to skip or repeat steps on the fly. But imagine a train station in which some trains are orders of magnitude larger than the others, and some travel at the speed of light and others at 60 mph. The digital microfluidic hub is designed to negotiate these differences, functioning like a train station that can shrink and enlarge trains as necessary and manipulate their speeds.
Instead of trains, droplets are the mode of transportation in this station and voltage serves as the engine. The sample is cargoed within a microliter-scale droplet that is spatially moved across the Teflon-coated surface of the hub when electrostatic forces are appropriately applied. The hub also manages the size of the sample, extracting the right amount for each process.
Reagents dispensed as needed
Size is only one variable that the microfluidic hub manages. Reagents and enzymes necessary for different manipulations are stored in reservoirs connected to the hub and dispensed as needed. “If we need to do a reaction at a set temperature, we can move the sample through a connector tube off the hub into a heated microreactor, perform the reaction at temperature with appropriate reagents, and then redispense the sample back onto the hub for the next processing step,” Ken says. “This is where AMB becomes very powerful — it allows you to connect multiple, different components together through a common flexible interface. All of the microreactors are replaceable, so contamination is not an issue.”
At the start of the Grand Challenge, the AMB team wasn’t sure how efficiently they could repeatedly move droplets on and off the hub. Turns out, the ability to move the droplets is one of the most powerful features of the device.
“We’ve concluded that this is one of the main contributions we’ve made to the field,” Ken says. “Interfacing to a droplet and other microfluidic chips is not just possible, it’s quite effective and a good path forward for processing samples.”
As the AMB team continues to refine the digital microfluidic hub, they are also working on a parallel project to culture cells within the droplets. “There are several exciting advantages to this approach — we can work with different microcultures of cells independently on the device. It is possible to study infections at the cellular level, working with small amounts of cells — thousands at best, not millions — in a hermitically sealed, safe environment,” Ken says. “The end goal is a device that could be used in Biosafety Level-3 containment, enabling safe diagnosis and research of infectious agents.”
Expanding the role of this technology, Ken was recently awarded additional funding from the US Army Criminal Investigations Laboratory to develop a microfluidic-based approach for genotyping in the field. Such a device would allow law enforcement to rapidly process forensic evidence at the crime scene for matching DNA, rather than sending a sample to the lab and waiting days for confirmation, generating immediate intelligence that can then be applied to the unfolding situation.
The digital microfluidic hub could have a wide range of applications — from crime scene investigators to first responders to a general practitioner’s office.
“An eventual goal might be to develop an all-in-one portable device, a sequencer with a sample prep front end. We have portable sensors, so why do not DNA sequencing in the field?” says Ken.