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Jun 1, 2012 (Vol. 32, No. 11)

Real-Time Sample Prep on the Horizon

  • No longer just science fiction, fully integrated molecular diagnostic devices are being developed to take raw sample loaded at the intake port, process it through the device, and deliver an answer at the other end. A number of companies have made significant progress toward this goal, and it seems, based on presentations at Knowledge Foundation’s recent sample-prep meeting, that hand-held devices will be a reality in the very near future, allowing scientists to take a real world sample, process it, and produce an accurate identification in real time.

    A droplet-based digital microfluidics technology is at the heart of the solution from Advanced Liquid Logic. Michael Pollack, Ph.D., and Vamsee Pamula, Ph.D., are co-founders of the company that, to date, has commercialized two products based on this core technology, one for genomic sample preparation and a second for newborn screening.

    The microfluidic device enables precise and programmable manipulation of droplets using an array of electrodes that obviate the need for traditional pumps, valves, and fixed channels, according to Dr. Pollack. The electrode array is fabricated on a printed circuit board with 1 mm2 electrodes. Droplets are processed within an oil-filled chamber formed by an injection-molded plastic top piece. Sample and reagents are loaded via dedicated ports.

    Volumes ranging from less than a microliter to hundreds of microliters can be processed with individual droplets being joined together, split apart, moved to locations adjacent to a magnet, or to a thermal region for PCR, etc. All movements through the device are defined by SpotLogic software scripts.

    “A couple of years ago we identified an opportunity to apply our technology to the problem of library preparation for next generation sequencing (NGS),” shared Dr. Pollack, CTO. “The flexibility of the technology was a key benefit because of the diversity of workflows that can be enabled and the ability to adapt as workflows evolve. We can essentially ‘reprogram’ the device to perform a wide range of protocols. Automation to improve reliability and consistency, and miniaturization to reduce reagent consumption are additional benefits.”

    The microfluidic device is a disposable cartridge about the size of a standard microtiter plate. The system is sold into the research market under the brand name Mondrian™ SP by NuGEN Technologies to perform fully automated NGS sample prep.

    Advanced Liquid Logic has also commercialized the LSD-100, a newborn screening system for lysosomal storage disorders (LSD). This automated system enables processing of up to 40 dried blood spot extracts, along with controls and calibrators, for multiplex testing of an LSD panel.

  • Human ID

    Click Image To Enlarge +
    According to IntegenX, the RapidHIT™ 200 System is a self-contained human identification system that automates and accelerates the process of producing standardized DNA profiles from mouth swabs and other human samples in less than 90 minutes.

    IntegenX was formed around microfluidic-valve technology developed in the lab of Richard Mathies, Ph.D., at the University of California, Berkeley. The technology is aimed at simplifying sample preparation for life science applications, addressing the issue of volume and scale, and ultimately developing complete small footprint “sample-to-answer” solutions.

    The IntegenX solution reportedly solves the problem of moving fluids at the macro to micro interface and mixing fluids in the microfluidic environment. The IntegenX solution is a chip made up of three layers: a fluid layer, a pneumatic layer, and a third layer that consists of a PDMS membrane, which is sandwiched between the two.

    “The process works like an aquarium pump with a diaphragm valve. With three valves in a row you can move fluids in any direction with close to zero dead volume in the transfer,” explained Dennis Harris, Ph.D., co-founder and CSO.

    “For example, using these on-chip pumps, we can pump in 2 to 3 mL of fluid into a chamber holding a buccal swab, lyse the cellular material from the swab, and capture all the DNA on magnetic beads. The magnetic beads are moved through the chip by virtue of the valve technology. We’re currently working on a human identification solution that can process a buccal swab and provide a CODIS profile in 90 minutes.”

    According to Dr. Harris, the criminal justice system is anxiously awaiting the RapidHIT™ solution, which will enable them to screen individuals in lockup to determine if the detainee’s DNA is in the system or associated with open cases before they have to move them from holding cells.

    IntergenX also has applied its technology for NGS sample preparation. Its Apollo 324 is not based on microfluidics, but rather uses the same chemistries implemented on a liquid-handling robot to provide library-prepped DNA for NGS. This product for genomic sample prep is targeted at sequencing centers in core labs with medium to low throughput.

  • Disposables

    Click Image To Enlarge +
    PureLyse® DNA Extraction Kits are capable of preparing PCR-ready samples from whole cells, without the use of harsh chemicals or bulky lab equipment, in less than two minutes, according to Claremont BioSolutions.

    The sample-prep solution from Claremont BioSolutions is said to be novel in that it is entirely disposable, fast, and it doesn’t require an instrument. The solution is based on mechanical lysis of cells and simultaneous extraction of nucleic acids or proteins depending on the buffers used.

    “The PureLyse® cartridge device contains a micromotor like the vibrator used in a cell phone, but in this case with a propeller. With the added beads, high shear forces are generated in the cartridge to enable rapid cell lysis. DNA is captured by binding to the lysis beads and then eluted to yield DNA within a four-minute protocol,” reported Bruce Irvine, CTO. “The flow-through configuration of the PureLyse cartridge enables its use with a wide range of sample volumes, typically 1 mL, but as high as 2 to 5 mL samples have been processed.”

    The PureLyse system has been used to lyse and extract nucleic acids from microbial sources, including E. coli, Bacillus subtilis, Mycobacterium bovis, and Clostridium difficile. The latter was the focus of a NIH-funded SBIR project where the PureLyse cartridge was embedded in the OmniValve fluidic system.

    The system has a valve with up to six ports connecting the lysis and extraction cartridge to other chambers. This set up facilitated integration of DNA extraction with sample, pre-filters, wash, elution, and amplification. This semi-integrated system enabled detection of C. difficile from human stool samples resulting in 100% specificity and 96% sensitivity as compared to EIA results, Irvine explained.

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