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

David Daniels, Ph.D.

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

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.


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.

Disposables

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.


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.

Hospital Labs

Idaho Technology’s solution is the single-sample FilmArray. Its first commercialized product, Respiratory Panel, has been approved by the FDA to test for viral pathogens from a nasal pharyngeal swab. The system is reportedly simple: all the reagents for sample processing and amplification are contained in a single-use, disposable “pouch”.

The first step is to hydrate the freeze-dried reagents in the pouch by injection of buffer under vacuum. The patient sample is then washed off the swab and resuspended in lysis buffer. Approximately 250 µL of that sample is then loaded at the other end of the pouch under vacuum.

The barcoded FilmArray pouch is processed by placing it into the FilmArray benchtop instrument. Any microbial material in the sample is then subjected to a bead-beating mechanical lysis using ceramic beads that can effectively bust open spores or other difficult to lyse pathogens. Nucleic acids are scavenged from the lysis mix by magnetic beads for subsequent processing.

“Respiratory pathogens, in particular viruses, are notoriously difficult to culture,” said Stephanie Thatcher, director of systems integration. “Given the effectiveness of the sample-prep step and sensitivity of the rtPCR and detection PCR reactions in the FilmArray, a culture step is not necessary. We are able to process the material routinely collected in the hospital when patients come in with a respiratory infection. Processed in the hospital lab, the pathogens are identified in less than 60 minutes so that the proper course of therapy can be initiated.”

The FilmArray system is easy to use, explained Thatcher. First, the FilmArray extracts and purifies all nucleic acids from the sample. Next, the FilmArray performs a nested multiplex PCR in a single large volume multiplexed reaction. Then individual singleplex second-stage PCR reactions detect the product from the first-stage PCR in each chamber of the array.

Using endpoint melting curve analysis with double-stranded binding dyes, which pathogens are present in the patient sample can be determined. The system is optimized for low-level detection of pathogens from clinical samples. A control organism is provided in the sample buffer, which serves to provide confidence that the sample signal, whether positive or negative, is valid.


FilmArray RP is Idaho Technology’s first clinical diagnostic test designed to run on the company’s Film Array system.

Enzyme-Based Technology

ZyGEM has taken a different approach to nucleic acid sample prep. Rather than using solid-phase extraction (SPE), ZyGEM offers an enzyme-based technology to strip the sample down to nucleic acid. While the technology is amenable to use on liquid handlers in the research lab, James Landers, Ph.D., was brought into ZyGEM as CSO to adapt the technology to microfluidics.

As professor of chemistry and mechanical engineering and assistant professor of pathology at the University of Virgina, Dr. Landers has abandoned SPE for ZyGEM’s enzymatic approach because of its simplicity and elegance, he said. With the adoption of microfluidics, the extraction process has been shrunk to sub-microliter volumes, which saves on enzyme and other reagent costs. In addition, the reaction kinetics are sped up; the entire process has been reduced to 35 minutes.

The reaction is controlled by temperature. The EA1 proteinase, a highly active metallo-endo-proteinase isolated from a thermophile in a volcanic vent, is inactive at room temperature. Peak activity occurs at 75°C where the enzyme works as a protease. Raising the reaction temperature to 94°C irreversibly kills enzyme activity.

Regarding the macro-to-micro interface, ZyGEM deals with real world samples; a buccal swab or blood droplet is collected in 100 µL volume. Of that starting material, only 1 µL is needed for downstream processing; the rest of the material can be stored for reanalysis, if necessary. Front-end concentration of the sample has not been determined to be necessary. From a buccal swab, ZyGEM routinely liberates purified DNA in the range of 6–12 ng/µL, which is in excess of the 4 ng/µL that is needed for PCR, Dr. Landers explained.

The ZyGEM approach has a narrow scope; the sample-prep method provides material that is “PCR-ready”, but is not limited to microfluidic devices. Kits are also available for use in microtiter plates on liquid handlers, where the enzyme approach is reportedly a dramatic improvement over SPE protocols as the number of sample-handling steps is reduced, avoiding loss of sample from transfer steps.

“It is important to remember that the ZyGEM process doesn’t help with finding a needle in a haystack. DNA that is liberated from the reaction comes from all sources in that sample. You must depend on the use of proper PCR primers to amplify up rare sequences in the mix or resort to SPE-based selection of target from the mix,” explained Dr. Landers.

“But with its simplicity and elegance, PoC applications are not far off. For example, where the challenge is to differentiate between viral and bacterial Encephalitis meningitis when the patient presents with similar symptoms, given the rapid kinetics of the sample prep of Cerebral Spinal Fluid, and subsequent PCR, you could determine the right course of treatment within the first two hours of hospitalization,” Dr. Landers claimed.


ZyGEM’s family of products, derived from its collection of microorganisms from extreme environments, enables nucleic acid extraction from diverse sample types.

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