October 1, 2009 (Vol. 29, No. 17)
More Efficient Ways of Collecting and Concentrating Samples Are Being Introduced
The Knowledge Foundation’s “Sample Prep Symposium” held recently in Baltimore focused on the mundane but critical aspects of sample preparation including collection, concentration, lysis, target extraction, and transfer to analytical and identification tools. “Sample prep remains a challenging area,” noted Anson Hatch, research scientist at Sandia National Labs. “Methods that work for DNA and RNA don’t necessarily work well for proteins. The best case is an integrated platform that can handle all kinds of samples. Preliminary approaches are on the horizon.”
Michael Connolly, Ph.D., president and CEO of Integrated Nano-Technologies (INT), agreed. “Customers need the ability to do DNA, RNA, and protein sample prep on one platform. When we tested our methodology against PCR, we found that we needed to change the conditions appreciably to make it work for protein sample gathering.”
INT focuses on lab-based diagnostics for defense, homeland security, and veterinary medicine applications. These areas require automated, robust diagnostics that are nonPCR based, and the firm is developing a universal sample-prep approach to prepare DNA or RNA from a variety of sample types to enable field diagnostics.
INT’s technology uses superparamagnetic nanoparticles to capture and concentrate target molecules. “We use nanomagnetic particles to magnetically separate and concentrate the targets from the sample, and we get better than 90% yields and can concentrate the material more than 100-fold,” said Dr. Connolly. “The captured target molecules can be washed to remove inhibitors. All steps of sample collection, processing, and analysis can be carried out automatically in a single disposable cartridge.”
INT is engineering a two-pound unit to perform sample prep in an integrated process. “We have combined and modified a number of existing techniques, and the result is reproducible,” Dr. Connolly added. “We use a traditional technique like sonication to disrupt the sample and to shear the nucleic acid molecules, but to make it work within a portable device and with high yields, we have developed different stabilizers that keep DNA from degrading past a certain point.”
INT’s approach also works for preparing protein samples. “Our customers asked whether we could also process proteins using this system, and our scientists found that the paramagnetic particles could capture proteins under different conditions than those used to purify nucleic acid molecules.”
The system has been tested using toxins in complex samples prior to immunoassays. The company has also tested a couple of different toxins to make sure the process is not denaturing.
Rapid Nucleic Acid Purification
Akonni Biosystems’ core technology is a gel-drop microarray licensed from Argonne National Lab. “We have developed new sample-preparation technologies for rapid cell/spore lysis and nucleic-acid purification,” said Phil Belgrader, Ph.D., vp of R&D. “These can serve as stand-alone devices or be used as components in integrated systems. We also engineer these technologies to allow fast-track to commercialization.”
The firm recently launched a sample-preparation device that performs efficient nucleic acid extraction, purification, and concentration from small or large volume clinical, environmental, and forensic samples in as little as three minutes, according to Dr. Belgrader. “TruTip purification started out as a component for an integrated, microfluidic, sample-to-answer system, but it had appeal as a stand-alone product,” he added.
“The modified pipette tip is suited for both field-portable and high-throughput applications. It works with single-channel pipettors, multichannel pipettors, and robotic pipetting workstations. It has also been incorporated as a sample-preparation module in a prototype microfluidic cartridge. The microfluidic cartridge integrates sample preparation, PCR, and gel-drop microarray hybridization for a complete, automated sample-to-answer solution for infectious disease testing that provides more sequence information than conventional real-time PCR-based tests without sample splitting.”
Dr. Belgrader said that TruTip has been used to successfully process swab extracts, whole blood, sputum, nasal wash, urine, and soil to detect bacteria cells, DNA viruses, RNA viruses, and human cells. “It works on DNA and RNA, and is compatible with a variety of backend amplification and analysis technologies.”
Lack of universal requirements is a significant challenge, according to Dr. Belgrader. “Customers have different needs, especially when you compare the life science, environmental testing, and diagnostic markets. Priorities with respect to cost, sample volume, sample type, and processing speed can vary across the fields of use.”
At the symposium, scientists from InSituTec showcased the firm’s solution for sample-concentration problems. According to Shane Woody, CEO, AccuWand enables rapid cellular collection and shepherding without degrading or lysing the cells. The device, a mechanical probe that modulates at an ultrasonic frequency, generates wide ranging vortices in a fluid environment, which create fluid velocities exceeding 1 m/s.
“This fiber, called a standing wave probe, is 7 µm in diameter, 1.5 mm in length, and modulates back and forth at 32 kHz,” Woody said. A pronounced mechanical wave is generated on the fiber with peak-to-peak amplitude of 45 microns. When inserted into a microsample liquid quantity, a complex flow field is generated with the general appearance of a quadrupole extending greater than 1 mm from the center of the probe.
“As it relates to diagnostics, when the fiber is placed in a liquid sample, cells and pathogens will be accelerated toward the vibrating fiber. The reason this is so compelling is the difficulty in concentrating enough sample to work with. For example, with S. aureus, it is difficult to localize pathogens in one place, he said. “We have found that vibrating the fiber creates whirlpools in which the cells get caught up and clustered together. So now we have a method for localizing pathogens.”
Woody believes that integrating that power on a chip with a detector can go a long way toward helping deal with the sample-concentration problem. By integrating this with another sensor on a chip, the detector can monitor what collects around the fiber.
In addition to concentrating cells, the fiber creates a field in which there are four vortices, and each vortex reaches 300 times beyond the fiber, which increases the influence over more of the sample volume.
“In addition, we have found that this technology doesn’t lyse cells—we thought the vibration of the fiber would break cells open, but the cells remain intact,” noted Woody. “We have found that we can use the device for mining information on DNA. You can maximize the sample and not devalue the DNA.”
Nucleic Acid Detection
Applied Biosystems, part of Life Technologies, presented an automated procedure and workflow for high-throughput sample preparation using its PrepSEQ Nucleic Acid Extraction Kit at the symposium. The procedure presented by Nan Liu, a senior scientist, consisted of sample lysis, nucleic acids binding to the magnetic beads, washing, and elution on a MagMax-Express automated magnetic particle processor.
Getting adequate quality and concentrations of nucleic acid is crucial, and Life Technologies’ magnetic particle chemistry is a key differentiator for nucleic acid recovery, noted Maxim Brevnov, senior staff scientist. “Our optimized magnetic particle chemistry provides effective capture on large surface area making a high nucleic acid recovery rate possible for a broad range of sample sizes, yielding from femtograms to micrograms of highly purified nucleic acids. Even with complex samples, we can deliver consistent and accurate results.”
Jenkui Liu, a senior staff scientist, noted that this technology can be used for both automated and manual systems. “Two major attractions to users are the performance of the magnetic particles in automation procedures and the ability of the chemistry to deal with diverse sample types. We have demonstrated that the PrepSEQ chemistry and purification procedures can be easily automated in different instruments up to 96 samples each time. Automated and manual procedures produced similar results in getting accurate reads and high DNA recovery from diverse sample types.”
Nan Liu noted that isolation and purification of nucleic acids at low levels from chemically complicated and PCR-incompatible samples can be done with the system. “Detection of low-level residual DNA from biological drugs has been challenging. The level of DNA in the samples can be in the picogram range, and we can achieve DNA recovery as high as >95% with our system.
“The resultant purified DNA shows high efficiency in TaqMan PCR assays, so we are able to detect residual DNA levels that are much lower than the limit in FDA regulations. Our system can also provide more accurate quantification of DNA in the drugs in diverse matrices.”
Preconcentration of Proteins
Addressing another angle of the preconcentration problem was Anson Hatch, Ph.D., research scientist in the biosystems research department at Sandia National Laboratories. “We are developing microfluidic medical diagnostic platforms but have realized that rapid point-of-care diagnostics demand attention in the sample-prep realm,” he said.
Dr. Hatch’s work focuses on lab-on-a-chip approaches for rapid detection of biomolecules in trace quantities in complex biofluids and matrixes such as blood, saliva, or extracted cells. “Our group is developing a number of on-chip capabilities including sample preparation.”
At the symposium, Dr. Hatch described a suite of microfluidic sample-prep modules in development that are specifically designed for isolating protein fractions from complex samples. “The modules include size-based filtration of cells and particulate matter, immunodepletion of high-abundance interfering proteins, isoelectric fractionation of proteins, and preconcentration membranes that mix and enrich proteins greater than 10,000-fold. All elements can be integrated in various arrangements for rapid, streamlined processing and analysis.”
“Sample prep is a crucial step for good results and good yields,” noted Romain Verollet, product manager at Bertin Technologies. “For toxins and pathogens, the key is to increase detection limits and linearity, which enables lower concentration detection and an easier decision-making process. Automation and standardization also increase reliability in the final analysis.”
At the symposium, Verollet’s colleagues presented studies that investigated mechanical lysis with the Precellys bead-beater versus manual, chemical, or sonicator methods. The Precellys system enables sample preparation in 0.5 mL, 2 mL, and 7 mL in a few seconds within a temperature-controlled environment, according to the Bertin scientists.
A high-speed multidirectional motion shakes the beads that grind and homogenize samples in sealed tubes. The system can be operated in multiple configurations in the same built-in tube holder, and it eliminates inefficient sample preparation such as a vortex, mortar and pestle, sonicator, or blender,” added Verollet.
Three applications were examined and reviewed at the meeting—uses of DNA/RNA extraction from microorganisms (such as Bacillus), DNA extraction from plants, and protein extraction from animal tissues. “Precellys can extract, detect, and quantify DNA, RNA, or proteins,” Verollet said. In these three case studies, bead-beating technology was shown to be efficient and reproducible without sacrificing time and labor.