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Feature Articles : May 15, 2010 (Vol. 30, No. 10)

Tiny but Powerful Tools for Sample Prep

Host of Technologies Born for Biodefense Improve Wide Range of Industry Workflows
  • Catherine Shaffer

Biological research instrumentation is getting smaller and smaller as genetic and biomarker analysis moves out of the laboratory and into the clinic or the field. Smaller technologies now available include handheld immunoassay strips, flow assays, PCR systems, and mass spec equipment, but sample-prep hasn’t always kept pace with the latest advances.

Centrifuges, chromatography columns, spectrometers, and other sample-prep tools are not quite so portable yet. Not surprisingly, the Knowledge Foundation’s “Sample Prep” meeting held earlier this month in Baltimore was packed with presentations featuring small and sleek sample-prep technologies.

Microfluidics offers one way around the sample bottleneck, automating and miniaturizing the same sample-prep steps users would carry out at the bench. Other approaches seek to skip the prep step entirely through direct detection.

InnovaPrep, which was formed last year to commercialize sample-prep technology developed at AlburtyLab, is trying to close the gap between sample-prep technologies and runaway miniaturization of biodetection technology. “We feel that what is missing in that trend and in the world is, of course, the sample prep,” said CEO David Alburty. “We think of it as a link between the real-world sample size and the microliter world that those systems operate in. A macro-micro interface.”

In developing its sample-concentration system, the InnovaPrep HSC-40, for the biodefense industry, AlburtyLab sought to address issues associated with collecting samples in the field. Although many detection devices are now small enough to be portable, even handheld, the size and dilution of many samples in nature remains a problem.

Taking 5 µL of water from a lake, for example, is probably not going to provide very good results, unless you can concentrate a larger volume of sample from the lake. The conventional methods are centrifugation and filtration, but Alburty thought there might be a better way.

“Centrifugation is hard to automate, and with most filtration it is easy to automate but difficult to extract the sample again from the filter. Once you trap the sample, our discovery was how to get it in and out of the hollow fiber.” The solution was a carbonated foam that can be designed to be compatible with the rapid assay. The foam sweeps through the fiber, extracting the sample.

The InnovaPrep HSC-40 was designed to concentrate airborne aerosols to a smaller volume that more closely matches the detector’s requirements. However, the method can be extended to any volume of liquid sample, including clinical diagnostic applications. For example, the device can be used to concentrate whole blood by first removing the blood cells in a single pre-treatment step, and then achieving 100-fold concentration using the filter device.

Lab on a Chip, Not Chip in a Lab

Rheonix’ CARD (chemical and reagent device) technology is a point-of-care assay product that is completely disposable and can be used by technicians with no special training and with minimal instrumentation, according to Peng Zhou, senior vp.

CARD is constructed of 1 mm thick polystyrene using a fabrication method called solvent elimination. The process takes 15 to 20 seconds, and when it is complete, all of the valves, channels, and networks are in place on the card, reported Zhou.

Rheonix is a spin-off from Kionix. At first, it used MEMS-based materials such as silica and glass wafers to create microfluidics devices. “We realized that the requirements for applications in the life science areas are so different that we switched to a different substrate and developed a totally different fabrication method,” said Zhou.

Sample preparation is included on one of the modules on the CARD, as well as amplification and endpoint detection, which is carried out by means of microarray technology. The CARD can identify 20–30 different subtypes of viruses. The main application is clinical testing, where a sample of blood or a cheek swab can be taken and analyzed immediately on the CARD.

The CARD is able to fully manifest the idea of lab-on-a-chip, which has not yet been fully realized by microfluidic technologies, Zhou said. “Instead of lab-on-a-chip, you have chips in a lab. People have heard about it a lot, but no one can actually deliver. I hope that what Rheonix has done is really demonstrate that we have indeed successfully built a highly functional and automated processes for molecular diagnostic around CARD technology.”

Autonomous Platform

Spyglass Biosecurity’s products are based on IP developed at the Monterey Bay Aquarium Research Institute (MBARI). Spyglass is using the technology to address environmental issues that threaten the safety of oceans, rivers, lakes, and any other aquatic environment.

The Spyglass ESP autonomous platform collects water samples and processes and analyzes them on board. Essentially, it is a self-propelled, robotic molecular biology lab that can be used for a variety of research or environmental quality applications.

According to Chris Melancon, president and CEO, ESP has been deployed over the past eight to nine years to collect data from a whale carcass at the bottom of Monterey Bay, showing the development of communities of organisms. The company has also monitored harmful algal blooms in the bay and fecal bacteria in Santa Cruz for beach water quality.

Electrochemical Setting

Many conventional sample-prep approaches are based on the idea of removing unwanted bulk from a sample, usually in progressive steps, until nothing remains except the analyte of interest. Many newer sample-prep methods are improvements on this strategy. However, advances in microfluidics technology have opened up new options in direct testing from crude samples.

Genefluidics has developed a direct-detection platform for biomolecules that it reported has  sensitivity in the sub-femtomolar range for DNA and RNA and sub-picogram range for protein. The company’s electrochemical sensor cartridge is designed to work with Asklepios’ point-of-care system. The sample matrix can be blood, saliva, urine, or a tissue sample, but matrix effects are minimized because of the unique nature of the capture technology.

All of the sample-prep steps that would normally be done at the bench happen inside the cartridge, which is modular with configurable components depending on assay requirements. The sensor is based on a self-assembling monolayer, which blocks nonspecific binding and provides a smooth anchor surface for probe attachment. Once the enzyme is immobilized onto the surface via the captured target, the enzymatic redox reaction generates 20,000 electrons per second from each enzyme molecule. This allows detection of a small number of target molecules.

The electrokinetic properties of the device can be customized for the sample. “We can optimize the AC frequency or amplitude to attract certain cells based on the dielectric property of the cells and also the size. It also depends on the connectivity of the specimen,” said Vincent Gau, Ph.D., CEO and president.

For example, when processing urine samples, the impedance of the sample will vary depending on whether the patient has had a lot of water to drink. Likewise, varying levels of protein in the blood will affect the impedance of the sample. Each assay begins with an impedance-matching step to select the correct current for the sample. “We identify the electrokinetic conditions to do the specific test we want to do,” Dr. Gau added.

Nanopores

Liviu Movileanu, Ph.D., assistant professor in the structural biology, biochemistry, and biophysics program at Syracuse University, talked about his group’s work with nanopores at the meeting. Using a combination of techniques from nanotechnology, biomolecular engineering, and surface chemistry his team is developing a chip platform for analysis of biomolecules. The sample-prep approaches previously discussed all depend on miniaturizing, automating, or simplifying the process. Dr. Movileanu said that sample prep can be effectively bypassed using an extremely sensitive detection method based on nanopores.

Natural ion channels that transport charged molecules through a potentiated membrane inspired the concept behind Dr. Movileanu’s research. Increasing understanding of the mechanisms of ion channels, and the ability to measure the current running through them, have enabled scientists to replicate them in the laboratory by creating nanometer-scale holes in a silicon nitride membrane.

When placed in an electrolyte solution with voltage across the membrane, these holes behave similarly to a natural ion channel. More importantly, the current measured when an analyte passes through a nanopore positively identifies that analyte—a technique called stochastic sensing.

“This is a technique for probing very minute, small quantities of biologic material, in this case, proteins or nucleic acids. It’s called stochastic sensing because each molecule interacting with a single nanpore will cause a current blockade. The nature of that current blockade is stochastic. The technique allows quantification, as well as identification of the analyte,” said Dr. Movileanu.

Applications for stochastic sensing include DNA sequencing and protein detection. For example, it’s possible to modify the nanopores for studying aptamers. Then, when the proteins bind to the aptamers, they create a current blockade that detects the presence of the proteins.

Microscale fabrication plays an important role in this year’s crop of sample-prep innovations. Smooth surfaces, high-tech materials, and precision design make it possible to fit an entire workflow on a chip, cartridge, or handheld device. Innovative approaches to filtration and concentration can bridge the gap between the real world and the microworld on the chip.

Although the end-user applications can be extremely different, sample-prep technologies overlap significantly between environmental, biodefense, and medical research fields. For this reason, biological researchers have benefitted tremendously by investment in sample-prep technologies for biodefense applications.