By Dr. Matt Wilkinson

US researchers have developed a microfluidic device for purifying and concentrating DNA directly from blood samples that could speed up the use of genomics in clinical trials.

Researchers from the University of Virginia in the US, have developed a microdevice, described in an early view article in the journal Analytical Chemistry, for extracting DNA samples from whole blood samples.

The device can capture DNA from whole blood samples as large as 10μL allowing the detection of low abundant disease biomarkers or infectious agents such as bacteria or viruses.

"DNA purification and preconcentration [of whole blood] is a requirement for most genetic analysis applications, primarily due to the complex nature of the biological samples," write the authors.

They continue to explain that whole blood is a complex mixture of nucleic acids, proteins, lipids, metabolites and inorganic ions and some of these can inhibit DNA amplification using PCR (polymerase chain reaction) techniques.

While other microdevices have been designed to process human whole blood, they often suffer from poor DNA extraction efficiency and tend to involve a protein wash that increases the number of steps involved in the process.

The poor DNA extraction efficiency arises due to the large mass of protein present in blood that can block DNA capture and lead to poor PCR results unless they are washed away prior to analysis.

These devices tend to only be able to process small sample volumes of blood, up to 1.5μL, making the need for multiple runs or parallel purifications necessary.

The new device makes use of a two-stage system extraction system that is made up of a C18 reverse phase column for protein extraction coupled to a monolithic column for DNA extraction.

"This provided not only an improvement in extraction efficiency over other chip-based DNA extraction solid phases but also the highest extraction efficiency reported to date for such sample volumes in a microfluidic device," write the authors.

"As an added bonus, the two-stage, dual phase microdevice allowed the 2-propanol wash to be completely eliminated, streamlining the process without affecting the PCR amplifiability of the extracted DNA."

Excerpt take from DrugResearcher.com

By Jeremy Shere

A University of Virginia chemistry professor has created a device that he says can very quickly diagnose certain diseases at the earliest stages of onset.

University of Virginia’s James Landers told Earth & Sky that this device – which resembles a glass microscope slide – uses nanotechnology. It works by analyzing the patient’s blood. Tiny, nanoscale pores embedded in the device allow it to examine DNA molecules in the blood for signs of disease.

James Landers: The whole purpose of the clinical diagnostic procedure is to essentially evaluate certain parts of DNA and see whether or not DNA sequences are normal or whether there are abnormal or mutated sequences in there.

Landers said abnormalities in particular sections of a DNA strand can be signs of early-stage cancer or other problems. While standard genetic analyses for cancer can take days and even weeks, this device can do the same work in a matter of hours, according to Landers.

James Landers: When comparing that to 3 days or 2 weeks, it’s a paradigm shift, and I think the MDs that we work with tell us that this changes how they do their job, which is what research is about.

And doctors know that the earlier they can detect cancer and other diseases, the better chance they have of treating and possibly curing them.

Excerpt take from Program #5141 of the Earth & Sky Radio Series

By Gail Dutton

At the University of Virginia, a lab, run by James Landers, Ph.D., is automating SPE for biological testing during extended space missions and has developed a fully integrated microfluidic genetic analysis system for unprocessed biological samples, like whole blood. “Astronauts take medications in flight for general pain, congestion, motion sickness, etc.,” said graduate student Daniel J. Marchiarullo.

Unfortunately the medications aren’t always as effective as on earth because space flight causes physiologic changes that alter a drug’s absorption, distribution, metabolism, and elimination properties. Therefore, a small, portable device to monitor the levels of medication in the body is needed, especially for long missions.

Marchiarullo, working with Lakshmi Putcha, Ph.D., at Johnson Space Center, is in the early stages of developing a prototype device to do just that. Ultimately, the device should integrate sample processing and analysis in one unit.

They have succeeded in using SPE to separate the anti-motion sickness medication promethazine and co-extract two hydroxyl free radical formation markers from saliva at recovery rates of 90–100%, reported Marchiarullo. “Concentration enhancements as high as 80-fold have been achieved by collecting only the fraction of eluent with the most analyte. Such high concentrations mean that solvent evaporation and reconstitution aren’t required because the eluent was compatible with electrophoretic separation.”

Landers’ lab already developed a glass microchip with three functional domains for genetic analysis—two for SPE and PCR and one for microchip electrophoresis—and are working on a gating device to control liquid transport through the microchip. The resulting device includes differential channel flow resistances, elastomeric valves, laminar flow, and electrophoretic mobility along with external fluid flow control using a syringe. Using this system, solid-phase extraction, PCR, and microchip electrophoresis, amplicon separation and detection takes less than 30 minutes. “This is one of the first systems with true sample-in-answer-out capability,” Marchiarullo said. It has detected Bacillus anthracis from 750 nL of whole blood from asymptomatic mice and Bordetella pertussis from 1 µL of nasal aspirate from a human patient. “Because the microfluidic device can use nanoliters or picoliters of fluid, quantities of reagents needed are dramatically reduced, which allows the device to be about the size of a shoe box.”

Integrating the microchannel with electrophoresis is in the beginning stages of development and is based on previous work with DNA analysis. Once a working prototype is developed, Marchiarullo said, it will be tested in collaboration with NASA, first in a hypergravity or microgravity environment and perhaps eventually in the space station.

Excerpt take from GEN. March 1, 2007, 27, 5.

"From Soup to Nuts" by MSL

A fully integrated microfluidic system has been developed that can detect a pathogen in whole blood and other bodily fluids in less than 30 minutes. Combining recent advances in microfluidic technologies, Easley and colleagues have created a system that successfully integrates three distinct functions: solid-phase extraction of DNA from complex samples, PCR amplification, and electrophoretic separation of the PCR product for size analysis. Their design overcomes the incompatibility between reagents used in solid-phase DNA extraction and those used in PCR through the clever manipulation of differential flow resistances, elastomeric valves and laminar flows. The authors demonstrated the chip’s utility by identifying the presence of Bacillus anthracis in 0.75 µl of whole blood from infected mice and of Bordetella pertussis, the causative agent of whooping cough, in 1 µl of human nasal aspirate. The integrated design substantially reduces the turnaround time for sample processing and genetic analysis, representing another step toward personalized medicine at lower cost. (Proc. Natl. Acad. Sci. USA, published online 11 December 2006, doi:10.1073/pnas.0604663103)

References from Proc. Natl. Acad. Sci. U.S.A. 103, 19272 (2006).

Excerpt from Science, 319, January 19, 2007

"Sample-to-readout on a chip" by AL

The promise of microfluidic systems, in which very small volumes of liquids are manipulated, processed, and interrogated, is that it may be possible to develop low-cost diagnostic systems, particularly for use under challenging field conditions. Although there has been tremendous progress in developing microfluidic components, creating an integrated system that can analyze an unpurified sample has remained a goal. 

Easley et al. describe a microfluidic system with three distinct functional domains. The first two are for sample preparation, consisting of solid-phase extraction (SPE) to pull out sample DNA from a crude specimen and for subsequent PCR amplification. After this, the amplified products are then injected along with a DNA standard into an electrophoretic detection domain. One key aspect of the device (3 x 6 cm) is a series of valves that are used to isolate each unit, thus keeping SPE reagents from reaching the PCR domain; these valves are also used in a diaphragm-like fashion to pump the amplified DNA into the analytical chamber. The authors demonstrate the detection of Bacillus anthracis in 750 nl of whole blood taken from infected but asymptomatic mice, and they also are able to measure Bordetella pertussis in 1 µl of nasal aspirate taken from a patient suspected of having whooping cough. — MSL 

References from Proc. Natl. Acad. Sci. U.S.A. 103, 19272 (2006).

Excerpt from Nature Biotechnology, 25, 1, January 2007

By Joe Alper

A major goal for microfluidics researchers is to develop a single, easy-to-manufacture device that takes in a blood sample at one end and yields diagnostic results at the other quickly and inexpensively. James Landers and colleagues at the University of Virginia and the U.S. Food and Drug Administration have built just such a device and used it to create a genetic analysis system capable of diagnosing infectious diseases and cancer in less than an hour from unprocessed clinical samples (Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19,272–19,277).

Excerpt from Analytical Chemistry, 79, January 1, 2007 

By Melissa Maki

Matthew Begley is among a growing community of researchers at the University of Virginia and worldwide that work and think on a scale so tiny that it is not even comprehensible to many of us.

The unifying theme of Begley’s research is to understand and utilize the behavior of materials at the nanoscale.  This means that the phenomena he studies typically occur at scales much smaller than the width of a human hair- which is about 80,000 nanometers. 

Conventional engineers have mastered the microfabrication of a wide variety of hard materials, such as ceramics and metals.  Begley’s work seeks to expand this capability to include organic materials, for new applications in the life sciences.  “The notion is that we can develop similar techniques for soft materials that are biocompatible and responsive to their chemical environment,” he explains.

Begley is an associate professor of mechanical and aerospace engineering (MAE) at U.Va., with appointments in the Departments of Materials Science and Engineering (MSE) and Electrical and Computer Engineering (ECE).  As is typical of today’s nanoscientists, Begley is involved in a number of collaborative and interdisciplinary research projects.  Some of his recent successes are strongly intertwined with James Landers, professor of chemistry at U.Va.  Landers and Begley are using funding from the National Science Foundation (NSF) to develop self-contained, fluidic microchips that detect the presence of specific molecules, such as DNA.  The researchers envision the chips as the foundation for handheld devices with the capability of providing inexpensive, portable and rapid analysis.  According to Begley, this “lab on a chip” technology would greatly expand “point-of-care health monitoring.”  Imagine having a biopsy taken and instantly being able to access the results rather than sending the sample to a distant laboratory and waiting days or more to find out if it is benign or malignant; the technology that Begley and Landers are creating could enable this possibility.

Excerpt from University of Virginia: Arts & Sciences Magazine, July 2006

By Charlie Feigenoff

This Chemistry professor James Landers is a master of compression.  He has reduced an entire laboratory for DNA analysis to a chip the size of a common everyday microscope slide.Asked about the advantages of his Lilliputian laboratory, he’s appropriately succinct: “With a lab-on-a-chip, it takes just 30 minutes to do the work it would take three technicians and three instruments to complete in a week.”

Excerpt from University of Virginia: Arts & Sciences Magazine, July 2006

By Celia Henry

A simple microfluidic method could substantially decrease the time required to prepare samples for forensic analysis of sexual assault evidence [Anal. Chem., 77, 742 (2005)]. Faster sample preparation could help eliminate the backlog of such evidence waiting to be analyzed.

A simple Current methods to prepare sexual assault evidence for analysis are based on a process called differential extraction. This technique relies on the ability of sperm cells (from the perpetrator) to survive chemical conditions that rupture the membranes of other cells (primarily epithelial cells from the victim). It is applied to the evidence still on the cotton swab used for collection.

Chemist James P. Landers and his coworkers at the University of Virginia take a different approach: Sort the cells before rupturing any. They have developed a microfluidic device that exploits the physical differences between the two types of cells to separate them.

The simple device consists of two reservoirs connected by a channel. The epithelial cells (cheek cells in the demonstration samples) settle to the bottom of the inlet reservoir, which takes four to five minutes. Then, a pressure-induced flow sweeps the sperm cells into the second reservoir. The separated cells can then go through normal DNA analysis.

Landers must address a number of issues before the method could become practical for real forensic samples. His team is finding that desorption of the cells from the cotton swabs could be an issue. In addition, because cells can rupture while the samples are dry during storage, free DNA in the samples could be another concern, which may have to be addressed by chromatography.

Susan Greenspoon, a forensic molecular biologist with the Virginia Division of Forensic Science, says that "the device is not yet up to the level of performance we can obtain using either semiautomated robotic or manual differential extraction." But she believes that, with further development, it "has real potential to supplant our current methods."

Excerpt from Chemical & Engineering News. January 17, 2005, 83, 3, 15.