University, West Lafayette, Indiana, is .... the Department of Chemistry, Indiana ... Courtesy of James P. Landers, University of Virginia, Charlottesville, VA.
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Laboratory Automation Faster, Cheaper, Better Laboratory automation involves much more than allowing a user to run tests with a minimum of hands-on activity. Goals of those working in the field include improving turn-around time and decreasing cost, which in turn depends on miniaturization to use smaller amounts of expensive reagents, as well as the development of cheaper platforms. Applications will ultimately be limited only by the user’s imagination.
FISH on a Chip Interphase fluorescence in situ hybridization (FISH) is more sensitive than conventional cytogenetic methods for detecting chromosomal changes, such as translocations and deletions, and can be applied to any cancer where there is a chromosomal abnormality. FISH can be used to predict disease aggressiveness and response to therapy for some types of cancers. Linda Pilarski, Department of Oncology, University of Alberta, Edmonton, Canada, and her group are importing FISH to a microfluidic platform that uses etched channels in glass to control the flow of individual cells. The current chip multiplexes 10 channels, allowing 10 different tests at once at one-tenth the cost of one conventional test, so that people can start using it as a routine clinical tool. Pilarski says they need to move to a disposable chip, and they need to develop the method as a stand-alone without extensive sample preparation and without the need for a dedicated, skilled technician to read the results. One goal is to improve on-chip cell sorting. Christopher Backhouse in the university’s Department of Electrical and Computer Engineering is addressing another goal by developing software (FISHnet) to read the results of the assay without extensive computer resources. His program has already been able to match the results obtained by Pilarski’s highly skilled technician. FISH analysis for one conventional test currently costs about $500 to $600. The estimated cost with glass chips is around $150 for 10 tests, with the cost reduction related to the reduced amount of probe Vol. 44 ı No. 1 ı 2008
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Courtesy of Vincent Sieben, University of Alberta, Alberta, Canada.
(Left) Chip with 10 channels for 10 different FISH tests. (Right) (A) Cells immobilized in a channel after staining with FISH probes. (B and C) Higher power images of stained cells. N = normal cells, T = tumor cells.
used in the microchip setting. The glass chip itself costs about $100. As soon as chips are developed that are disposable, the cost per chip may decrease to $10 or even $1.
“Then the user would just add the patient sample, turning a fairly sophisticated technique into a routine test.” Pilarski predicts it may take a year and a half to develop a fully automated system that could go from a drop of blood to purifying intact cells of interest to hybridization to analysis. Then clinical trials will be needed to compare FISH on a chip to conventional FISH, so it may be 5 years to regulatory approval. The actual assay takes 12 hours, but Pilarski says it could be faster. “It can be done in 4 hours with less intense staining. It’s possible to figure out what the constraints are. For commercial use, almost everything needed will be lyophilized on the chip, maybe even the probes. It is possible to have a chronic lymphocytic leukemia (CLL) chip, a multiple myeloma chip, or an acute leukemia chip, with the controls built into the chip. Then the user would just add the patient sample, turning a fairly sophisticated technique into a routine test.”
Good Enough to Eat?
Arun K. Bhunia, Professor, Department of Food Science, Purdue University, West Lafayette, Indiana, is interested in detecting pathogens and toxins in food. His group, in collaboration with the School of Mechanical Engineering, has developed a light scattering sensor that can identify the species of bacteria in colonies growing on an agar surface. Each bacterial species produces a unique scatter
Courtesy of Arun K. Bhunia, E.D. Hirleman, J.P. Robinson, B. Rajwa, E. Bae, and P.P Banada, Purdue University, West Lafayette, IN.
The light scatterometer can be used to distinguish colonies of different species of bacteria via analysis of the different scatter patterns generated. www.biotechniques.com ı BioTechniques 27
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mentation, and doesn’t want to pay a lot of money for tests,” Bhunia says, “yet when an outbreak [of food-borne illness or toxicity] occurs, it can bankrupt a company.” He observes that inexpensive, user-friendly technologies developed for the food industry could benefit the healthcare industry, too.
Microminiaturization Courtesy of James P. Landers, University of Virginia, Charlottesville, VA.
Integrated microchip analysis of a positive Tcell lymphoma (TCL) sample. (A) An integrated TCL chip with the DNA extraction, PCR, injection, and separation domains. (B) Analysis time for performing both PCR and the electrophoretic separation. (C) Exploded view of the chip-based electrophoretic trace magnified to distinguish the primer and amplified monoclonal products.
signature pattern that can be analyzed using image analysis software developed in collaboration with the School of Biomedical Sciences. This technology is still dependent upon taking a sample of the food to be tested and growing the colonies, which can take 12 to 16 hours. However, identification by light scattering takes only a few seconds, which is much faster than other means of identification, such as biochemical tests or PCR, and does not destroy the culture. Bhunia acknowledges that other technologies will need to be developed for real-time detection and identification of pathogens in other settings, for example, meat processing plants. One challenge is that the number of organisms may be quite low relative to the sample volume. His group is working on a microfluidic chip using antibody capture to extract organisms from food matrices, followed by growth and polymerase chain reaction (PCR) identification. Multiplexing the sensors will be another goal. One advantage of the food setting is that the group knows what organisms to look for, and is developing reagents to support the growth of those target organisms. One disadvantage of working with the food industry is that profit margins are low, which is a driving force in research and development. “The food industry is not interested in sophisticated instruVol. 44 ı No. 1 ı 2008
James P. Landers holds positions in the Chemistry, Mechanical Engineering, and Pathology Departments at the University of Virginia, Charlottesville, Virginia. He says that development of capillary electrophoresis led the way in microminiaturization, in which technologies that were first developed in the computer industry, including microetching and miniaturized electronics, have been leveraged for use in the laboratory. Improvements in gel electrophoresis, he notes, were only incremental; that is, although the time to run a slab gel may have decreased from several hours to 15 minutes, sample preparation time was still an obstacle, particularly for clinical diagnostics. The development of microfluidic chips provided what capillary electrophoresis could not, namely the ability to integrate sample preparation with separation technology. Further integration of PCR technology on the same chip has not only decreased assay time, but lowered the cost by a factor of 100, because the amount of Taq polymerase, the most expensive reagent, is greatly reduced. This is analogous to the cost reductions seen with FISH on a chip technology, where the probe is the costly reagent. The Achilles’ heel of combining sample preparation, including DNA extraction technology, with PCR, was that the guanidine and isopropanol used in the extraction are both potent PCR inhibitors, explains Landers. “Valving devices developed at University of California, Berkeley, solved the problem of bringing two disparate chemistries together,” he says. With a system using 400 picoliters of amplified sample, and a separation time of
160 seconds, multiple analyses of one sample can be performed quickly and cheaply for confirmation. For example, Landers’ group can detect a sequence specific to Bacillus anthracis in whole blood in 30 minutes, whereas this would take days using conventional technology. This system can detect “anything that can be amplified” in many types of samples, for example, Bordetella pertussis in nasal secretions, or viruses or bacteria spinal fluid, for making a clinical diagnosis more rapidly than having to wait for cultures to grow. The same technology can be applied to cancer diagnosis, where a 1-hour test of a biopsy sample could either relieve a patient from worry, or allow the physician to begin treatment much sooner. Lander notes that there are engineering and other problems to solve. These include one identified by Pilarski, namely how to replace the glass slides or chips with plastic.
Even Smaller Michelle Kovarik, a graduate student in Stephen C. Jacobson’s group in the Department of Chemistry, Indiana University, Bloomington, Indiana, is working on nanofluidics, with a goal of further miniaturizing microfluidic-based separation systems. Although microfluidic separations are now fairly wellcharacterized, at nanoscale dimensions physical properties of components that were absent or insignificant on a larger scale become more important. The Jacobson group focuses on fundamental device development, and some of their work involves incorporating microfluidic channels for sample preparation and transport to nanopores for detection. A potential application might use signals at the pores to distinguish a small molecule or component generating a small signal from that same molecule bound to another molecule or complex, which would generate a larger signal. Such a system could be used for an assay or for molecular assembly.
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Courtesy of Michelle Kovarik and Stephen C. Jacobson, Indiana University, Bloomington, IN.
(A) Schematic and (B) white light image of a multilayer device that contains integrated microchannels and nanopores. (C) Fluorescence image of 1 micron, fluorescent, and polystyrene microspheres that have been trapped at the nanopores, allowed to accumulate, and then released.
“The collaboration with individuals in other fields changes how people talk about science.” Kovarik says that one of the exciting things about working in laboratory automation is the interaction their group has with colleagues in other departments, for instance, biology and physics. Their group recently moved to a new interdisciplinary science building with a nanoscale characterization facility. “I’ve had to diversify,” she says. “The collaboration with individuals in other fields changes how people talk about science.” She is hoping to develop a system that can detect a single analyte passing through a single pore in a single microfluidic channel both electrically and optically. Currently working with latex particles and bacteria as models, Kovarik would like to move to a real-world system of biologic interest, for example, detection of DNA strands annealing. An interdisciplinary program at the university could provide collaborators for another potential application, analysis of viral particle assembly. Lynne Lederman is a freelance medical writer in Mamaroneck, New York.
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