The original GeneXpert system could be configured with 1 to 4 modules, each of which runs one cartridge at a time. To meet the demands of high-throughput labs, Cepheid introduced the GeneXpert 16-site system earlier this year. Users can choose to configure the GX-16 system with 4 to 16 modules, offering the opportunity to run up to 16 different tests in parallel and throughput of up to 388 samples/day. Configurations with even higher throughput are in development, but all GeneXperts, whether for large or small labs, are designed to run the same set of cross-compatible test cartridges.
For maximum versatility, sample prep for clinical diagnostics requires a macrofluidic component, since “many kinds of clinical samples, such as whole blood, swabs, stool, sputum and pus would likely clog up a microfluidics system,” says David Persing, M.D., Ph.D., chief medical and technology officer. Automated processing of these specimens requires building in a macro/micro-fluidics interface upfront.
Dr. Persing points to the “sheer viscosity” of DNA as another challenge of nucleic acid analysis. Detection of rare organisms in several milliliters of blood, for example, necessitates concentrating the sample, which would yield large amounts of genomic DNA that would overwhelm most microfluidics. To overcome this problem, the GeneXpert pumps a specially treated sample through a filter, thus concentrating the microorganisms, and then lyses the retained material by using glass beads powered by sonic energy.
Self-contained microfluidic systems such as in the GeneXpert will also facilitate nested PCR applications without the risk of sample contamination that has limited the use of this technique in the past, suggests Dr. Persing.
The company’s CE-marked test for the BCR-abl oncogene, for monitoring leukemia patients, “is the first commercial PCR test that harnesses the power of nested PCR,” Dr. Persing says.
By miniaturizing diagnostic tests onto credit card-sized disposable devices called lab cards, Micronics(www.micronics.com) is applying its patented microfluidics platform to enable rapid, point-of-care diagnostics testing. Earlier this year, Micronics received a Phase I SBIR grant from the NCI for the development of a point-of-care diagnostic system for early detection of colon cancer in blood, work being done in collaboration with the Fred Hutchinson Cancer Research Center. The microfluidic detector would identify and enrich tumor cells from blood, extract their nucleic acid, and perform molecular analysis to confirm a diagnosis of cancer in an automated process performed on a disposable device.
Micronics’ technology and product-development experience incorporates surface chemistries and materials science with integrated circuits that combine multiple microfluidic elements, including sample collection, mixers, and reactors on a lab card. Micronics has developed lab card applications for drug discovery, protein crystallization, hematologic analysis, immunoassays, and molecular assays. The company is developing a combined immuno-molecular assay lab card as part of the diagnostics team effort funded under the Grand Challenges in Global Health program from the Bill and Melinda Gates Foundation.
“Micronics controls a patent estate in the application of laminar flow diffusion and micro pumps and valving to control fluidics in the micro domain,” says Karen Hedine, president and CEO of Micronics. “Our focus is not on simply reducing the size of an instrument required to run an assay, but rather on re-optimizing existing chemistries and assays for reduced use of sample, reagents, time, and labor to produce an outcome.”
Hedine points to molecular diagnostics as a key strength of Micronics. “We reduced PCR on card to enable a rapid, doctor’s office-based test,” she says. “This combines with our ability to process biological samples directly on card, such that tests may be run at the patient’s bedside directly from sample collection to on-card processing.”
Nanogen’s (www.nanogen.com) NanoChip® electronic microarray technology uses electric fields to attract molecules in solution to an array spot. A positive electric current draws negatively charged, biotinylated DNA molecules to individual test sites on the microarray where they bind to the streptavidin/hydrogel permeation layer on the chip. Rapid hybridization (within 1–2 minutes) to complementary DNA capture probes and subsequent washing away of unbound DNA strands is followed by fluorescent detection of target DNA using fluorescently labeled reporters. Individual test sites can be stripped and re-probed, and an aliquot from a single sample well can be bound to multiple test sites for multiplexed analyses.
The NanoChip 400 system has 400 test sites; the company’s Molecular Biology Workstation cartridge has 100 reactions sites. Each site is represented by an electrode, and the chip individually controls the current/voltage of each electrode.
At present, sample prep is a separate process, but Nanogen is working to integrate sample prep with detection, and Dalibor Hodko, Ph.D., director of advanced technology at Nanogen, describes this as a general trend in the industry. Although nanoscale processing volumes are a common goal, “you have to develop the systems that can interface nanofluidics with microfluidics for larger sample volumes,” and that is one of the current challenges, says Dr. Hodko.
For example, for applications such as detection of pandemic flu virus or biological warfare agents, you need to achieve a sensitivity of about 10 copies/mL or lower in a diagnostic system. To do this, Hodko explains, you need to start with 1–2 mL sample volumes, so you need macroliter processes upfront to extract the analyte of interest from your sample.