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Sep 1, 2006 (Vol. 26, No. 15)

Fluidics for qPCR, Diagnostics & Biodefense

Nanofluidics Applications Evolve Toward Integrated Sample Processing

  • Micro- and nanofluidics technologies are enabling highly parallel, rapid analytical processes in drug discovery. In addition to research applications, microfluidics is playing an increasingly important role in the development of highly sensitive point-of-care diagnostics and in devices designed to detect pathogens and toxins in environmental samples for the biodefense sector. Microfluidics minimizes sample volumes and conserves resources across a range of applications including real-time quantitative PCR for nucleic acid analysis, high-throughput genotyping, and lab-on-a-chip technology for highly sensitive detection and quantification of DNA, RNA, and proteins.

    In July Gyros (www.gyros.com) sold two of its microfluidics platforms to AstraZeneca(www.astrazeneca.com) and Cambridge Antibody Technology (which has been acquired by AstraZeneca). Gyros’ microfluidics technology platform for miniaturizing and integrating protein-quantification applications centers around the company’s Bioaffy® compact disk (CD)-based microlaboratories, controlled by the Gyrolab Workstation.

    The Gyrolab Bioaffy enables parallel processing of sandwich immunoassays at nanoliter scale in hundreds of identical microstructures present on the CD. Samples are processed simultaneously under the same conditions as the CD spins in the workstation. According to the company, the system requires less than 200 nL of sample per data point and can yield 112 data points in less than one hour. Later this year, the company will introduce the Gyrolab Bioaffy 20 HC, which will accommodate 20-nL sample volumes, according to Jan Wurtz, CEO of Gyros.

    Nanoliter sample volumes “increase the possibility of automation with a smaller footprint,” says Wurtz. The new 20-nL CD will eliminate the time-consuming step of sample dilution, he notes, adding that with the option to select from two different CDs that operate on the Gyrolab workstation, users can quantify protein samples in a broader concentration range.

    Agilent (www.agilent.com) applies microfluidics across multiple technology platforms including in gas and liquid chromatography systems and lab-on-a-chip technology, which is at the heart of the company’s 2100 bioanalyzer platform. Agilent recently introduced second-generation 2100 bioanalyzer Series II Chip kits for RNA, DNA, and protein analysis. The new RNA 6000 Nano and Pico kits feature improvements in the RNA ladder, higher sensitivity, enhanced quantification specification, and increased assay stability for samples with a high salt content, according to the company.

    Improved sizing and quantification and streamlined protocols characterize the new DNA 1000, 7500, and 12000 kits, while the Protein 230 and 80 kits offer an extended size range with more precise sizing and improved, automated marker assignment. Kits in development will target siRNA analysis and include a Pico DNA assay for genome analysis.

    New technologies are enabling expanded genomics applications, such as gene-location analysis, microRNA studies, and DNA characterization. Marc Valer, microfluidics program manager for genomics at Agilent, describes the use of the bioanalyzer for the quantification of complex PCR reactions, enabling the detection and measurement of DNA methylation sites using the COBRA technique.

    Valer says the main challenges in advancing microfluidics-based assay systems are twofold: the complexity of the assay development platform (with the need for interconnected wells and the ability to run samples in parallel or sequentially) and the natural timeline for acceptance of a new technology by the market. On the positive side, microfluidics enjoys “a huge opportunity in all areas,” he says, because the technology is not dedicated to any one application area.

    As an example, Valer points to long-term goals such as microfluidics-based, high sensitivity protein detection—Western blots on a chip—which still requires fundamental technology development.

  • Compound Screening

    Andrea Chow, Ph.D, vp microfluidics R&D at Caliper Life Sciences (www.caliperls.com), has seen growing acceptance of microfluidic technology in drug discovery research as users explore areas where microfluidics can resolve bottlenecks. Caliper works closely with its partners and customers, encouraging them to communicate their wish lists to identify areas of need. Caliper has active co-development programs with companies such as Agilent, Bio-Rad (www.bio-rad.com), Affymetrix (www.affymetrix.com), and Wako Pure Chemicals(www.wakousa.com)—partners helping to leverage the technology into genomic, proteomic, and diagnostic applications.

    “We see our customers moving toward high-content screening assays,” says Dr. Chow, “and they are looking for chip-based assay systems.”

    Dr. Chow identifies three main goals of technology development: improved integration of reagent components; enhanced robustness of microfluidic devices; and reduced cost of devices, which can be achieved through larger volume production (economies of scale) and more efficient (higher yield) production.

    The LabChip® 3000 drug discovery system employs microfluidic technology for high-throughput compound screening using a range of enzymatic and cellular assays. Caliper’s mobility shift enzymatic assays rely on electrophoretic separation of the product and substrate. Proprietary sipper chips enable automated sampling from microtiter plates, with parallel processing of up to 12 samples at a time. Movement of samples through the 100-µm dimension microfluidic channels is controlled using a combination of pressure and/or voltage change. All processing and detection operations take place within the system; unattended run times to screen tens of thousands of compounds range from 8–16 hours.

    Caliper’s chips have been used for chemical synthesis, according to Dr. Chow, in a collaboration with a major pharma company to synthesize families of compounds. Eventually, the goal is to couple compound synthesis with chip-based screening, with direct transfer of the newly synthesized compounds from one chip to the other. For more downstream applications, the company is exploring microfluidic chip technology for performing secondary screening, kinase profiling, and toxicology studies.

  • SNP-based Genotyping

    One of BioTrove’s (www.biotrove.com) main goals in developing the OpenArray™ nanoliter-scale fluidics platform for high-throughput SNP genotyping was “to make the interaction with the nanoworld as conventional as possible,” says Bob Pedersen, business unit manager for OpenArray. The OpenArray NT Imager Genotyping System can process more than 3,000 genotyping assays on a single, disposable plate, according to the company.

    Each plate contains 3,072 through-holes. Samples and reagents for PCR-based TaqMan® assays are loaded onto the plate via passive fluidics. The surface of the plate is hydrophobic and the interiors of the holes are hydrophilic, enabling self-loading. BioTrove loads the plates with dried-down assays supplied by the customer. The user then adds sample and master mix and processes the plates in the OpenArray NT Cycler (or other compatible thermal cycler) or NT Imager.

    An advantage of this platform is the ability to perform spatial multiplexing by loading different assays in subarrays of 8x8, 64-hole grids. By testing multiple samples in each subarray, the user can take advantage of the system’s flexibility and generate genomic information at rates of up to hundreds of thousands of data points per day, with consumption of sample and reagents at nanoliter scale.

    Pedersen believes that the system offers particular advantages in human genetics research, enabling large-scale genotyping studies that were previously cost prohibitive, and in agriculture, where the higher throughput systems currently available can interrogate either large numbers of SNPs or large number of samples but not both.

    BioTrove is expanding the capabilities of the initial system, “moving the low end lower and the high end higher,” says Pederson, allowing users to test for as few as 16 and as many as 3,072 SNPs, optimizing the technology for individual markets.

    BioTrove’s RapidFire™ Lead Discovery system performs high-throughput mass spectrometry via a computer-controlled fluidic robot for label-free compound screening.

  • PCR Systems

    Cepheid (www.cepheid.com) recently received FDA approval of its Xpert GBST (Group B Streptococcus) diagnostic test for use on the GeneXpert® real-time PCR platform. The cartridge-based system combines sample preparation with real-time quantitative PCR and detection in a closed, fully integrated macro/microfluidic system. The test became available on the European market in July.

    XpertGBS yields results in about 75 minutes, according to the company, and is intended for testing pregnant women just before childbirth to detect bacterial infection in the mother that could be passed to the baby during birth. A test for enterovirus for identification of meningitis is also before the FDA, and a methicillin-resistant Staphylococcus aureus assay will soon begin clinical testing.

    Cepheid’s Smartcycler system is a standalone real-time amplification system that can run 96 independently programmable reactions in parallel and is compatible with a range of fluorescent assay systems such as Molecular Beacons, TaqMan probes, and Amplifluor® and Scorpion® primers.

    In June, Cepheid announced an expanded licensing agreement for its thermal cycler technology with Applera(www.applera.com) to include quantitative detection, characterization, and monitoring of HIV and hepatitis C infections. The amended agreement relates both to Cepheid’s SmartCycler and GeneXpert instruments.

    Other tests available on the SmartCycler include CE-marked assays for cytomegalovirus, varicella zoster virus, and Epstein-Barr virus, and a variety of analyte-specific reagents.

  • High-throughput Demands

    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.

  • Leveraging Biodefense Applications

    Building and expanding on the microfluidics-based systems it is designing for pathogen and toxin detection in air samples under contract with the U.S. Department of Homeland Security, Microfluidic Systems (www.mfsi.biz) is leveraging its technology for applications outside of the biodefense sector, including nucleic acid concentration and detection for molecular diagnostics and flow-through immunoassays.

    One product, the Biolyser, performs rapid lysis of cells, viruses, bacteria, and spores using ultrasonic energy. The instrument uses a disposable, capped vial. Samples do not come in direct contact with the sonication device, minimizing the risk of cross contamination among samples.

    The Bioagent Autonomous Networked Detectors (BAND) air-borne pathogen detection system that Microfluidic Systems is developing for the U.S. government consists of an air collector, sample-processing microfluidics, a flow-through disposable thermal-cycling device, and a minifluorimeter for detecting bacteria, viruses, and toxins. It is currently in pre-production, according to M. Allen Northrup, Ph.D., the company’s CEO and CTO. As part of another program for DHS, called Instantaneous Bio-Aerosol Detection System (IBADS), MFSI has developed a device that within five minutes can confirm the presence and identification of a variety of microorganisms, as well as toxins including ricin, botulinum toxin, and Staphylococcal enterotoxin B.

    Bio-Rad’s (www.discover.bio-rad.com) Experion automated electrophoresis system applies microfluidic technology to help researchers perform protein and RNA electrophoresis, according to William Gette, marketing manager, electrophoresis. The product combines Bio-Rad’s expertise in electrophoresis with Caliper Life Sciences’ LabChip technology to deliver highly reproducible analyses in much less time than that required with conventional gel electrophoresis, says Gette.

    Purity and molecular weight for up to 12 samples are computed automatically and reported at the end of the 30 minute run. Tools for US FDA 21 CFR Part 11 compliance and installation qualification/operational qualification are also available.



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