January 1, 2010 (Vol. 30, No. 1)
New Types of Arrays, Novel Combinations of Capabilities, and Increased Speeds Revitalize Field
A new approach to high-throughput screening has been developed by microPEP. Rather than imprint reagents onto microarrays, microPEP’s approach injection-molds microwell arrays to be used with microbeads. These microarrays contain 40 million locations that are 3 µm in diameter and 3 µm deep, making them the smallest microwell arrays in the world, according to the company.
“This molded slide is meant to be an option to the 25 x 75 mm microarrays,” according to Scot MacGillivray, business development manager. “We think we’re the only company in the world to offer this,” MacGillivray reported at the Knowledge Foundation conference on “Detection Technologies” late last year.
“This molded microarray has the potential to change the way healthcare is delivered and assays are performed,” and it certainly makes high-throughput screening more cost effective. For example, traditionally microbead assays could only be performed by first etching away a silicon wafer. That’s expensive, costing hundreds of dollars. Molding that substrate, however, costs less than $30, MacGillivray says. “When we get into the area of thousands of slides, the cost is under $10 per microwell slide.” The microwell slides meet Class 10,000 cleanroom specifications.
The tooling is based upon standard injection technology and some proprietary technology. The next step for microPEP is to explore the submicron fabrication limits. “We’re interested in trying nanofeatures,” MacGillivray said. Additionally, the company has developed 50 and 100 µm well slides. The 3 µm microwell slides are available now.
While microPEP is advancing miniaturization for biodetection, other presenters discussed new types of arrays, expanded capabilities, increased speeds, new combinations of capabilities, and novel molecular methods to immobilize and label DNA.
Platypus Technologies is developing protein-detection assays based upon liquid-crystal technology to rapidly detect and report the presence of target molecules bound to specially designed surfaces.
As Richard Schifreen, Ph.D., president and CEO, explained, liquid-crystal molecules are rod-shaped and tend to align parallel with each other. That alignment changes, however, when target molecules bind to receptors that are immobilized on the surface of liquid crystal-coated substrate. The standard analogy compares the alignment to upright bowling pins, and the binding to the scattered pins after the bowling ball—the target molecule—has struck.
The dramatic difference makes direct detection of viruses captured by surface-bound antibodies possible with low- or no-power readouts based upon interrogation with polarized light. For a simple “yes/no” readout, a light image indicates presence of the target molecule and a dark image indicates absence of the target.
“The work is at the precommercialization phase,” Dr. Schifreen said. “The sensor can be packaged into an instrument, a badge, a handheld device, or incorporated into an alarm system.”
Platypus is currently developing a protein-detection assay for influenza exposure and water-borne pathogens, and gas sensors to detect nitric oxide and pesticides and to monitor asthma. “The advantages,” he said, “are low- or no-energy requirements, the inherent flexibility of a single format for multiple analytes, and the simplicity of a solid-state system. It’s very inexpensive, costing a couple of dollars at research scale,” versus about $100 for an electrochemical sensor.
“Detection speed depends on the particular system,” Dr. Schifreen said, but ranges from a few seconds to one or two minutes. “The assay can be either qualitative or quantitative. We’re working to optimize the system so it’s more specific, more reproducible, and, in some applications, reversible.”
Multiplexing assays are another speed-booster. GenArraytion’s microsequencing method, Sniper Sequencing, allows researchers to perform multiple analyses in parallel that generally are performed sequentially, according to R. Paul Schaudies, Ph.D., CEO.
Sniper Sequencing provides a functionally complete characterization of a broad spectrum of microorganisms within hours, he said. It combines information about the functional elements of pathogens with information about their specific signatures to provide a detailed fingerprint that accurately characterizes organisms to strain and isolate levels on platforms such as the CombiMatrix microarray. That capability allows scientists to genetically characterize unknown strains and to determine whether those strains have been genetically manipulated.
The company has an SBIR grant to develop the technology for characterizing strains of drug-resistant Staphylococcus aureus (MRSA) bacteria. That project uses genetic sequences from a wide range of staph strains to generate detailed hybridization profiles for organisms with no published sequences.
In the second phase of the project, “GenArraytion has an agreement with two major medical centers to provide clinical isolates with antibiotic growth profiles for genomic characterization using Sniper Sequencing. GenArraytion plans to select a subset of 4,400 oligonucleotides that correlate with antibody susceptibility profiles to provide rapid, accurate treatment,” said Dr. Schaudies.
Working with the Luminex beaded array platform, GenArraytion will use functional and unique genetic sequences to provide a more relevant signature subset that will be transferred to smaller, faster beaded arrays for point-of-care diagnostics, he explained. The goal is to provide an assay to identify and determine the antibiotic susceptibility of MRSA viruses for the hospital critical-care market to identify hospital-acquired and community-acquired MRSA strains and to determine antibiotic susceptibility.
Speed is one of this system’s key benefits. Normally, in treating septic infections, traditional microbiological methods for identifying pathogens and determining their sensitivity to particular antibiotics requires four or more days. GenArraytion’s approach will require less than six hours, added Dr. Schaudies. Therefore, physicians can treat immediately with an effective antibiotic rather than using a broad-spectrum cocktail while awaiting test results. That difference speeds patients’ recoveries and extends the useful life of existing antibiotics.
Sniper Sequencing technology can be applied to veterinary diagnostics, biological defense, epidemiological monitoring, environmental testing, and food and water safety. GenArraytion has worked with several U.S. government agencies to develop detailed signatures for organisms that pose a threat to human and animal health, including an effort with the U.S. EPA to identify multiple oligonucleotide differences among strains of Cryptosporidium parvum and hominis.
Atlas Genetics has developed a portable, integrated cartridge and instrument system that combines sample preparation, DNA amplification, and detection to deliver results within 25 minutes. “The first test is for chlamydia; it will be in clinical trials in 2011,” according to John Clarkson, Ph.D., CEO. “The first clinical trials showed a sensitivity of 96% and specificity of 98% against the gold standard. Limit of detection for chlamydia is approximately 10 cells.”
The system runs on the Velox platform, which detects DNA and RNA from clinical samples using electrochemistry. Optical clarity is not an issue. It is designed initially for clinical diagnostics but can be used for other analytes, multiplexing up to 20 analytes per cartridge. Because this is designed as a CLIA-waived system, it can be used at point of care.
“The next test will be chlamydia and gonorrhea combined, and we are also developing a test in collaboration with the U.K.’s Animal Health Trust for equine strangles, which is caused by Streptococcus equi,” Dr. Clarkson said. Other tests are in development for MRSA, group B streptococcus, and Norwalk viruses in humans.
At the conference, Amy L. Altman, Ph.D., director of extramural research office for Luminex, discussed the development of xMAP® technology. This technology, she explained, “enables laboratories to multiplex bioassays, reducing time, labor, and costs over traditional methods.” Thanks to its open-architecture platform, xMAP technology can be applied across a wide range of markets.
“Our xMAP technology is the underlying foundation for all our systems,” Dr. Altman said, including the recently released Flexmap 3D system, with 500-plex capabilities for high-volume academic and pharmaceutical research applications.
The smaller, deployable MagPix biological analyzer is for academic research labs and decentralized diagnostic laboratories. The MagPix is a low-cost, rugged, compact, biological testing device designed as a deployable biological analyzer. The MagPix instrument simultaneously detects up to 50 analytes in a single reaction. Because it can include multiple signatures per target agent, false positives are greatly reduced, Dr. Altman pointed out.
“In addition, each microsphere essentially represents a single reaction. Reading up to 100 microspheres per reaction further validates the result by allowing for statistical analysis of each sample.”
A 96-well plate can be analyzed within one hour, Dr. Altman said, and can take advantage of an extensive menu of assay panels built on Luminex xMAP beads. “This bead-based format allows for improved kinetics compared to planar arrays.” Furthermore, “the ability to multiplex both proteins and nucleic acids saves the end-user time, labor, and sample, and is a flexible and scalable solution.”
Rather than interrogating analytes via a flow cell illuminated with focused laser excitation light, “the MagPix employs light-emitting diodes and a charged coupled device imager, coupled with an enhanced magnetic microsphere-based array,” she explained. “The design is rugged with no need for complex laser alignment or hydrodynamic focusing.”
Biotami is using molecular lock technology licensed from The Gene Pool to develop certain highly specific, simple format nucleic acid tests.
Molecular locks are molecular assemblies that bind nucleic acids as specifically as antibodies bind antigens. “Diagnostic molecular-lock components cooperatively and selectively assemble onto the target nucleic acid and lock to form highly stable handles on the nucleic acid. A handle can be used to selectively immobilize the target in a crude lysate. A second handle with incorporated labels can be used to count copies of the locked target without target amplification such as PCR,” explained Susan Weininger, founder and CEO.
“Molecular locks can lock a nucleic acid target directly, without the use of a hybridizing probe. Direct engagement of the nucleic acid target simplifies sample preparation and may have IP advantages. Sequence patents regarding specific targets may not be relevant when no copy of the nucleic acid has been made or is used in the test.”
Weininger pointed out that “molecular locks are relatively temperature- and contaminant-insensitive as compared to other technologies, making them ideal for crude sample preparations.”