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Feature Articles : Nov 15, 2009 (Vol. 29, No. 20)

Taking Multiplexing to the Next Level

Increased Speed and Efficiency and Decreased Costs Have Further Boosted Powerful Technology
  • K. John Morrow Jr., Ph.D.

Multiplexing technologies are essential for the rapid screening of large numbers of samples in proteomic and genomic profiling. Tool providers are increasingly offering new solutions that feature ramped-up speed and efficiency. Other advances include disposable labware with improved plates for microarray screening, nanoparticle-based diagnostic tests, label-free systems, and multiplexing assays that identify small molecule drug candidates.

Corning Life Sciences manufactures a range of microwell plates for different multiplexing technologies. “The wide selection of products allows customers to evaluate their kits against different plates in order to choose that which will provide the optimum performance for a specific project,” says Mark Rothenberg, Ph.D., applications manager.

As current microarray technology typically takes advantage of either fluorescent and luminescent cell-based assays, investigators usually choose a black plate for fluorescent assays and a white plate for luminescent assays. Some protocols, however, call for a mixed fluorescent/luminescent combined assay, which is referred to as a multiplex kit. For this use, Dr. Rothenberg recommends a black plate in order to obtain minimal background noise, greater signal, and fewer false positives in both modes.

Tight budgets have forced many companies to search out the economic benefits of improved multiplexing devices. To this end, Corning has been adapting its product line to the demands of miniaturization, allowing for more rapid processing of samples at lower cost and with reduced sample requirements. According to Mike Briggs, Ph.D., product line manager, the 1,536-well plate is the flagship product this year. With its low volume requirements it can be used for a range of investigation, he says. 

Ultrasensitive Detection

Nanosphere is focused on the development and commercialization of molecular diagnostic systems for ultrasensitive genomic and protein detection, says Winton Gibbons, vp for business development. “Our nanotechnology-based platform, the Verigene System, provides sensitivity for direct genetic and protein detection that is orders of magnitude higher than existing products.”

The Verigene System provides assays for flu, cancer, and cardiovascular and autoimmune diseases with a high multiplexing capability. “We believe that our system provides simple, inexpensive tests for genetic disorders as well as early detection of protein disease markers.” 

The company has a number of different targets in the R&D stage, as well as FDA-approved tests for Warfarin metabolism, cystic fibrosis, and a first generation infectious respiratory disease assay. Additionally, Nanosphere is working with pharmaceutical and biotechnology firms to develop potential pharmacogenetic assays or companion diagnostics.

The Verigene equipment uses disposable test cartridges with a microarray format for high-count multiplex assays that insert into the microfluidics processor. The results are rapidly generated and there is no interpretation required, Gibbons notes.

The detection system is based on gold nanoparticle probes that bind to samples immobilized on microarrays. The nanoparticles are complexed with antibodies or with DNA probes depending on the particular test being performed. The protein-detection system can detect as low as zeptomolar levels of target.

“Through our nanotechnology approach, we are able to address the major limitations of existing technologies, providing lower cost and faster turnaround time with an easy-to-use platform. We have an extensive pipeline and portfolio with substantial rights to intellectual property from Northwestern University’s International Institute for Nanotechnology.” 

Isothermal Titration Calorimetry

“The Auto-iTC300 is a powerful tool for small molecule biotherapeutics,” says Eric Reese, Ph.D., head of business development at Microcal, now part of GE Healthcare. According to Dr. Reese, an isothermal titration calorimetry system is used in Microcal instruments to study the binding of small molecules to larger macromolecules.

The Auto-iTC300 system is an upgraded version of the Auto-iTC200. It consists of a sensitive isothermal titration calorimeter combined with full automation for unattended operation. With the included software it is possible to program the device for a variety of experimental parameters.

“The platform is ideal for a high-throughput format. It can be used to measure binding affinity for small molecules in drug discovery programs as well as assist in designing innovative drug candidates.”

Isothermal calorimetry can also be used to study protein-metal, protein-protein, receptor-nucleic acid, antibody-antigen, protein-carbohydrate, and protein-lipid interactions.

Automating Sequencing

Agilent Technologies is working with a number of industrial and academic clients, including Los Alamos National Laboratory and the UCLA School of Public Health, to speed up genomic workflow, reports Marc Beban, Ph.D., director, integrated systems and software marketing. 

The company also has several initiatives under way in the agricultural biotechnology sector to automate sequencing of crop and pathogen genomes. The sequencing data can be used to discover favorable genetic traits that can then be inserted into crop plants either through conventional breeding or genetic engineering.

In the field of microbial detection, Agilent uses its platform to sequence genomes from single cells. This is an especially useful approach to gene mapping in hard-to-culture strains of bacteria and could lead to unique markers that could be integrated into new diagnostic probes. Because of the vast amount of DNA that must be screened, this project must be executed with a rapid screening methodology, Dr. Beban adds.

Agilent moved into automation of genomic workflow two years ago through the acquisition of Velocity 11. The process of large-scale sequencing feeds upon itself, as more and more genomes are sequenced  the process becomes more automated, accelerating the rate of sequencing.

“We have a team of application scientists, including biologists and engineers that work with customers to build solutions adaptable to complex scientific problems,” said Nitin Sood, division manager, integrated systems and software marketing.

SPR Platform

The SPRimager®II array reader is a label-free, SPR-based platform developed by GWC Technologies. Voula Kodoyianni, Ph.D., CSO, reports that the system “delivers a compelling combination of efficient throughput and experimental robustness.”

She says that the technology is among the most versatile of molecular detection systems, given that it is label free, monitors multiple interactions simultaneously, and acts independently of the chemistry of the molecules or the reactions that they may enter into.

“There is no need to modify the molecules with fluorescent or other tags that might compromise their function,” she adds. “Furthermore, and this is not generally appreciated, the technology provides real-time data, enabling the investigator to monitor the progress of binding without disturbing the molecular interaction.”

As is standard practice in such systems, the SPRimager II reads interactions occurring on gold-coated chips to which molecules may be covalently attached or bound through affinity interactions such as streptavidin-biotin complexes.

Producing the arrays is straightforward and can be done through hand-pipetting using the company’s SpotReady™ chips, or, for higher density arrays, with the aid of a robotic spotter using plain gold SPRchip™ substrates. Solutions containing analytes are washed through the flow cell of the reader over the arrayed molecules and molecular associations can be monitored as changes in reflectivity as a function of time.

GWC Technologies is collaborating with Lloyd Smith, Ph.D., professor in the department of chemistry at the University of Wisconsin, who has developed an amorphous carbon covering to protect the gold surface of the chip.

Conventional gold-coated chips are acknowledged to be fragile and can delaminate under harsh conditions such as with the use of strongly alkaline or acidic solutions. The amorphous carbon layering technology provides a much more robust surface and allows a wider variety of chip modifications to be performed, Dr. Kodoyianni says, including direct on-chip synthesis of oligonucleotides and other oligomeric biomolecules of interest. Such approaches dramatically reduce costs and increase efficiency of array fabrication. 

Evolving Microarray Platforms

Graffinity’s lead product is its drug-target screening service on its chemical microarray screening platform, which consists of a library of 23,000 small molecule fragments and 87,000 lead-like compounds. These compounds are immobilized on chips and are scanned in the presence of drug-target proteins using SPR imaging technology. “Because our library is large, it is an effective screening tool for identifying novel, diverse structures that bind to a customer’s drug target,” explains Mathias Woker, CBO.

“We work with various large pharmas and biotechs to increase their chances to discover NCEs,” he continues. “Since the platform is label- and assay-free, we can perform this service every time within the same four-month time frame—including resynthesis of hit molecules.”

The platform offers access to a range of drug discovery insights, which Kristina Schmidt, Ph.D., CEO, says sets it apart from other lower-throughput SPR technologies with much smaller libraries. “This gives the platform an unprecedented level of diversity,” she adds.

Graffinity has started applying its technology to the identification of customized ligands for affinity chromatography in protein purification. Using immobilized ligands already at the primary-screening stage simulates how they will behave on affinity chromatography columns. Hence, once identified, a validated hit will most likely enable a unique purification process for a biological. More importantly such small molecule alternatives open up opportunities for new purification process patents, which are often unavailable with peptide- or protein-based methods. “Our small molecule ligands are robust and can undergo numerous cycles,” Dr. Schmidt says.

There appear to be no limits to the ingenuity of multiplexing gurus as they strive to ratchet up the speed and sensitivity of molecular detection systems. The resultant powerful technologies generate data that may lead to better diagnostic tests and earlier and more effective treatment of infectious diseases, cancer, and other illnesses.

Sidebar: PCR Multiplexing

Scientists at Biosearch Technologies say that dramatic improvements in detecting pathogens in food and agricultural products and the environment can be obtained by multiplexing PCR assays. Spectrally distinct fluorophores and quenchers provide the ability to detect genetic signatures and distinguish closely related strains, all within the same reaction chamber, according to the researchers.

In a poster entitled “Intricacies of PCR Multiplexing as Revealed through a Pathogen Detection Assay,” the Biosearch team reported the development of a quadruplexed assay that distinguishes several strains of Bacillus anthracis from a similar organism, B. cereus. The scientists relied on careful selection of target sequences, fluorescent reporters, and a real-time PCR instrument for detection.

They used RealTimeDesign™ software to generate TaqMan® assays to the species-specific sequences. To multiplex these assays together the scientists characterized dye crosstalk, confirmed that each assay had a high amplification efficiency, and identified their detection limit, especially in the context of disproportionate targets.

“For situations where one sequence may be in vast excess over the others, we show the benefit of supplementing master mix components so that depleted reagents don’t limit detection sensitivity,” they wrote. “Multiplexing demands increased effort to characterize amplification performance but is ideally suited to interrogate multiple genetic signatures from small quantities of sample DNA."