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Sandia Lab Creates Quadraplex Immunoassay on SpinDx System, Developing Isothermal MDx

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NEW YORK (GenomeWeb) – Researchers at Sandia National Laboratories have developed a multiplex immunoassay that detects four enteric pathogens in less that 20 minutes using the group's SpinDx platform and are also developing the system for nucleic acid detection.

Chung-Yan Koh, corresponding author on a Biosensors study of the immunoassay and a senior research and development scientist at Sandia, told GenomeWeb in an interview that the group is pursuing both recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) for molecular testing using its diagnostics platform.

The SpinDx system consists of a cube-shaped instrument and small disc-shaped cartridges containing channels that can be preloaded with reagents. It requires a small amount of sample, which is loaded into the center of the cartridge and then manipulated through the chambers using centrifugal force.

The platform has been in development for immunoassay approaches since at least 2012, as reported by GenomeWeb. But now, Sandia scientists have made the immunoassays multiplexed, and are also pursuing molecular diagnostics using the device.

The enteric pathogen immunoassay described in Biosensors detects E. Coli, Listeria, Salmonella, and Shigella directly from small amounts of diluted stool samples, traditionally considered a very difficult sample type. The group also tested the assay by diluting these bacteria into other standard matrices, such as blood and urine, and found "a robust, linear response regardless of sample matrix."

The enteric pathogen test is multiplexed spatially, Koh said, such that reagents for each pathogen of interest are sequestered in different channels and the sample is split upon spinning of the cartridge.

The group has also developed a method for in-channel multiplexing, he said, by using different densities of beads, and a manuscript describing this method has been submitted for publication.

Centrifugal diagnostics have certain advantages over more traditional lateral flow methods for immunoassays. Primarily, they aim to be as easy to use, but with less room for misinterpretation of the instructions for use. They are also more easily multiplexed, and are also an improvement over ELISAs in that they don't require a lab or trained personnel.

Additionally, centrifugal systems tend to have just a single moving part, as opposed to valves and pressure systems required for other kinds of microfluidics. Indeed, techniques like sample prep and amplification can be performed on the same disc-shaped cartridge, with the speed of rotation determining timing of sample movement from one chamber to the next.

There are a number of firms that currently use centrifugal methods for diagnostics, Koh noted. Abaxis makes the Piccolo Xpress, a portable clinical chemistry analyzer that is exclusively distributed by Abbott, primarily in the veterinary space. Samsung has developed a platform and was one of the first firms to demonstrate an immunoassay on a centrifugal system, Koh said. Gyros Protein Technologies also has a centrifugal device and may be a bit further ahead in getting into the human diagnostics space than other firms, Koh added.  And a compact analyzer called LabDisk is being developed by Qiagen.

Meanwhile, research into centrifugal microfluidics is being pioneered in labs like those of Marc Madou at the University of California, Irvine, Roland Zengerle at the University of Freiburg, and Jens Ducrée at Dublin City University, Koh said.

SpinDx is an approximately 4.5-inch cube that weighs about one kilogram, Koh said. It is thus very portable, and centrifugal techniques are especially forgiving of environmental jostling, he said.

For the enteric pathogen assay in particular, a single battery charge supplies the system for two eight-hour work days, or about 50 runs of the 20-minute assay, processing one patient sample per run in the current disc configuration.

In general, point-of-care and handheld diagnostic systems are becoming more common. Koh said that Abbott's iSTAT is a good example of a system that is easy to use and interpret, with a range of tests, which are all qualities that customers seem to be seeking.

SpinDx is also easy-to-use, requiring only a single user step with data interpretation referenced to a calibration curve done on the device, Koh said. The researchers have also attempted to design it so that it is easy to manufacture, using as many off-the-shelf components as possible.

The Sandia SpinDx is now being developed to run nucleic acid amplification-based tests, and the scientists will debut its molecular diagnostics capabilities at the International Conference on Miniaturized Systems for Chemistry and Life Sciences, also known as µTAS, in Dublin, Ireland next week.

The group has implemented both RPA and LAMP on the platform. It has also tried PCR, but found it was more energetically costly and the required battery size limited the portability of the device, Koh said.

For molecular testing, the scientists have also enhanced the LAMP reaction using a Sandia-developed technique called quenching of unincorporated amplification signal reporters, or QUASR. That visualization method has also been used by the researchers in a multiplexed reverse-transcription LAMP-based, field-deployable assay for differential diagnosis of malaria and viral febrile illness.

The QUASR-LAMP method provides enhancement over traditional LAMP because intercalating dyes tend to give false-positive results, while with the quencher-based method the false positive rate is "on par with qPCR for most targets," Koh said.

LAMP is also resistant to inhibitors and other problems found in clinical matrices, so the group is able to do detection from whole blood, as well as saliva and urine, with no preliminary cleanup steps, Koh said.

The scientists also liked RPA, particularly because some of the new RPA-based kits that have come out from TwistDx allow for use of qPCR primers without modification, Koh said. "We were really intrigued by the idea that we can validate qPCR in four wells of a plate and then take those same primer sets, put them into RPA, and have a working diagnostic," he added.

Sandia has out-licensed aspects of the SpinDx platform to "more than a handful" of licensees already in different declared fields of use, Koh said, particularly in the fields of fertility, neonatal health, and water quality monitoring.

The organization also has a dedicated business development team for other groups who wish to explore licensing and commercialization of the assays or platform.