By Ben Butkus
Scientists from France's Université Paris-Diderot and Centre National de la Recherche Scientifique have created a startup company called Easy Life Science to commercialize a quantitative real-time PCR method that uses electrochemical detection instead of traditional fluorescence-based detection to monitor the amplification of target sequences.
Having recently demonstrated proof of concept of their method, the scientists are currently seeking industrial partners to help further develop and commercialize a device that uses the technology to perform real-time PCR, isothermal PCR, and melting curve analysis, the researchers said this week.
In addition, the scientists are trying to incorporate their technology into an inexpensive, handheld, point-of-care device for PCR-based nucleic acid testing, they said.
"The main idea here is that electrochemical methods are much simpler and easier to miniaturize and integrate than optical methods," Damien Marchal, assistant professor at Université Paris-Diderot and a co-founder of Easy Life Sciences, told PCR Insider.
Marchal developed the new method in collaboration with Benoit Limoges, research director at CNRS; director of the CNRS-Université Paris-Diderot joint laboratory of molecular electrochemistry; and co-founder and scientific advisor for Easy Life Science.
The technique is based on electrochemical monitoring of a DNA intercalating redox probe that, upon binding to amplified double-stranded DNA, becomes harder to electrochemically detect compared with its free counterpart, thus leading to an exponential decrease of the electrochemical signal as DNA product increases during PCR amplification.
Other researchers have demonstrated electrochemical, as opposed to the more commonly used fluorescence, detection of PCR amplification product, but these efforts involved immobilizing primers on the surface of an electrode, which is laborious and time-consuming, according to Limoges and Marchal.
In order to create their electrochemical-based detection scheme in a homogenous solution, Limoges and Marchal had to first identify a suitable intercalating redox probe with a host of characteristics, including the ability to strongly and specifically bind to amplified dsDNA; to not significantly inhibit PCR; to remain chemically stable during cycling; and to be sensitively detected using a standard laboratory potentiostat in a matter of seconds.
After investigating several such probes, the researchers found that the probe Os[(bpy)2 DPPZ]2+, an osmium bipyridine-based complex, satisfied all of these requirements, work that was detailed in a paper published in January in Analytical Chemistry.
In that paper, the scientists demonstrated that their method was comparable in terms of sensitivity and detection limit (about 100 copies in 50 µL) to optical-based real-time PCR methods, including TaqMan-based real-time PCR performed on a Qiagen Rotor-Gene system.
The researchers noted that another aspect of their method is the possibility of generating a post-PCR melting analysis curve by heating the amplicon using a linear temperature ramp and measuring changes in the electrochemical response resulting from the release of intercalated redox probe upon double-strand DNA dissociation.
This feature would allow users to differentiate full-length amplicons from shorter products and thus perform genotyping analyses, the researchers said.
Limoges and Marchal helped found Easy Life Science about a year ago, when it became apparent that their method worked. Thus far, the company has been awarded two grants under a technology competition held by France's Ministry of Research; and has received undisclosed investments from Paris Biotech Santé, the Centre Francilien de l'Innovation, and Scientipôle Initiative, Marchal said.
Easy Life Science's first product, dubbed LEO (light electrochemical detection), is a plastic consumable with an embedded quantitative DNA-detection system based on the electrochemical method, which the researchers have named DetScan.
The consumable is designed to be used on a dedicated block adaptable to any conventional thermal cycler, and will initially be available in a 48-well format, though it can be developed in 8- and 96-well formats, according to the company.
"At the moment, the company is looking for an industrial partner to develop and industrialize a device," Marchal said, adding that Easy Life Science has already initiated discussions with several companies that play in the real-time PCR space. Marchal declined to disclose their identities, citing confidentiality agreements.
Longer term, through their academic lab work, Limoges and Marchal hope to develop a point-of-care device that uses the DetScan technology combined with isothermal DNA amplification, Marchal said.
Due to the "relative insensitivity of electrochemical methods to downscaling and their intrinsic low instrumental cost compared with optical ones," the DetScan technology is "promising for the development of real-time PCR instruments that would be much cheaper" than fluorescence-based systems; as well as for integration in a handheld miniaturized system such microchips or microfluidic devices for point-of-care diagnostics, the researchers said in a statement.
Thus far, "proof of concept has been demonstrated for the detection of different DNA targets such as human virus, plasmid bacteria, bacteria, and genomic DNA," they added.
Marchal said that the group soon plans to submit a research paper for publication detailing the point-of-care device integration.
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