DNA detection, reagent-free

A team of researchers at the University of California, Santa Barbara (USCB), has developed a reagentless, electrochemical DNA detection system, according to a report in the early online edition of PNAS.UCSB postdoctoral fellow Chunhai Fan, assistant professor of chemistry and biochemistry Kevin Plaxco, and 2000 Chemistry Nobel Laureate Alan Heeger modeled their electrochemical DNA sensor on fluorescent molecular beacons. Molecular beacons are stem-loop–forming DNA molecules tagged at one end with a fluorescent dye and at the other with a fluorescent quencher, with a target-specific sequence in between. In the absence of target DNA, the beacon adopts a closed, hairpin-like structure that keeps the dye and quencher in close proximity, dousing fluorescence. When it binds its target, however, the probe unfolds, separating the two ends and producing a measurable light emission.The team's electrochemical variant involves a stem-loop–forming piece of DNA tethered to a gold electrode to which an electroactive compound called ferrocene is attached. In its closed configuration, this DNA's hairpin structure keeps the iron-containing ferrocene near the electrode surface, where it can undergo a measurable electron transfer with the electrode by electron tunneling. Harry B. Gray, Arnold O. Beckman professor of chemistry at CalTech and one of the paper's peer reviewers, told The Scientist that the efficiency of electron tunneling diminishes exponentially with increasing distance. By separating the reporter from the electrode surface, DNA hybridization events reduce the current.Like molecular beacons and unlike existing electrochemical detection methods, Plaxco's approach is reagentless—that is, no chemicals need to be added following hybridization for detection, according to the paper. But unlike fluorescent approaches, electrochemical methods require no expensive hardware such as light sources, optics, and photodetectors.The authors report detecting target DNA at as low as 10 picomolar concentration, which Plaxco told The Scientist is "pretty much state of the art." Plaxco said that under ideal laboratory conditions, optical systems can detect target at concentrations as low as 10 femtomolar. But in the real world, he said, such systems typically report in the nanomolar to picomolar range, in line with his system. "We're competitive with optical in the real world, and we're absolutely competitive with any other electronic technique."The authors report recovering about 80% of the original signal strength after washing, suggesting that the system is reusable. They attribute the 20% loss to the instability of ferrocene in aqueous solutions at high temperature. According to Holden Thorp, professor of chemistry at the University of North Carolina, this is one of the system's strengths. "There's nothing that happens in the reaction that does anything to the DNA or to the target or to the sensor," Thorp, a consultant for electronic biosensor developer Clinical Microsensors and who was not involved with the paper, told The Scientist, "So this would be a great way to monitor a solution continuously for the presence of a specific DNA sequence."Plaxco, who has applied for a patent on the method, said that the system could pave the way for passive bioweapons sensors and point-of-care devices but that the system's sensitivity would first have to be increased by three to four orders of magnitude. The team is investigating various approaches for doing that, either by boosting the signal or by reducing background.Another barrier to biosensor development: fashioning an in-line sample preparation system to extract nucleic acids from potential pathogens in the sample. "That's the bugaboo of a lot of this stuff," Thorp said. "I think all of us who work on these sensors would be really excited to find a great way to get DNA and RNA out of cells conveniently."The team is working on other modifications to the technology, including decreasing the sensor element "pixel" size and enabling multiplexing on each electrode element.According to Plaxco, others have demonstrated electrochemistry sensitive enough to detect a single molecule. "There's no fundamental physics that stands between our present detector and a detector that's good enough that your doctor would carry it around to see if you've got SARS or whatever," he said. "We're not there yet, but there's no fundamental speed-of-light barrier between us and that. There's difficult technology, perhaps, but it physically should be doable."

DNA detection, reagent-free

Electrochemical system may give rise to better microbiologic sensors, say study authors

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