NEW YORK (GenomeWeb) – Scientists from the University of California, Berkeley, have married basic physics with molecular biology to enable PCR nucleic acid amplification in less than five minutes.
In a proof-of-concept paper published last week in Light: Science and Applications, the researchers, led by UC-Berkeley professor Luke Lee, describe how they were able to combine light-emitting diodes (LEDs), gold film, and microfluidic chips to get ultra-fast thermocycling with potential for point-of-care diagnostics.
As part of their work, the scientists took advantage of the basic physics that happens when light strikes a metal film. Light can activate electrons at the surface of the film, generating heat that the scientists used to drive thermal cycling.
In their study, they were able to heat the reagent mixture at a rate of more than 12 °C per second and cool it at a rate of 6 °C per second. But since submitting the paper, Lee told GenomeWeb his lab has made it even faster. "Now we are approaching even three minutes," he said. "It can go even faster but the limiting factor at this moment is not light any more, it's enzymes. The enzymes cannot keep up with that speed."
The idea to make thermal cycling faster came out of a project Lee was working on for the Bill & Melinda Gates Foundation to improve the separation of plasma from blood in sample preparation.
"I realized, what's the point of reducing sample prep time if downstream PCR is so slow?" Lee said. While he had been working with microfluidics to improve sample preparation, he had spent most of his career working in the field of photonics — research involving light, lasers, and diodes — including a decade-long stint in the private sector.
With that experience, he was able to merge physics with life science. Lee's lab created microfluidic chips that were coated in a film of gold atoms about 120 nanometers thick. Light of a certain frequency can excite electrons in the film. "If I'm a violinist, I can break a glass of water from far away by matching the resonant frequency of the cup with my instrument," Lee said. It's the same principle that he used to generate heat at the boundary of the gold film and the PCR reaction mixture. "I use light to oscillate electrons far away on the chip at the interface between the DNA solution and metal layer."
The metal also works in cooling the mixture, since it conducts and disperses heat. Metal electrodes have been used as a heat source for thermal cycling in PCR before, Lee said, but his technique is different, and much faster than the heat blocks used in traditional PCR.
How Lee might differentiate this technology from other super-fast PCR methods, such as capillary tubes with high surface area-to-volume ratios, is unclear. Besides speed, Lee says his method has several bonus features. It's high-throughput and can save on reagent costs, since it uses tiny wells of about 10 microliters. And it's cheap: the LEDs are not special equipment but available off the shelf; and the gold film costs less than a dollar per chip — and it doesn't even have to be gold, it could be other metals, something Lee is currently evaluating. Ultimately, Lee has plans to integrate this fast PCR on a chip along with RNA and protein analysis.
Lee noted that his bigger vision is for a point-of-care molecular diagnostic chip. "In particular I was interested in making a sample-to-answer biochip, where you draw blood, and then you analyze protein and nucleic acid info in one chip as a molecular diagnostic for developing countries," he said. He declined to provide additional details on other technologies related to this chip.
Lee said he has applied for a patent on the thermocycling method described in the recent study and while he hasn't yet started a company based on the idea, he has plans to do so. The paper was a first step, a proof of concept to get the idea out there, but he has some ideas for applications, including POC diagnostics. The authors pointed out that their method meets several important criteria for use in a POC device: it's inexpensive, compact, fast, and doesn't need a lot of power.
In the time since the study was submitted for publication, Lee said his lab has further developed the technology so that it performs better when compared with existing PCR and has already submitted a second paper detailing a 3D nanostructure that he says will improve performance.
In addition to POC molecular diagnostics, potential applications for the technology include drug screening and long-term evaluation of biomarkers in infectious disease, cancer, neurodegenerative diseases, and cardiac diseases.