November 15, 2011

Using ionized plasmas as cheap sterilizers for developing world

University of California, Berkeley, scientists have shown that ionized plasmas like those in neon lights and plasma TVs not only can sterilize water, but make it antimicrobial – able to kill bacteria – for as long as a week after treatment.

Devices able to produce such plasmas are cheap, which means they could be life-savers in developing countries, disaster areas or on the battlefield where sterile water for medical use – whether delivering babies or major surgery – is in short supply and expensive to produce.

“We know plasmas will kill bacteria in water, but there are so many other possible applications, such as sterilizing medical instruments or enhancing wound healing,” said chemical engineer David Graves, the Lam Research Distinguished Professor in Semiconductor Processing at UC Berkeley. “We could come up with a device to use in the home or in remote areas to replace bleach or surgical antibiotics.”

A brief spark in air produces a low-temperature plasma of partially ionized and dissociated oxygen and nitrogen that will diffuse into nearby liquids or skin, where they can kill microbes similar to the way some drugs and immune cells kill microbes by generating similar or identical reactive chemicals. (Courtesy of Steve Graves)

Journal of Physics D: Applied Physics - Long-term antibacterial efficacy of air plasma-activated water

Low-temperature plasmas as disinfectants are “an extraordinary innovation with tremendous potential to improve health treatments in developing and disaster-stricken regions,” said Phillip Denny, chief administrative officer of UC Berkeley’s Blum Center for Developing Economies, which helped fund Graves’ research and has a mission of addressing the needs of the poor worldwide.

“One of the most difficult problems associated with medical facilities in low-resource countries is infection control,” added Graves. “It is estimated that infections in these countries are a factor of three-to-five times more widespread than in the developed world.”

Graves and his UC Berkeley colleagues published a paper in the November issue of the Journal of Physics D: Applied Physics, reporting that water treated with plasma killed essentially all the E. coli bacteria dumped in within a few hours of treatment and still killed 99.9 percent of bacteria added after it sat for seven days. Mutant strains of E. coli have caused outbreaks of intestinal upset and even death when they have contaminated meat, cheese and vegetables.

Based on other experiments, Graves and colleagues at the University of Maryland in College Park reported Oct. 31 at the annual meeting of the American Vacuum Society that plasma can also “kill” dangerous proteins and lipids – including prions, the infectious agents that cause mad cow disease – that standard sterilization processes leave behind.

In 2009, one of Graves’ collaborators from the Max Planck Institute for Extraterrestrial Physics built a device capable of safely disinfecting human skin within seconds, killing even drug-resistant bacteria.

Diagram of dielectric barrier discharge, which generates a plasma (pink) that diffuses into a nearby liquid and kills bacterial contaminants. (Graves lab, UC Berkeley)

In the study published this month, Graves and his UC Berkeley colleagues showed that plasmas generated by brief sparks in air next to a container of water turned the water about as acidic as vinegar and created a cocktail of highly reactive, ionized molecules – molecules that have lost one or more electrons and thus are eager to react with other molecules. They identified the reactive molecules as hydrogen peroxide and various nitrates and nitrites, all well-known antimicrobials. Nitrates and nitrites have been used for millennia to cure meat, for example.

Indirect air dielectric barrier discharge in close proximity to water creates an acidified, nitrogen-oxide containing solution known as plasma-activated water (PAW), which remains antibacterial for several days. Suspensions of E. coli were exposed to PAW for either 15 min or 3 h over a 7-day period after PAW generation. Both exposure times yielded initial antibacterial activity corresponding to a ~5-log reduction in cell viability, which decreased at differing rates over 7 days to negligible activity and a 2.4-log reduction for 15 min and 3 h exposures, respectively. The solution remained at pH ~2.7 for this period and initially included hydrogen peroxide, nitrate and nitrite anions. The solution composition varied significantly over this time, with hydrogen peroxide and nitrite diminishing within a few days, during which the antibacterial efficacy of 15 min exposures decreased significantly, while that of 3 h exposures produced a 5-log reduction or more. These results highlight the complexity of PAW solutions where multiple chemical components exert varying biological effects on differing time scales.

Redefining clean

Aiming to take "clean" to a whole new level, researchers at the University of California at Berkeley and the University of Maryland at College Park have teamed up to study how low-temperature plasmas can deactivate potentially dangerous biomolecules left behind by conventional sterilization methods. Using low-temperature plasmas is a promising technique for sterilization and deactivation of surgical instruments and medical devices, but the researchers say its effectiveness isn't fully understood yet.

"Bacteria are known to create virulence factors – biomolecules expressed and secreted by pathogens – even if they have been killed," says David Graves, a professor working on the research at UC Berkeley's Department of Chemical and Biomolecular Engineering. These molecules are not always inactivated by conventional sterilization methods, such as heating surgical equipment in an autoclave, and can cause severe medical problems.

The misfolded proteins called "prions" that are thought to cause mad cow disease are one well-known example of harmful biomolecules, Graves says. "These molecules may not be inactivated by conventional autoclaves or other methods of disinfection or sterilization," he says. "In some cases, expensive endoscopes used in the brain must be discarded after a single use because the only way to reliably decontaminate them would destroy them."

Another harmful biomolecule is called lipopolysaccharide (LPS), which are found in the membranes of E. coli bacteria. In humans, LPS can initiate an immune response that includes fever, hypotension, and respiratory dysfunction, and may even lead to multiple organ failure and death.

Graves' research team, in conjunction with a group led by Gottlieb Oehrlein at the University of Maryland in College Park has focused their attention on Lipid A, the major immune-stimulating region of LPS. The researchers exposed Lipid A to the effects of low-temperature plasmas using a vacuum-beam system.

"Low-temperature plasma generates vacuum ultraviolet photons, ions/electrons, and radicals that are known to be able to deactivate these molecules even at low temperature," notes Graves. "However, the mechanisms by which they do this [are] poorly understood, so we can't be sure when they work and when they don't. Our measurements and calculations are designed to reveal this information."

One of the biggest challenges, Oehrlein says, was producing samples of lipopolysaccharide and Lipid A that were compatible with the equipment typically used to study plasma-surface interactions. "The collaboration of Professor Joonil Seog, who is an expert on biological assay methodologies and characterization, has been crucial in this respect," Oehrlein notes. The scientists' results suggest that plasma-generated vacuum ultraviolet light can reduce the toxicity of Lipid A. "We have been surprised by the high sensitivity of endotoxins to UV or vacuum UV irradiation," says Oehrlein. The results mean that the ability of plasma to sterilize equipment might strongly depend on what the plasma is made of, since plasma optical emissions vary based on plasma compositions. As a next step, Oehrlein says that his group plans to focus their efforts on understanding the influence of plasma-generated radicals on the deactivation of biomolecules.

Both groups' results are a good indication that "clean" can indeed be redefined.

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