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Essential sterilisation needs

Hospital infection by multidrug-resistant Staphylococcus Aureus (MRSA) and transmissible spongiform encephalopathy diseases through protein contamination of surgical instruments are just two examples of an unprecedented challenge currently facing the sterilisation service in hospitals and other health-care facilities. Conventional procedures such as autoclaving and ethylene oxide are generally impractical in tackling MRSA contamination in hospital wards. They are similarly ineffective in removing prion proteins of Creutzfeldt-Jakob diseases (CJD) from surgical instruments. There is clearly a fundamental need to support and enhance the current portfolio of sterilisation procedures with new and novel strategies. With the exploration and development of new sterilisation strategies comes naturally an opportunity to evaluate their potential benefits in the manufacture of medical devices.

Gas plasma technology

One sterilisation strategy can be developed from the low-temperature gas plasma technology. In the semiconductor industry, low-temperature gas plasmas have been used to fabricate computer chips by selectively destroying organic and inorganic matters on a submicrometre scale. There exists a wealth of substantial scientific evidence that these gas plasmas are also capable of destroying pathogenic bacteria and proteins. These gas plasmas are normally generated in a vacuum chamber and it is, in practice, difficult to deliver them to contaminated sites, whether at the end of a long lumen, inside a drug-synthesis chamber or in a hospital ward. If low-temperature gas plasmas are capable of accessing and sterilising structures of scales ranging from micrometres to tens of centimetres, they could potentially facilitate a new range of spatial scales and structures employed for the design and manufacture of future medical devices.

Applying cold atmospheric plasmas

Figure 1: A cold plasma torch.

Thanks to a sequence of revolutionary innovations and advances in the gas plasma technology over the past 15 years or so, the restriction of the vacuum chamber has been lifted and it is now possible to generate room-temperature open-air gas plasmas, also known as cold atmospheric plasmas. Figure 1 shows an example of cold atmospheric plasma delivered to a human hand. These open-air and low-temperature gas plasma systems can be engineered to treat three-dimensional objects and therefore offer an invaluable opportunity to be developed into a major sterilisation solution for the health-care service and industry. A new sterilisation capability to sterilise intricate structures of micrometres facilitates the design of innovative and smaller surgical instruments with reduced invasiveness and improved functionality. Furthermore, it is possible to develop industrial-scale cold atmospheric plasma systems and thus enable opportunities for decontamination of, for example, a manufacturing production line of sterile products. It could also be used to reduce the down time of drug production.

Gas plasma is formed when sufficient energy is added to a gas to release electrons from a significant number of gas atoms and molecules. This process is known as gas ionisation and is often facilitated with electrical energy, but sometimes with optical or thermal energies. Significantly, the ionisation process can, at a point of demand, turn an originally inactive and environmentally friendly gas into a mixture of highly reactive plasma species. These chemically reactive species include positive-charged particles (ions), negative-charged particles (electrons) and various uncharged, but chemically reactive particles such as free radicals, all of which can destroy living organisms. Charged particles can etch or rupture cell membranes, whereas free radicals may irreversibly modify the chemical composition and structure of cell mem- branes. Both are, therefore, capable of potentially overwhelming the natural defence of living organisms, leading to their destruction. Another product of gas ionisation is ultraviolet (UV) photons that can damage the deoxyribonucleic acid (DNA). In summary, gas plasma produces a wide range of biocidal agents capable of causing damage from the cell membrane to the intracellular structure.

Mode and efficiency of kill

Figure 2: SEM of Bacillus Subtilis spores following treatment with cold atmospheric plasma.

Cold atmospheric plasmas can inactivate Gram-negative, Gram-positive and biofilm-forming bacteria growing on a variety of surfaces including glass, polymer, paper filter and stainless steel. Depending on the type of gases used and the electrical characteristics of the plasma-generating power sources, bacterial reduction of over six orders of magnitude can be achieved in less than 60 seconds.¹ To illustrate this, Figure 2 shows a scanning electron micrograph (SEM) of Bacillus Subtilis spores on a membrane filter after exposure to the cold atmospheric plasma. There is clear evidence of cellular denaturation and destruction. As expected, inactivation efficiency of cold atmospheric plasmas against Gram-negative bacteria such as E. coli is greater. Numerous and independently performed inactivation kinetics studies suggest that an important factor in bacterial resistivity to cold atmospheric plasmas is the strength of the bacterial membrane. Gram-positive bacteria are more resistive than Gram-negative bacteria because they possess a much thicker bacterial membrane. The microbiological, biochemical and physical environment in which the bacteria grow also influences their ability to resist plasma treatment. For example, biofilm-forming bacteria add further resistance by producing a three-dimensional polysaccharide structure in which to embed themselves.² Although these factors are important in deciding the timescale over which effective bacterial reduction can be achieved, it is now widely accepted that cold atmospheric plasmas can inactivate Gram-negative, Gram-positive and biofilm-forming bacteria. Given that they easily produce over half a trillion (that is, 500000000000) chemical reactive plasma species per cubic centimetre volume, cold atmospheric plasmas can effectively inactivate many common hospital-significant bacteria. The challenge is how to engineer seamless delivery of such abundant plasma species to the sites of bacterial colonies.

Figure 3: Removal of protein coating on a stainless steel ball.

Cold atmospheric plasmas can also destroy and remove protein from stainless steel surfaces and from many surgical instruments. This is significant because conventional sterilisation procedures have been known to be incapable of satisfactorily containing and removing prion contamination of surgical instruments. A protein removal capability is certainly welcome news. Figure 3 shows a SEM of a stainless steel ball; the top-right half of which is plasma treated and the bottom-left half is untreated to highlight the effect of cold atmospheric plasmas. Electron energy dispersion X-ray spectroscopy suggested that the plasma-treated region had little trace of carbon and oxygen whereas the untreated region had substantial carbon and oxygen lines. Currently, cold atmospheric plasmas can reduce protein below several tens of femto-mole, which is the current protein-detection limit. Protein removal using cold atmospheric plasmas is a new field of research and most activity to date is seemingly performed at Loughborough University, UK. More advances are anticipated when new data from other plasma sterilisation research groups become available. The considerable challenge posed by destruction prion proteins and its profound implication for health would most certainly benefit from sustainable scientific endeavours on a larger scale than the current level of activities.

Application possibilities

Finding the identity in cold atmospheric plasmas of the main species responsible for bacterial inactivation and protein removal is important both to understand relevant biocidal mechanisms and to enable design guidance when cold atmospheric plasmas are scaled-up or -down to cater for different application requirements. Spectroscopic studies and computer simulation suggest that oxygen atoms, hydroxyl radicals, and excited oxygen-containing or/and nitrogen-containing species are most likely to be major enablers of the observed bacterial inactivation and protein removal. A systematic study of spectroscopic, electrical and biological diagnostics is currently being undertaken to correlate the production of the above-mentioned plasma species with the efficiency of protein removal and thereby establish a hierarchy of their relative importance. This is clearly a challenging task because the observed protein removal or/and bacterial inactivation could well be a result of synergistic effects of many of the above-mentioned plasma species. Nevertheless, the challenge could result in an invaluable reward in the form of a potentially quantitative guidance to future development and optimisation of cold atmospheric plasmas for medical product sterilisation.

From an application standpoint, cold atmospheric plasmas could be introduced into hospitals and other health-care facilities as an additional step to the current sterilisation procedure. This would allow them to take advantage of the considerable decontamination efficacy of conventional techniques and to enable synergistic decontamination with conventional sterilisation steps. Hence, decontamination techniques based on cold atmospheric plasmas could be integrated in a complementary fashion to the existing sterilisation procedure. Their deployment in hospitals presents little, if any, environmental hazard, because most cold atmospheric plasmas are generated in nontoxic gases such as oxygen, nitrogen and helium. Their products and by-products are harmless to the environment and personnel in a hospital setting.

In the context of medical device manufacturing, appropriately scaled-up cold atmospheric plasmas could be used as an added contamination-controller in the manufacturing process of sterile products or as a versatile tool to rapidly decontaminate the manufacturing production line and minimise its downtime. These and other opportunities should benefit from a close collaboration between the technology provider and the user community. Significantly, almost all reactive species of cold atmospheric plasmas can be controlled to diffuse into tiny crevices, internal cavities and channels that are difficult to access, which suggests they are not only an effective, but also thorough decontamination solution.

Cold atmospheric plasmas offer a substantial and unprecedented opportunity as a complementary sterilisation technology. Before they can become a common feature in tomorrow’s sterile service departments, however, many challenges ranging from scientific issues, through engineering, to deployment and integration must be overcome. The prospect for cold atmospheric plasmas to offer a genuine sterilisation option is, however, justifiably bright.

Michael Kong is Professor of Bioelectrics Engineering and Head of Energy Research Division, Dept of Electronic and Electrical Engineering, Loughborough University, Loughborough LE11 3TU, UK, tel. +44 1509 227 075, e-mail: m.g.kong@lboro.ac.uk, www.lboro.ac.uk/departments/el/staff/kong.html.