1. Dr. Ariël de Graaf, ariel.degraaf@tno.nl Meeting VUMC 17-03-2009 Plasma sterilization

2. Contents Sterilization applications Common sterilization methods Plasma sterilization What is a plasma? Plasma sterilization agents Advantages & drawbacks plasma sterilization Commercial plasma sterilization systems Experiments endoscope sterilization Conclusions

3. Sterilization applications Medical Surgical tools In vivo diagnostic tools (e.g. endoscopes) Packaging material (i.e. bottles, sterile bags) Human tissue (e.g. surgeon’s hands) Surgery room (e.g. tables, floors, etc.) Food processing Bacteria on surface of foods (e.g. fruits and meats) Toxic fungal contamination (e.g. in seeds, grains and spices) Food packaging and conservation Biological warfare defence Anthrax spores and smallpox virus Nerve and blister agents

4. Sterilization applications Public health Water disinfection Air purification (e.g. against “sick building syndrome”) Processing industry of natural materials (e.g. Bacillus spores in raw cotton and fabric manufacture) Space Decontamination of life-searching probes on planetary missions

5. Common sterilization methods Sterilization by heat (autoclave) Moist or dry heat (120-170°C, 20-60 min.) Not applicable for temperature-sensitive materials Sterilization by reactive gases Mostly used are ethylene oxide, formaldehyde, ozone and hydrogen-peroxide Concerns about toxicity and carcinogenicity of residual gas Sterilization by membrane filtration For liquids sensitive to heat, chemical interaction or radiation Pore size: < 0.2 µm for bacteria, < 20 nm for viruses Prions (proteinaceous infectious particles) cannot be removed Recycling (i.e. cleaning) of filters is cumbersome

6. Other sterilization methods Sterilization by radiation UV light: applicable for UV resistant surfaces and UV transparent objects Gamma rays: good shielding of hazardous radioactive radiation is necessary X-rays: long exposure times required Electromagnetic fields: microwave, RF Sterilization by electron beams Plasma sterilization Plasma-assisted sterilization: germicidal liquids and gases (such as H 2 O 2 and aldehydes) in combination with plasma Plasma-based sterilization: gases that become biocidal only when a plasma is ignited (e.g. air, He/air, He/O 2, O 2 /N 2 )

7. What is a plasma? Plasma is an ionized gas consisting of free electrons, ions, reactive atoms, neutral molecules and photons The plasma state can be reached by supplying sufficient energy (heat or electric power) to a gas or mixture of gases Plasmas can be operated both at low and atmospheric pressure SolidLiquidGasPlasma Add energy H 2 O (s)H 2 O (l)H 2 O (g) H,H 2,H +,e -,H 2 - O,O 2,O 3,O -,O 2 - H2OH2O

8. What is a plasma? H2OH2O e-e- H-H- H2H2 H2-H2- H+H+ O2O2 O 3 (ozone) OH OH - O2-O2- H2O2H2O2 O+O+ OH + photon UV/VIS H O H2OH2O e-e- H H2OH2O e-e- O Plasma + ++

9. Plasma sterilization agents Heat Sample will receive more heat in direct exposure mode as compared to remote exposure However, temperature increase due to plasma is usually too low (<100 °C) to have a significant effect UV radiation In the plasma UV photons are generated (wavelength depends on plasma species) 200-300 nm radiation causes damage to the cell’s DNA and RNA, inhibiting the bacteria to replicate properly Charged particles Plasma contains both ions and electrons Charging of cell membrane may cause disruption

10. Plasma sterilization agents Reactive species Dissociation of molecules and plasma chemistry leads to reactive species (depending on plasma composition) Air plasma: O, O 2 *, O 3, OH, NO, NO 2, etc. Radicals chemically attack the walls, coats, and membranes of cells of microorganisms Radicals may even remove killed bacteria and their debris (particularly pyrogens)

11. Advantages plasma sterilization Plasma is more reactive than normal gas or liquid phase chemistry due to presence of free radicals Plasma combines a number of important sterilization agents UV light Reactive species Electric fields and charge In some occasions also heat This will lead to lower sterilization times as compared to standard techniques (several seconds instead of minutes to hours) Complete removal of organic material due to plasma reactivity! Low sample temperatures for temperature-sensitive tools No solvents  no toxic residue or hazardous emission Hazardous radiation is avoided Operation could be very cheap (when operating on air)

12. Drawbacks plasma sterilization Reactive plasma species may also attack instrument materials Material degradation due to UV exposure Plasma can not enter autoclave bags or non-permeable materials  plasma must be created from the inside Plasma does not enter very narrow crevices and fissures  requires new plasma techniques

13. Commercial plasma sterilization systems Based on H 2 O 2 plasma H 2 O 2 sterilization followed by plasma treatment Sterilization of materials sensitive to heat and humidity Gentler cleaning than steam or peracetic acid No toxic residue or hazardous emission Sterrad, Stericool, Steriplas Based on peracetic acid (C 2 H 4 O 3 ) plasma Plazlyte (not sold anymore due to medical incident)

14. Commercial plasma sterilization systems Plasma-label for food conservation (“PlasEt” by JE PlasmaConsult) Plasma created inside packaging via antenna on label Few minutes plasma generation Ozone generation Label can be equipped with sensors

15. Feasibility study – Endoscope sterilization Endoscopes have several narrow (down to 0.5mm diameter) channels for insertion of instruments Sterilization of these narrow channels is cumbersome Idea: create a plasma inside the narrow channels  plasma will remove bacteria Without plasma 25 cm

16. First experiments with PTFE model system

17. Two concepts Axial wireSandwich

18. Experimental results Removal of Geobacillus Stearophillus spores 97% spores removed within 5 minutes Settings not yet optimized

19. Experimental results Removal of Geobacillus Stearophillus spores and Enterococcus hirea (ATCC 8043) vegetative cells Geobacillus ATCC 8043 untreated sandwich axial wire

20. Conclusions There is sufficient evidence that plasma sterilization works, and that dead bacteria and pyrogenic debris are removed as well Plasma allows sterilization of heat- and humidity-sensitive materials without toxic residue or hazardous emission Most important sterilizing agents have been identified Possibly a combination of techniques (e.g. liquid, UV and plasma treatment) will be the best choice Sterilization of crevices and fissures with plasma requires more research, but seems not impossible The resistance of the sterilized materials to plasma exposure should also be examined