Katharina Stapelmann

On the application of inductively coupled plasma discharges sustained in Ar/O2/N2 ternary mixture for sterilization and decontamination of medical instruments

Non-equilibrium low pressure-plasma discharges are extensively studied for their high potential in the field of sterilization and decontamination of medical devices. This increased interest in plasma discharges arises from, among other reasons, their capability not only to inactivate bacterial spores but also to eliminate, destroy or remove pathogenic biomolecules and thus to provide a one-step process assuring safety of treated instruments. However, recent studies have shown that optimal conditions leading to inactivation of spores and physical removal of pathogens differ significantly—the efficiency of spores sterilization is above all dependent on the UV radiation intensity, whereas high etching rates are connected with the presence of the etching agent, typically atomic oxygen. The aim of this contribution is to discuss and demonstrate the feasibility of Ar/N2/O2 low-pressure inductively coupled plasma discharges as an option to provide intense UV radiation while maintaining the high etching rates of biomolecules.

Utilization of low-pressure plasma to inactivate bacterial spores on stainless steel screws.

A special focus area of planetary protection is the monitoring, control, and reduction of microbial contaminations that are detected on spacecraft components and hardware during and after assembly. In this study, wild-type spores of Bacillus pumilus SAFR-032 (a persistent spacecraft assembly facility isolate) and the laboratory model organism B. subtilis 168 were used to study the effects of low-pressure plasma, with hydrogen alone and in combination with oxygen and evaporated hydrogen peroxide as a process gas, on spore survival, which was determined by a colony formation assay. Spores of B. pumilus SAFR-032 and B. subtilis 168 were deposited with an aseptic technique onto the surface of stainless steel screws to simulate a spore-contaminated spacecraft hardware component, and were subsequently exposed to different plasmas and hydrogen peroxide conditions in a very high frequency capacitively coupled plasma reactor (VHF-CCP) to reduce the spore burden. Spores of the spacecraft isolate B. pumilus SAFR-032 were significantly more resistant to plasma treatment than spores of B. subtilis 168. The use of low-pressure plasma with an additional treatment of evaporated hydrogen peroxide also led to an enhanced spore inactivation that surpassed either single treatment when applied alone, which indicates the potential application of this method as a fast and suitable way to reduce spore-contaminated spacecraft hardware components for planetary protection purposes.

Understanding of the importance of the spore coat structure and pigmentation in the Bacillus subtilis spore resistance to low-pressure plasma sterilization

Low-pressure plasmas have been evaluated for their potential in biomedical and defense purposes. The sterilizing effect of plasma can be attributed to several active agents, including (V)UV radiation, charged particles, radical species, neutral and excited atoms and molecules, and the electric field. Spores of Bacillus subtilis were used as a bioindicator and a genetic model system to study the sporicidal effects of low-pressure plasma decontamination. Wild-type spores, spores lacking the major protective coat layers (inner, outer, and crust), pigmentation-deficient spores or spore impaired in encasement (a late step in coat assembly) were systematically tested for their resistance to low-pressure argon, hydrogen, and oxygen plasmas with and without admixtures. We demonstrate that low-pressure plasma discharges of argon and oxygen discharges cause significant physical damage to spore surface structures as visualized by atomic force microscopy. Spore resistance to low-pressure plasma was primarily dependent on the presence of the inner, and outer spore coat layers as well as spore encasement, with minor or less importance of the crust and spore pigmentation, whereas spore inactivation itself was strongly influenced by the gas composition and operational settings.

FTIR spectroscopy of cysteine as a ready-to-use method for the investigation of plasma-induced chemical modifications of macromolecules

A rapid screening method for the investigation of plasma-induced chemical modifications was developed by analyzing cysteine using Fourier Transform Infrared (FTIR) spectroscopy. Cysteine is a key amino acid in proteins due to the presence of a thiol group which provides unique structural features by offering the possibility to form disulfide bonds. Its chemical composition makes cysteine a well-suited model for the investigation of plasma-induced modifications at three functional groups—the amino, the carboxyl and the thiol group—all highly abundant in proteins. FTIR spectroscopy is present in most physical laboratories and offers a fast way to assess changes in the chemical composition of cysteine substrates due to plasma treatment and to compare different treatment conditions or plasma sources with each other. Significant changes in the fingerprint spectra of cysteine samples treated with a dielectric barrier discharge (DBD) compared to untreated controls were observed using a FTIR spectrometer. The loss of the thiol signal and the simultaneous increase of bands originating from oxidized sulfur and nitrogen species indicate that the thiol group of cysteine is modified by reactive oxygen and nitrogen species during DBD treatment. Furthermore, other plasma-induced modifications, such as changes of the amino and carbonyl groups, could be observed. Complementary mass spectrometry measurements confirmed these results.

Improvement of Biological Indicators by Uniformly Distributing Bacillus subtilis Spores in Monolayers To Evaluate Enhanced Spore Decontamination Technologies.

ABSTRACT Novel decontamination technologies, including cold low-pressure plasma and blue light (400 nm), are promising alternatives to conventional surface decontamination methods. However, the standardization of the assessment of such sterilization processes remains to be accomplished. Bacterial endospores of the genera Bacillus and Geobacillus are frequently used as biological indicators (BIs) of sterility. Ensuring standardized and reproducible BIs for reliable testing procedures is a significant problem in industrial settings. In this study, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of Bacillus subtilis 168 spore monolayers on glass surfaces. A detailed description of the structural design as well as the operating principle of the spraying device is given. The reproducible formation of spore monolayers of up to 5 × 107 spores per sample was verified by scanning electron microscopy. Surface inactivation studies revealed that monolayered spores were inactivated by UV-C (254 nm), low-pressure argon plasma (500 W, 10 Pa, 100 standard cubic cm per min), and blue light (400 nm) significantly faster than multilayered spores were. We have thus succeeded in the uniform preparation of reproducible, highly concentrated spore monolayers with the potential to generate BIs for a variety of nonpenetrating surface decontamination techniques.

A H2 very high frequency capacitively coupled plasma inactivates glyceraldehyde 3-phosphate dehydrogenase(GapDH) more efficiently than UV photons and heat combined

Plasma sterilization is a promising alternative to commonly used sterilization techniques, because the conventional methods suffer from certain limitations, e.g. incompatibility with heat-sensitive materials, or use of toxic agents. However, plasma-based sterilization mechanisms are not fully understood yet. A low-pressure very high frequency capacitively coupled plasma is used to investigate the impact of a hydrogen discharge on the protein glyceraldehyde 3-phosphate dehydrogenase (GapDH). GapDH is an enzyme of glycolysis. As a part of the central metabolism, it occurs in nearly all organisms from bacteria to humans. The plasma is investigated with absolutely calibrated optical emission spectroscopy in order to identify and to quantify plasma components that can contribute to enzyme inactivation. The contribution of UV photons and heat to GapDH inactivation is investigated separately, and neither seems to be a major factor. In order to investigate the mechanisms of GapDH inactivation by the hydrogen discharge, samples are investigated for etching, induction of amino acid backbone breaks, and chemical modifications. While neither etching nor strand breaks are observed, chemical modifications occur at different amino acid residues of GapDH. Deamidations of asparagines as well as methionine and cysteine oxidations are detected after VHF-CCP treatment. In particular, oxidation of the cysteine in the active centre is known to lead to GapDH inactivation.

Elucidation of Plasma-induced Chemical Modifications on Glutathione and Glutathione Disulphide

Cold atmospheric pressure plasmas are gaining increased interest in the medical sector and clinical trials to treat skin diseases are underway. Plasmas are capable of producing several reactive oxygen and nitrogen species (RONS). However, there are open questions how plasma-generated RONS interact on a molecular level in a biological environment, e.g. cells or cell components. The redox pair glutathione (GSH) and glutathione disulphide (GSSG) forms the most important redox buffer in organisms responsible for detoxification of intracellular reactive species. We apply Raman spectroscopy, mass spectrometry, and molecular dynamics simulations to identify the time-dependent chemical modifications on GSH and GSSG that are caused by dielectric barrier discharge under ambient conditions. We find GSSG, S-oxidised glutathione species, and S-nitrosoglutathione as oxidation products with the latter two being the final products, while glutathione sulphenic acid, glutathione sulphinic acid, and GSSG are rather reaction intermediates. Experiments using stabilized pH conditions revealed the same main oxidation products as were found in unbuffered solution, indicating that the dominant oxidative or nitrosative reactions are not influenced by acidic pH. For more complex systems these results indicate that too long treatment times can cause difficult-to-handle modifications to the cellular redox buffer which can impair proper cellular function.

VUV absorption spectroscopy of bacterial spores and DNA components

Low-pressure plasmas can be used to inactivate bacterial spores and sterilize goods for medical and pharmaceutical applications. A crucial factor are damages induced by UV and VUV radiation emitted by the plasma. To analyze inactivation processes and protection strategies of spores, absorption spectra of two B. subtilis strains are measured. The results indicate, that the inner and outer coat of the spore significantly contribute to the absorption of UV-C and also of the VUV, protecting the spore against radiation based damages. As the sample preparation can significantly influence the absorption spectra due to salt residues, the cleaning procedure and sample deposition is tested for its reproducibility by measuring DNA oligomers and pUC18 plasmid DNA. The measurements are compared and discussed with results from the literature, showing a strong decrease of the salt content enabling the detection of absorption structures in the samples.

Implications of electron heating and non-uniformities in a VHF-CCP for sterilization of medical instruments

A capacitively coupled plasma driven at a frequency of 81.36 MHz from the VHF-band is investigated by means of optical emission spectroscopy (OES) and multipole resonance probe (MRP). The discharge is operated with hydrogen, yielding an electropositive discharge, as well as oxygen, yielding an electronegative discharge, and mixtures of both. Pressure is varied from Pa to Pa. Homogeneity of the discharge is investigated by CCD camera recordings as well as spatially resolved multipole resonance probe measurements. The results indicate the presence of electromagnetic edge effects as well as standing wave effects. Furthermore, a largely homogeneous discharge can be achieved with hydrogen as process gas at a pressure of –10 Pa. With increasing pressure as well as with increasing oxygen content, the discharge appears less homogeneously. The transition from an electropositive to an electronegative discharge leads to a change in electron heating mechanisms, with pronounced local maxima of electron density at the sheath edges. A comparison of OES and MRP results reveal a significant difference in electron density, which can be explained by a non-Maxwellian distribution function of electrons.

Phase resolved analysis of the homogeneity of a diffuse dielectric barrier discharge

Cold atmospheric pressure plasmas have already proven their ability of supporting the healing process of chronic wounds. Especially simple configurations like a dielectric barrier discharge (DBD), comprising of one driven electrode which is coated with a dielectric layer, are of interest, because they are cost-effective and easy to handle. The homogeneity of such plasmas during treatment is necessary since the whole wound should be treated evenly. In this investigation phase resolved optical emission spectroscopy is used to investigate the homogeneity of a DBD. Electron densities and reduced electric field distributions are determined with temporal and spatial resolution and the differences for applied positive and negative voltage pulses are studied.