WO2013037008A1

WO2013037008A1 - Cleaning and sterilising method for biomedical surfaces

Info

Publication number: WO2013037008A1
Application number: PCT/AU2012/001107
Authority: WO
Inventors: Sunil Kumar
Original assignee: University Of South Australia
Priority date: 2011-09-15
Filing date: 2012-09-14
Publication date: 2013-03-21
Also published as: AU2011224068A1

Abstract

The present invention relates to methods of cleaning and sterilising biomedical surfaces such as those associated with surgical instruments and medical devices. The methods involve the application of a low temperature non-reactive gas plasma treatment, preferably an air plasma treatment, to the surface of a surgical instrument or medical device for a period sufficient for the effective removal of at least any proteinaceous contamination.

Description

CLEANING AND STERI LISING METHOD FOR BIOMEDICAL SU RFACES

FIELD OF THE INVENTION

The present invention relates to a method of cleaning and sterilising biomedical surfaces such as those associated with surgical instruments and medical devices.

INCORPORATION BY REFERENCE

This patent application claims priority from:

- Un ited States Provisional Patent Application No 61 /535326 filed 1 5 September 201 1 , and - A ustral ian Standard Patent Application No 201 1 224068 filed 1 5 September 201 1 .

The entire content of these applications is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Conventional cleaning and sterilisation processes of surgical instruments and medical devices employ wet chemical methods involving surfactants, enzyme reagents, puri fied deionised water, autoclaves, hydrogen peroxide (H2O2) or ethylene oxide. These processes are routinely used in hospital sterilisation centres and generally remove visible residues of blood, proteins and other organic matter due to irreversible inactivation or breakdown of vital structural components of micro-organisms. However, despite the use of strong surfactants, h ighly puri fied water and several rinses, these processes are well known to often leave behind molecular levels of contamination (mainly proteinaceous) of surfaces. Such remaining proteinaceous contamination may be invisible to conventional inspection but may be quite significant (reported to be as much as 0.42-4.2 μg per instrument even after cleaning by the recommended cleaning and decontamination procedures') and poses a serious health risk if, for example, the proteinaceous contamination includes prions. Accordingly, the potential of prions to be present in the proteinaceous contamination of surfaces of surgical instruments (particularly

neurosurgical and ophthalm ic instruments) and thereby cause cross-contamination and iatrogenic transmission of pathogens, is of widespread and great concern to health service providers.

Unfortunately, not only has it proven difficult to remove the remainder of the proteinaceous contamination remaining on surgical instruments fol lowing conventional c leaning and sterilisation processes, but any contaminating prions themselves show marked resistance to removal or inactivation. Indeed, it has been found that prions are extremely resistant to conventional gaseous disinfectants (eg ethylene oxide and formaldehyde) as well as physical processes such as ionising, ultraviolet (UV), microwaves and other types of rad iation, steam heat at 1 34°C, and dry heat up to 600°C^2"*\ Further, chemical disinfectants such as alcohols, ammonia, glutaraldehyde and formalin are also ineffective⁵; the main effective reagents are sodium hydroxide and sodium hypochlorite⁶, however these "aggressive" agents have been found, in some cases, to adversely affect some surfaces found on surgical instruments ^' and medical devices. Accord ingly, there is a considerable need for the identi fication and development of alternative methods for c leaning and sterilisation.

Low temperature gas plasma treatments are known for their efficient and low cost clean ing capacity for a variety of surfaces^{7' 10}. Further, owing to their low thermal load, such plasma treatments have been recognised as being wel l-suited for cleaning heat-sensitive instruments and devices. One example of a low temperature plasma treatment is known as rad io frequency glow discharge (RFGD). RFG D is generally described as a non-thermal process that results in the formation of neutral and ionic spec ies, free electrons and UV radiation. It has, amongst other things, been used for surface cleaning of both polymeric and inorganic materials; for which application, it is thought that the combined effect of plasma-induced processes such as radical chemistry, UV absorption and ion bombardment causes the breakdown and removal of organic matter (such as proteinaceous contamination and micro-organisms) and also inorganic matter"¹. However, while low temperature gas plasma treatment is an establ ished method for cleaning surfaces of things such as microelectronic devices, its use in the medical context has been very l imited.

As described hereinafter, the present applicant has investigated whether a low temperature gas plasma treatment may be employed for c leaning and, more particularly, sterilising surfaces of surgical instruments and medical devices. By using the highly surface-sensitive analytical method of X-ray photoelectron spectroscopy (XPS), he has recognised that reliable and effective removal of proteinaceous contamination from surfaces of surgical instruments and medical devices can be achieved with a low temperature gas plasma generated from a non-reactive gas, namely air. Moreover, this air plasma treatment has been found to also remove inorganic contamination which may additional ly contribute to the potential success of the treatment for prion removal since it has been suggested that such contamination may provide an inorganic molecular template that causes the nucleation and aggregation of prion proteins, thereby possibly explaining why the removal of prions has been hitherto so difficult; even resisting treatments under intense dry heating conditions (up to 600°C)\

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method of cleaning and sterilising a surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non- reactive gas plasma treatment to said surface for a period sufficient for the effective removal of at least any proteinaceous contamination. In a second aspect, the present invention provides a method of removing biological contaminants from the surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non-reactive gas plasma treatment to said surface for a period sufficient for the effective removal of at least any proteinaceous contamination.

In a th ird aspect, the present invention provides a method of clean ing and sterilising a surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non- reactive gas plasma treatment to said surface for a period sufficient for the effective removal of at least any proteinaceous contam ination, wherein the gas plasma of said treatment comprises plasma of air and argon (or another heavy inert gas). In a fourth aspect, the present invention provides a method of removing biological contam inants from the surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non-reactive gas plasma treatment to said surface for a period sufficient for the effective removal of at least any proteinaceous contamination, wherein the gas plasma of said treatment comprises plasma of air and argon (or another heavy inert gas).

BRI EF DESCRIPTION OF THE FIGURES

Figu re 1 provides a schematic diagram of an air plasma treatment system suitable for use in the method of the present invention (RF, radio frequency); Figure 2 provides X-ray photoelectron spectroscopy (X PS) survey spectra, atomic concentration values (at.%), water contact angle and surface roughness values (root mean squared, Rrms) of silicon wafer samples: (A) blank; (B) bovine serum albumin (BSA)-adsorbed; and (C) BSA-adsorbed fol lowed by an air plasma treatment according to the present invention. For clarity, only the relevant photoelectron peaks are labelled in the spectra;

Figure 3 provides the X-ray photoelectron spectroscopy atomic concentrations of protein-adsorbed si l icon wafer samples air plasma-treated for di fferent periods (data represent an average of two samples in each case); Figure 4 provides a graphical comparison of the nitrogen concentrations measured by X-ray photoelectron spectroscopy for stainless steel coupons after various treatments: (A) blank; (B) A cleaned with air plasma; (C) A soaked in whole human blood; (D) C followed by sequential washing in water and H:0 ; and (F,) D cleaned with air plasma. The water contact angle values determined for the samples are given in the parentheses. The surface of the blood-contaminated sample was too rough to be measured for contact angle (data represent an average of two samples in each case); and

Figure 5 provides a graphical comparison of nitrogen concentrations of various dental bur samples: (A) as-received burs ready to be used (blank); (B) burs used on a patient, followed by cleaning and sterilising using a conventional cleaning and steri lisation process; and (C) B treated with an air plasma treatment accord ing to the present invention (data represent an average of two samples in each case). DETAILED DESCRI PTION OF THE INVENTION

In a first aspect, the present invention provides a inethod of cleaning and steril ising a surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non- reactive gas plasma treatment to said surface for a period sufficient for the effective removal of at least any proteinaceous contam ination.

The present applicant has found that reliable and effective removal of proteinaceous contamination from surfaces of surgical instruments and medical devices can be achieved with a low temperature non- reactive gas plasma treatment. Preferably, the low temperature non-reactive gas plasma treatment is a non-reactive, non-inert gas plasma treatment such as an air plasma treatment. Apart from the effectiveness of such an air plasma treatment, as observed by the present applicant, in the removal of proteinaceous contamination, the use of air as a process gas offers advantages over other plasma cleaning treatments (eg such as those using vaporised H2O2 and complex gas mixtures⁹' ' ³) in that it provides a gas plasma treatment method that can be considered "green" and is relatively simple to implement and control. More particularly, since air is a stable mix of oxygen and nitrogen (-21 :79), it is safer to handle and contain compared with corrosive H2O2 and, further, offers the potential of reduced costs as compared to the use of complex gas mixtures. By using the highly surface-sensitive analytical method of X-ray photoclcctron spectroscopy (XPS), the present applicant has found that lo

temperature air plasma treatment of surfaces rel iably and e ffectively removes proteinaceous contamination as well as inorganic contam ination. This is considered to be an important outcome of the treatment in the context of desirably achieving the removal of any prion contamination.

The term " low temperature" as used herein in connection to gas plasma (eg air plasma) will be understood by persons ski lled in the art as referring to a gas plasma with low electronic energy of < 1 0 eV (corresponding to a low temperature load of 1 0⁵ ) and a relatively low electron density (typically in the order of 10¹ °- 1 0~⁵ rrr ) generating a plasma pressure of < 1 .0 Torr.

The gas plasma may be generated using, for example, any of the standard AC or DC power sources well known to person skilled in the art (eg radio frequency (RF) power source in glow discharge (RFGD) plasma generators, and microwave sources). However, preferably, the gas plasma is activated with RFGD, wherein the RF Power applied is in the range of 25 to 200 W, more preferably 50 to 1 75 W, and most preferably, in the range of 75 to 1 25 W. In one preferred embodiment, the RF Power appl ied is - 100 W. Preferably, the plasma pressure of the gas plasma treatment is in the range of 0.25 to 1 .0 Torr, more preferably 0.35 to 0.75 Torr, and most preferably, in the range of 0.4 to 0.6 Torr. In one preferred embodiment of the invention of the first aspect, the plasma pressure of the gas plasma treatment is -0.5 Torr.

As mentioned above, preferably, the non-reactive gas plasma treatment is an air plasma treatment.

A ir plasma used in the method of the first aspect may be generated from the atmosphere or from industrial air, but more preferably, is generated from medical air.

In one part icu larly preferred embodiment of the invention of the first aspect, the air plasma is generated from industrial or medical air with an RFGD generator ( 1 00 W) and has a plasma pressure of 0.5 Torr.

In accordance with the method of the first aspect, the gas plasma treatment is applied to the surface for a period sufficient for the effective removal of at least any proteinaceous contamination.

As used herein, the term "effective removal of at least any proteinaceous contamination" means that any residual (ie unremoved) proteinaceous contamination is below the detection limit by X-ray photoelectron spectroscopy (XPS) performed as described hereinafter in Example 1 . This detection limit roughly corresponds to 1 0 ng/cm² of fibrinogen adsorbed onto a flat mica substrate²⁶.

The period sufficient for the effective remova l of at least any proteinaceous contamination will be of > 2 minutes in duration and, preferably, no more than about 30 minutes in duration. More preferably, the period will be in the range of 4 to 1 5 minutes in duration, and still more preferably in the range of 5 to 1 0 minutes in duration. Most preferably, the period that the gas plasma treatment wil l be applied to the surface for the effective removal of at least any proteinaceous contamination will be about 5 minutes.

In addition to proteinaceous contamination, the method of the first aspect results in the removal of inorganic contamination, particularly residual alumina and/or magnesium, that may provide an inorganic molecular template that causes the nucleation and aggregation of prions.

In any case, the method of the first aspect preferably results in the removal of infectious and/or pathogenic agents such as: micro-organisms including bacteria (eg Staphylococcus, Streptococcus. Mycobacterium and Pseudomonas), fungi (Candida and Aspergil lus) and spores thereof, viruses (eg hepatitis C virus (HCV) and human immunodeficiency virus (HIV)) and prions (eg causative of Creutzfeldt-Jakob disease (C.I D) and scrapie); and/or other undesirable agents such as nucleic acids and tox ic molecules. The method of the invention of the first aspect is preferably performed as a single step cleaning and steri l ising treatment (ie the method consists of the single cleaning and sterilising step of applying the low temperature non-reactive gas plasma to the surface). However, optionally, the surface may also be pre-treated with a conventional cleaning and steri lisation process (ie employing wet chemical methods such as those mentioned above). Further, the method may optionally comprise a subsequent treatment step (ie after the said gas plasma treatment) selected from one or more of the following:

( i) treatment with alkal i (NaOH) or Na hypochlorite; and

( i i ) treatment with argon plasma or plasma of another heavy inert gas (preferably for a period to remove residual inorganic contam ination, particu larly residual alumina and/or magnesium (eg by sputtering), that may provide an inorganic molecular template that causes the nucleation and aggregation of prions).

In a second aspect, the present invention provides a method of removing biological contaminants from the surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non-reactive gas plasma treatment to said surface for a period sufficient for the effective removal of at least any proteinaceous contamination.

The biological contaminants that be removed in accordance with the method of the second aspect, may be selected from infectious and/or pathogenic agents such as micro-organisms including bacteria, fungi and spores thereof, viruses and prions, and/or other undesirable agents such as nucleic acids.

In one particularly preferred embodiment of the invention of the second aspect, the method is for removing prions from the surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non-reactive gas plasma treatment to said surface for a period sufficient for the effective removal of at least any proteinaceous contamination.

Preferably, the low temperature non-reactive gas plasma treatment is a non-inert gas plasma treatment such as an air plasma treatment. In one particularly preferred embodiment of the invention of the second aspect, the air plasma is generated from industrial or medical air with an RFGD generator ( 100 W) and has a plasma pressure of 0.5 Torr.

The method of the invention of the second aspect is preferably performed as a single step treatment (ie the method consists of the single biological contaminant-removal step of applying the low temperature non-reactive gas plasma to the surface). However, optionally, the surface may also be pre-treated with a conventional cleaning and steri lisation process (ie employing wet chemical methods such as those mentioned above) and, further, the method may optional ly comprise a subsequent treatment step (ie after the said gas plasma treatment) selected from one or more of the following:

(i) treatment with alkal i (NaOH) or Na hypochlorite; and

(i i) treatment with argon plasma or plasma of another heavy inert gas (preferably for a period to remove residual inorganic contamination, particu larly residual alumina and/or magnesium, that may provide an inorganic molecular template that causes the nucleation and aggregation of prions).

The surface to be treated in accordance with the methods of the invention may comprise all, or only a part, of a surgical instrument or medical device. The surface may comprise a biocompatible material such as certain metallic, ceramic and/or polymeric materials. Further, the surface may comprise a hard surface (eg of a surgical instrument such as a scalpel) or may be a sem i-solid or soft surface (eg a surface of a soft medical device comprising soft or gel-like polymeric surfaces such as acryl ic hydrogel polymers and siloxane hydrogel polymers). The term "medical device" as used herein is intended to have a broad definition and may include any apparatus for permanent or temporary use on or in a body, for use in human or veterinary applications, or associated apparatus such as, for example, vessels for cleaning or storing such apparatus, and other items such as synthetic scaffold materials for cel l/tissue culture. Preferred medical devices for treatment in accordance with the method of the invention include implantable and non-implantable devices such as, for example, orthopaedic implants such as replacement joints (eg hip and knee prostheses), external fixation (ex-fix) pins, internal fixation screws, urinary catheters, percutaneous access catheters, catheter tips, stents and dental implants, as well as non-implantable devices such as contact lenses and masks and apparatus for breath ing medical air and oxygen. The term "surgical instrument" as used herein is intended to have a broad defin ition and may inc lude any tool or apparatus for single- or multi-use in surgery (inc luding dental and ophthalmic surgery), for use in human or veterinary applications, or associated apparatus such as, for example, vessels for cleaning or storing such apparatus. Preferred surgical instruments for treatment in accordance with the method of the invention include, for example, neurosurgical and ophthalmic instruments (which present a particular risk for iatrogenic transmission of prions), dental instruments (eg dental burs), reusable anaesthetic equipment, and general surgical instruments such as forceps, clamps and occluders for blood vessels and other organs, retractors, distractors, positioners and stereotactic devices, cutting tools (eg scalpels, lancets and dril l bits etc), di lators, suction tips and tubes for the removal of bodi ly fluids, seal ing devices (eg surgical staplers), irrigation and injection needles, tips and tubes for introducing fluid, powered devices (eg drills and dermatomes), scopes and probes, and measurement^' devices such as rulers and calipers. As mentioned above, the surface may comprise biocompatible metall ic, ceramic and/or polymeric materials (including biopolymers). Suitable metallic materials may include iron, copper, zinc, titanium, tantalum and al loys such as stainless steel, brass and cobalt-chrom ium alloys. Suitable ceramic materials may inc lude silica ceramic and oxide ceramic materials. A further example of suitable ceramic materials is bioactive hydroxyapatite (HA) which can encourage bone growth and may be used as, for example, a coating on ex-fix pins to improve bonding to the bone. Suitable polymeric materials include polypropylene titanium, polyethylene (which is useful for orthopaed ic implants), polyurethane. organosi loxane polymers, perfluorinated polymers (which are useful for catheters, soft tissue augmentation implants, and blood contacting devices such as heart valves), acrylic hydrogel polymers and siloxane hydrogel polymers (eg for contact lens and intraocular lens applications), and the like, and any combination thereof. Other surfaces suitable for treatment in accordance with the present invention may comprise inorganic materials such as tungsten carbide.

Optional treatment ( ii) of the methods of the first and second aspects, may be particularly suitable for a surface comprising a hard surface of, for example, stainless steel, but may be less suitable for surfaces comprising a hard (or soft) surface of a polymeric material.

Where it is desirable to include optional treatment (ii), it may be convenient to include argon or another heavy inert gas in the low temperature non-reactive gas plasma treatment. For example, the low temperature non-reactive gas plasma may comprise plasma of air and argon (or another heavy inert gas).

Thus, in a third aspect, the present invention provides a method of cleaning and sterilising a surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non- reactive gas plasma treatment to said surface for a period su fficient for the effective removal of at least any proteinaceous contamination, wherein the gas plasma of said treatment comprises plasma of air and argon (or another heavy inert gas).

Similarly, in a fourth aspect, the present invention provides a method of removing biological contaminants from the surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non-reactive gas plasma treatment to said surface for a period sufficient for the effective removal of at least any proteinaceous contamination, wherein the gas plasma of said treatment comprises plasma of air and argon (or another heavy inert gas). In a particular embodiment of the methods of the invention, the surface may comprise al l, or part of, a catheter or other tubular or elongated component of a surgical instrument or medical device, and is conducted using an RFGD generator w herein said catheter or component is located within coils of an RF power source.

The present invention also extends to low temperature non-reactive gas plasma-producing apparatus for use in the methods of the first, second, third and fourth aspects. Such apparatus is preferably adapted for table-top usage.

The present invention is hereinafter further described by way of the following, non-limiting examples and accompanying figures.

EXAMPLES

Whereas previous studies have investigated plasma clean ing and sterilisation, oxidative process gases such as hydrogen peroxide can adversely affect the integrity of biomedical devices, particularly polymeric components' In the fol lowing example, the present applicant reports a much more straightforward approach comprising the use of plasma establ ished in an air process atmosphere; based upon the hypothesis that when activated by an RFGD, air plasma may generate chemical species that can effect the removal of organ ic (proteinaceous) contamination without attacking medical device materials. Example 1

The following systems were studied: (i) a model surface (silicon wafer) with model proteinaceous contamination (ie bovine serum albumin); (i i) a real-life metallic biomaterial surface (ie surgical stainless steel) with proteinaceous contamination (ie adsorbed human blood) remaining after cleaning with Milli-Q water and hydrogen peroxide; and (iii) a practical surface (ie a used metall ic dental bur, DB) with proteinaceous contamination remaining after conventional cl inical cleaning.

Methods and Materials

Preparation of samples

Polished semiconductor-grade silicon wafers (SWs), 475-575 μιη thick (MMRC Pty Ltd, Malvern, VIC, Australia) and 1 mm thick stainless steel (SS) sheet (SS3 1 6L; Sheet Metal Supplies Pty Ltd, Adelaide, SA, Australia) were cut into 1 0 1 0 mnr coupons. The coupons were then cleaned with the detergent Decon 90, followed by thorough washing with Milli-Q water (ie high purity water; Millipore Corp, Lane Cove, NSW, Australia). Finally, the coupons were ultrasonically cleaned with ethanol and acetone. This solvent cleaning is a surface preparation process that is well-suited to the removal of grease and oil. Coupons thus cleaned were treated with air plasma ( 1 00W, 0.5 Torr, 5 m in) using a Harrick Plasma Cleaner (Model PDC-32 G; Harrick Plasma Co, Ithaca, NY, United States of America) in order to remove adventitious hydrocarbon from their surfaces. This air plasma system, described below, was also used for removing the proteinaceous contam ination from all of the samples. The following three types of samples were prepared: Case study I: .silicon wafer (SW) samples

These samples, representing a model protein as proteinaceous contamination on a model surface, were prepared by immersing the cleaned and plasma-treated SWs in BSA solution (bovine serum album in fraction V (9048-46-8), 1 5.4% (w/vv) n itrogen content; Sigma-Aldrich Pty Ltd, Castle H i ll. NSW, Australia) in water (3.5 g/dL) for 30 min, followed by thorough washing with water to remove the unabsorbed protein from the surfaces.

Case study II: stainless steel (SSj samples

These SS samples (grade 2), representing a real-life proteinaceous contamination on a real-l ife substrate (although not a surgical instrument), were prepared by dipping the cleaned and plasma-treated SS coupons into whole human blood for 30 min and then washing thoroughly and sequentially with water and hydrogen peroxide (H₂0₂ ). H₂0_: was used as a cleaning agent, being a strong and wel l-known reagent for removing protein layers.

Case study III: dental bur (DR) samples

These samples represented a practical dental instrument loaded with practical (clinical) proteinaceous contamination. These included: new, as-received burs ready to be used on patients; and burs used on patients and subsequently cleaned by using a standard cleaning process fol lowed routinely by the providing clinic's sterilisation centre. The standard cleaning process consisted of washing the burs in running cold water, fol lowed by ultrasonic treatment for 5 m in with a commercial kit (Rapid Cleaner, 70500-B; 3 M Australia Pty Ltd. Pymble, NSW, Australia). The burs were then immersed into a reagent ("M ilk" solution, Surgislip; Ruhof Corp, Mineola, NY, United States of Americia) and washed with demineral ised water. Finally, these heat-stable metallic instruments were bagged and autoclaved at 1 34°C. A ir plasma treatment

The air plasma treatment system (a table-top set-up) consisted of a horizontal glass plasma chamber 1 80 mm in length and 70 mm in diameter with a radio frequency induction (RF) coil wrapped around it for applying variable power up to I 00W from an in-built RF power generator operating at 1 3.56 MHz (Figure 1 ). Samples to be cleaned were positioned on a clean glass substrate holder located 50 mm from the front of the chamber and paral lel to the centre of the air inlet (leak) valve located on the chamber lid. A Javac DD I 50 rotary pump (Javac Pty Ltd, noxfield, V IC, Austral ia) was used to evacuate the chamber and the pressure recorded with an MKS pirani gauge. The base pressure in the chamber was of the order of 1 x 1 0^" Torr. Typically, a working pressure of about 0.5 Torr was obtained in the plasma chamber by leaking compressed air (instrument-grade compressed air: BOC Gases Australia Ltd. Rocklea. QLD, Australia) from the bottle, followed by igniting the plasma by applying RF power at I OOW for a specified time period. The following procedure reflects a typical air plasma treatment used in these studies:

(0 Sample introduced into the plasma chamber on top of a clean glass slide;

(ϋ) Chamber evacuated to its base pressure;

(ii i) Bottled air introduced and working pressure obtained;

(iv) RF power applied to generate the air plasma;

(v) Sample exposed to plasma for the specified time period;

(vi) RF power switched off and sample left to cool down under vacuum for a few m inutes;

(vii) Vacuum pump turned off and chamber brought to atmospheric pressure; and

(vi i i) Sample taken out of the chamber and stored in a vacuum desiccator for subsequent analysis. The SW, SS and DB samples were exposed to air plasma ( 100W. 0.5 Torr) for up to 5 min. Cleaned SW and SS coupons and DB were used as controls (blanks). A time-course study was conducted; air plasma treating the samples for different time periods (2, 4 and 5 min).

Characterisation of samples

Surface compositional analysis of the samples was performed by X-ray photoelectron spectroscopy (XPS) using a Perkin-Elmer Physical Electronics. PHI, 5600 instrument with Mg a radiation source operated at 300W. The high surface sensitivity of XPS (sampling depth of 8- 10 nm) makes this technique a useful method for detecting and quantifying protein films adsorbed onto a sol id surface. Atomic concentration values were measured for key elements present on the surface, analysing their detai led photoelectron spectra using the software CASA XPS (Casa Software Ltd, Teignmouth, Devon, United Kingdom). The detection limit of the instrument was 0.1 atomic percent (at.%; that is, 1 000 parts per million) for the elements detected in these studies. The presence of the element nitrogen (the N l s photoelectron signal in the XPS spectra) was taken as the marker of protein, including residual^2'1. The degree of wettability (hydrophi licity/hydrophobicity) of the protein-contaminated and air plasma- cleaned sample surfaces was investigated by measuring the contact angle with water (M illi-Q water). The sessile drop technique was used for this purpose^"4. The contact angle values were measured, using SCA-20 software (Dataphysics Instruments GmbH. Filderstadt, Germany), immediately after sample preparation,

A muitimode atomic force microscope (A FM) with Nanoscope III control ler and E scanner (Veeco Instruments Inc, Plainview, NY, United States of America) was used to image sample surfaces for topography and roughness (root mean squared, Rrms). Tapping mode A FM was appl ied to obtain the surface morphology of the BSA-adsorbed substrates²⁵.

Results and Discussion

Case study I (SW samples)

The samples were prepared to investigate the simplest scenario, that of removal of a well-known model protein ( BSA) from a substrate (silicon) using XPS. Figure 2 shows the X PS survey spectra of three SW samples: (A) cleaned SW (blank); (B) plasma-cleaned SW coupon after BSA exposure and thorough washing with Mill i-Q water; and (C) sample B after air plasma treatment for 5 m in.

The blank SW was observed to have only silicon (Si) and oxygen (O) and some carbon (C, mainly adventitious) (spectrum A), indicating no biocontamination after wet chemical cleaning. The

BSA-exposed SW coupon after thorough washing with Mil li-Q water showed the presence of nitrogen (N), centred at a binding energy value of -400 eV (spectrum B) consistent with amide nitrogen. This clearly demonstrated the adsorption of the protein on SW, which remained after thorough washing. However, after air plasma treatment for S min ( 1 00W, 0.5 Torr), the SW coupon showed no N l s photoelectron signal, indicating complete removal of the protein down to the detection limit of the XPS instrument (0. 1 at.%). As discussed below, this X PS detection limit rough ly corresponds to 1 0 ng/cm² of fibrinogen adsorbed on to a flat mica substrate²⁶. It is to be understood that this detection limit applies throughout the specification whenever the term "effective removal of at least any proteinaceous contamination" or "complete removal/elimination" of proteinaceous contamination is used.

Brevig el al. investigated albumin adsorption onto hydrophobic and hydrophilic surfaces of polystyrene and SW, respectively² ' . Album in proteins in aqueous solutions fold spontaneously so that hydrophobic amino acid side-chains become internalised in the molecule and polar residues become exposed at the surface. In general, for adsorption to hydrophilic surfaces, water-soluble proteins bind via their polar (surface) residues and a layer of water molecules is embedded between the protein and the substrate. In these studies, the adsorption of albumin onto the SW surface could be due to the above mechanism as the plasma-treated SW was found to be hydrophilic.

The X PS multiplex spectra were used to obtain the atomic concentrations of carbon, oxygen, silicon and nitrogen ( Figure 2). The blank sample (A) showed no nitrogen. Upon BSA adsorption, both the carbon and nitrogen concentrations increased significantly (B), The measured level of nitrogen at 1 6.8 at.% suggests complete coverage of the substrate by BSA . The observed silicon signal (9.6 at.%) suggests that the effective thickness of the BSA coverage is below the XPS sampl ing depth limit of 1 0 nm. This complete BSA coverage can be understood in terms of the shape and dimensions of the BSA molecule (molecular weight 66.4 kDa) measured by A FM to be an oblate ellipsoid of ~ I 4 x 4 x 4 nm\ It was estimated that a close-packed monolayer of BSA molecules adsorbed side-on onto the S W substrates would result in a surface loading of 400 ng/cm². Sim i larly, using the reported prion protein (PrP) molecular weight" (-30 kDa for the monomer) and dimensions (-3 x 3 3 nm '). it was estimated that the PrP loading of a flat surface would be 500 ng/cm². For the most infectious PrP particles of molecular weight in the range 300-600 kDa, reported by Si lveira et al. to consist of 1 4-28 PrP molecules with spherical/ellipsoidal dimensions in the range of 1 7-27 nm, a surface loading value of 4000 ng/cm² was estimated²⁹. Al l of these estimated protein load ing levels are comparable to the level observed by Wagner et al. to be 1 000 ng/cm' for a relatively large protein (fibrinogen) monolayer adsorbed onto mica, with the associated XPS detection limit determined to be 1 0 ng/cm² (equivalent to about one hundredth of the protein monolayer). It is to be noted that the X PS detection l imit is dependent on both the chemical and morphological characteristics of the adsorbing surface²". In view of this, the detection limit of 1 0 ng/cm" observed for fibrinogen can serve as a practical XPS detection limit for other proteins such as BSA and PrP (and its infectious units). In XPS, quantifying protein film thickness requires assumptions about protein film density, protein film coverage and the mean free path of photoelectrons²⁶. Air plasma treatment of the BSA-adsorbed sample reduced nitrogen concentration to zero and carbon concentration to 1 1 .5 from 44. 1 at.% (C). This indicated the complete elimination of BSA from the SW surfaces upon air plasma treatment. Since it is quite difficult to speculate on the possible infectivity potential of air plasma-treated PrP-contaminated sol id surfaces (with possible contamination < 1 0 ng/cm² that remains undetectable by XPS), it is suggested that in vivo bioassays should be performed on these surfaces as the "gold standard" of prion infectivity assessment⁴. It is. however, expected that such assays would yield favourable results, as the air plasma-treated PrP- contaminated surfaces are likely to be less contaminated ( 1 0 ng/cm²) compared with those treated by a range of commerc ial cleaning methods (< 1 000 ng/cm²)^",°.

In order to determine the minimum time required to remove adsorbed BSA completely from the SW surface by air plasma treatment, a time-course study was conducted (Figure 3). A 5 min exposure to air plasma under the conditions ( I 00W, 0.5 Torr) was found to be sufficient to remove the adsorbed protein completely from the SW surfaces. This time period is longer than that estimated as < 1 min from the data published by Staplemann et al.. but this difference can be understood in view of the relatively low concentration of oxygen in the plasma used in this case⁹.

As XPS is an expensive and specialist analysis technique, a more accessible method of assessing efficacy of plasma clean ing is desirable for field verification. Air/water contact angle values of the BSA-adsorbed SW coupons were determined before and after the air plasma treatment in order to assess the removal of the adsorbed protein layer (Figure 2). The blank sample exhibited an average value of 27.5" (N=2), and as expected this value increased to 7 1 .6° upon BSA adsorption. The removal of adsorbed protein by air plasma reduced the angle to < 1 0°, a value that was observed for an air plasma- cleaned SW. Surfaces with such low water contact angle values are seen as highly hydroph ilic and devoid of organic contam ination. A FM was employed for imaging the plasma-treated surfaces before and after BSA adsorption.

Compared with the image of a blank SW, the BSA-adsorbed SW clearly exhibited the protein and the surface appeared to be relatively high in roughness. The image obtained after the plasma treatment demonstrated the removal of adsorbed BSA. The removal was also supported by the roughness (Rrms, nanometers) values obtained from these images using Multimode II I A FM software (Veeco

Instruments) (Figure 2).

Case study 11 (SS samples)

These samples were prepared to investigate the removal of practical proteinaceous contamination (residue from human blood) on a real-l ife metallic biomaterial surface (surgical stainless steel). Five types of SS samples (A-E) were prepared and analysed by X PS. The samples were: cleaned coupons (A, blank); A cleaned with the air plasma (B); A soaked in whole human blood (C); C followed by sequential washing in water and H₂0₂ (D); and D c leaned with the air plasma (E). In terms of the nitrogen (N l s) signal, the X PS survey spectra of the SS samples exhibited a pattern similar to that of the SW samples shown in Figure 2, except that the cleaned SS blank sample exh ibited an inherent nitrogen concentration of 2.9 at.%; this nitrogen is part of the alloy composition. Therefore, the XPS survey spectra for the SS samples are not shown, but instead their nitrogen atomic concentration values are given in Figure 4. As expected, the key feature was the presence of nitrogen upon protein (mainly) adsorption from blood and its removal upon air plasma cleaning. The blood-contaminated SS coupon showed the presence of strong N I s photoelectron signal after pre-cleaning with both water and H₂0₂. It was evident from this analysis that H₂0₂, a chemical well known to be effective for bioburden removal, did not remove the proteinaceous contamination completely when analysed at the molecular level; hence its use may entail residual prion contamination on the "cleaned" device. This observation is in line with a recent study, where the effectiveness of H₂0₂ in its liquid and gaseous forms was investigated as an alternative low temperature method for steri lising prion-contaminated stainless steel wires' ^; in contrast to the gas form, liquid H₂0₂ was not effective. In particular, it was found that the l iquid H Oi treatment showed a degree of protein clumping and full resistance to protease degradation.

The air plasma treatment ( 1 00W, 0.5 Torr and 5 m in) of the blood-contaminated, pre-cleaned sample brings down the N l s photoelectron signal to a level similar to the one exhibited by a clean blank SS, indicating the complete removal of adsorbed protein within the X PS detection limit (Figure 4). The nitrogen concentration of 2.9 at.% inherent to the cleaned SS coupons was observed even' after the air plasma treatment. The blood-adsorbed coupons showed the presence of 1 8.5 at.% of residual nitrogen after pre-cleaning with water. Sequential cleaning with water and H2O2 removed the protcinaccous contamination to some extent as observed by the reduction of nitrogen to 1 3.6 at.%. The air plasma treatment brought down the n itrogen concentration to 1 .7 at.% from 1 8.5 at.% of pre-cleaned SS coupon with water, indicating complete removal of the adsorbed proteins.

Water contact angle measurements were carried out on SS samples in order to verify the removal of adsorbed proteins from blood-contaminated surfaces after pre-cleaning (Figure 4). The average advancing contact angle was found to be 82° for the blank SS coupon (A). Upon air plasma treatment, the angle was reduced to <10° (B). For the plasma-cleaned SS coupon with adsorbed proteins from the blood, the contact angle was found to be elevated to 70.6° (D), The water contact angle then reduced to < 1 0" (E) after the plasma treatment. As mentioned above, metal oxide surfaces with such low values of water contact angle values are devoid of organic contamination.

Case study III (DB samples)

These samples were prepared to investigate the removal of clinical proteinaceous contamination

(residual, after standard clinical cleaning) on a practical dental instrument (burs). Three batches of DB samples were analysed by XPS: (A) as-received burs ready to be used (blank); (B) burs used on a patient, fol lowed by cleaning and sterilising using conventional processes; and (C) burs of type B treated with the air plasma. In terms of the N l s signal, the XPS survey spectra for al l these DB samples exhibited a pattern similar to that of the SW samples shown in Figure 2, except that the cleaned DB blank sample exhibited an inherent nitrogen concentration of 3.3 at.%: again the binding energy and shape of the high resolution l s signal (not shown) indicated that this nitrogen is part of the alloy composition rather than protein on unused samples. The XPS survey spectra for the DB samples are not shown, but their nitrogen atomic concentration values are listed in Figure 5. As expected, the key feature was the presence of 7.7 at.% nitrogen on sample B due to residual proteinaceous contamination that remained after the conventional clinical cleaning. This level of nitrogen represents a sub-monolayer of the residual protein, and it also accounts for the nitrogen concentration inherent to the substrate (3.3 at.%). A lthough this nitrogen concentration value (7.7 at.%) is significantly lower than those observed for the corresponding residual proteinaceous contamination on the SW and SS samples, it can be concluded that the conventional c leaning processes used did not yield bur surfaces free of residual proteinaceous contamination. The air plasma-treated ( 1 00W, 0.5 Torr and 5 min) DB sample (sample C), on the other hand, exhibited a nitrogen concentration of 3.3 at.% (ie the level exhibited by the as- received DB sample) indicating complete removal of residual protein within the XPS detection limit. The geometry of the DB samples was not suitable for performing water^' contact angle measurements. Whittaker et al, also demonstrated contamination on used endodontic files cleaned and steri lised by hospital cleaning procedures³¹. The files were exposed to low-pressure oxygen-argon plasma for 10 m in. Scann ing electron m icroscopy and energy-dispersive X-ray (EDX) analyses were performed to determine the level of contamination before and after plasma cleaning. In all cases, the amount of organ ic material was reduced to a level below the detection limit of the instrument. However. EDX is far less surface sensitive and can only detect proteinaceous contamination at much higher amounts than X PS can ; the latter has a sensitivity to sub-monolayer levels, which is required when dealing with the possi bil ity of agents that are dangerous at very low amounts, such as prions. The present studies using a high ly surface-sensitive analytical technique (X PS) indicates complete elimination of total residual adsorbed protein from contam inated SS surfaces and dental burs by the air plasma treatment and confirmed the efficiency of air plasma in removing residual proteinaceous contamination from contaminated surfaces.

Gas plasma treatment has previously been found to have some efficacy towards prions" . Two plasma- based sterilisers have been commercialised, namely Starrad® (Advanced Sterilization Products, Johnson & Johnson, New Brunswick, NJ, United States of America) and the Plazlyte® Sterilisation system (AbTox, Mundelein, IL, United States of America). The Starrad® system has been shown to elim inate prion infectivity when used in conjunction with an alkaline cleaner^{1 2}. A technology based on gaseous H2O2, developed by Steris Corporation (Mentor, OH, Un ited States of America), has also been shown to eliminate prion infectivity^{1 '1}. Essentially, these methods combine the use of an oxidative chemical agent with a plasma cleaning step. However, some materials of interest, such as many polymers, are adversely affected by oxidative agents, and hence such devices may then be sterilised effective ly but become too degraded mechanical ly upon repeated re-use⁷. Metal oxide surface layers can also be changed by oxidative agents such as peroxides. Thus, the air plasma treatment of the present invention offers a sign ificant advantage for such devices. A lthough appl ied plasma cleaning and sterilisation of biomedical device surfaces has been studied, it is not well understood at the molecular level and many studies have not employed analytical techniques sensitive enough to probe complete removal of residual proteinaceous contamination. Thus, investigations continue on plasma-based sterilisation technologies"^5, '^{4" 16}; in particular, inductively coupled plasmas have been studied extensively ^17'21 . Typically, mixtures of non-toxic gases such as oxygen, nitrogen, argon or hydrogen are used to generate such plasmas, for example O2 N2 and

Ar/0_: N_: mixtures have been shown to be effective in removing biomolecules such as proteins from model surfaces^{8' q}. Removal of highly adherent pyrogens such as endotoxins seems to be a real possibility with such plasmas, thus offering an effective tool capable of el iminating the complete pathogenic bioburden from surfaces²², but further fundamental understanding needs to be developed before such a strategy can be used effectively. For example, the gas mixture 0;:N₂ ( 1 :4), commonly used for plasma steril isation, is known to yield very high levels of UV radiation that causes effective steri lisation, but this gas m ixture is not as effective for etching biomolecules or bacterial spores⁹. However, it has been overlooked that far UV radiation damages many polymeric materials, causing polymer chain scissions, and therefore such c leaning protocols may be unsuitable for some med ical devices^'"'. In a predominantly O; gas plasma, with on ly a smal l amount of N₂. high etching rates can be achieved with reduced U V radiation ^{1 7}. The use of even more complex gas mixtures such as 0₂ N₂/Ar has been suggested as a way to overcome this problem where the right balance between steri lisation and etching can be achieved^'5. However, the use of complex gas mixtures for plasma cleaning and steril isation may not be desirable, or may even be unnecessary, as such complex plasmas are relatively hard to control and their behaviour difficult to predict due to factors such as differential pumping of the constituent gases and the random quenching of the reactive plasma species^'^,

Air as a process gas leads to a gas plasma treatment method that is relatively simple to implement and control. Air plasma treatment offers advantages over other methods for prion decontamination such as vaporised H₂0₂ and complex gas m ixtures'^' ' \ Since air is a stable mix of oxygen and nitrogen

(-21 : 79), it is safer to handle and contain compared with corrosive Η₂0₂ and is more economical compared with specifical ly m ixed individual gases. Air plasma also offers a single step, completely dry ("green") surface cleaning method and compared with H₂0₂ is more effective for removing hydrophobic surface contaminants. The data presented in this example establishes the feasibility of the air plasma treatment for high quality proteinaceous contamination removal with a readily implementable method. A complete air plasma clean ing cycle wil l take <30 min. Air plasma cleaning can be viewed as an ablation/ashing technique, where the surface contaminant is removed as molecular fragments after plasma cleavage of bonds within molecu les, irrespective of how the contaminant is attached to the surface. Both hydrophobic and hydrophilic surface contaminants can be removed simultaneously by this technique. H ighly surface-sensitive techniques such as XPS and AFM can be used to assess the efficiency of plasma cleaning and val idate procedures for reprocessing of used biomaterials and medical devices. In particular, these surface-sensitive techniques are well-suited for detecting extremely low levels of residual proteinaceous contamination that normally exists on surfaces cleaned by conventional hospital cleaning methods.

In conclusion, a simple table-top air plasma cleaning system has been shown to be effective in removing proteinaceous contamination from model (silicon) and practical surfaces (surgical stainless steel and dental burs). X PS confirmed complete removal of this proteinaceous contamination, within the XPS detection lim it of the order of one hundredth of a monolayer of protein.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. A l l publ ications mentioned in this speci fication are herein incorporated by reference. Any d iscussion of documents, acts, materials, devices, articles or the l ike wh ich has been inc luded in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or al l of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this appl ication.

It wi l l be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in al l respects as illustrative and not restrictive.

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Claims

1 . A method of cleaning and sterilising a surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non-reactive gas plasma treatment to said surface for a period sufficient for the effective removal of at least any proteinaceous contamination.

2. A method of removing biological contaminants from the surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non-reactive gas plasma treatment to said surface for a period suffic ient for the effective removal of at least any proteinaceous contamination.

3. The method of claim 2, wherein the biological contaminants are prions.

4. The method of any one of claims 1 to 3. wherein the low temperature non-reactive gas plasma treatment is a non-rcactive, non-inert gas plasma treatment.

5. The method of claim 4, wherein the low temperature non-reactive gas plasma treatment is an air plasma treatment.

6. A method of cleaning and steri lising a surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non-reactive gas plasma treatment to said surface for a period sufficient for the effective removal of at least any proteinaceous contamination, wherein the gas plasma of said treatment comprises plasma of air and argon (or another heavy inert gas).

7. A method of removing biological contaminants from the surface of a surgical instrument or medical device, wherein said method comprises applying a low temperature non-reactive gas plasma treatment to said surface for a period sufficient for the effective removal of at least any proteinaceous contamination, wherein the gas plasma of said treatment comprises plasma of air and argon (or another heavy inert gas).

8. The method of any one of claims 1 to 7, wherein the gas plasma is generated with RFGD.

9. The method of claim 8. wherein RF Power in the range of 75 to 125 W is applied.

1 0. The method of claim 9, wherein the RF Power applied is - 1 00 W. The method of any one of claims 1 to 1 0, wherein the plasma pressure of the gas plasma treatment is in the range of 0.4 to 0.6 Torr.

The method of claim 1 1 , wherein the plasma pressure of the gas plasma treatment is -0.5 Torr.

The method of any one of claims 1 to 1 2, wherein the period sufficient for the effective removal of at least any proteinaceous contamination is in the range of 4 to 1 5 m inutes in duration.

The method of claim 1 3, wherein the period that the gas plasma treatment will be applied to the surface for the effective removal of at least any proteinaceous contamination is about 5 minutes.

The method of any one of claims 1 to 1 4, wherein the method is performed as a single step surface treatment.