Surface Sterilization and Modification of Medical

精品论文Surface Sterilization and Modification of MedicalPTFE by Remote Argon Plasma1Liu Hongxia, Chen Jierong*Department of Environmental Science and Engineering and Engineering, Xian JiaotongUniversity, Xian, P.R. China (710049)E-mail：hxliu72AbstractThis paper studies remote argon plasma sterilization to medical Poly(tetrafluoroethylene)(PTFE) film surface contaminated with E.coli and characterized surface structure, performances of PTFE film by the water contact angle, mass loss and platelet adhesion measurements as well as X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Results show that both remote and direct argon plasmas can sterilize effectively (GE 3.769) at the conditions of RF power 100W, exposure time 120s and argon flux 20 cm3/min. But the remote argon plasma sterilization contributes more effectively to the defluorination(F/C = 2.2425) from the PTFE film surface than that of the direct one contributes(F/C = 2.4853) and introduces more oxygen-containing groups (e. g. C=O) into it,makes this surface higher hydrophilicity and better antithrombogenicity while degradation and damages least, biocompatibility most excellent. Both the optimal inactivation and surface performances of PTFE film for use in medicine can be obtained simultaneously by remote argon plasma sterilization. The essential reason is that remote argon plasma sterilization can enhance interaction reactions with argon radicals relative to those with electron and argon ions.Keywords: Remote argon plasma, Sterilization, PTFE, Escherichia coli, Surface modification1. IntroductionMedical sterilization technology pays attention to higher stability and security than other ones. Plasma sterilization can overcome limitation of traditional methods and realizes green technology by its many merits, such as low temperature, fast and cost effective, dry and environmentally sound, and producing no toxic by-products, so it is regarded as one of the most promising sterilization technologies1-2. But when plasma acts on microorganisms, the surface of substrate is impacted consequentially, especially on polymers, which are used in modern medical devices and transplantable materials more and more. Certain functions, mechanical character and good biocompatibility with living tissues are required for polymers in medical use3. How effects do the impacting to medical using performances of these polymers and whether both the optimal sterilization and surface performances can be obtained at one time?The main reactions occurring on polymer surface during plasma sterilization are etching, cleaning, crosslinking, grafting and other chemical reactions (addition, substitution, formation of functional groups) dependent on the presence of active species in plasma 4. However, so far, researches about plasma surface sterilization have merely been limited to a mixed atmosphere constituted by all active species2, here, surface modification of polymer is hardly controlled5. So supported by National Natural and Science Foundation Council of China, we put forward an innovative idearemote plasma sterilization, that is, taking advantage of the different life-spans of various active particles such as electrons, ions and freeradicals (the rate constants of electron-positive ion recombination and radical recombination are 10-7 cm3/sand 10-33 cm3/s, respectively6) to make them separated at a special plasma field and attain super pure and high free radicals concentration at a distance from the plasma discharge region.1 Support by the National Natural Science Foundation of China (No.30571636), the Specialized Research Fund for the Doctoral Program of Higher Education (No.20060698002), the Key Scientific Technique Project of Shaanxi Province (No.2003K10-G61) and the Key Scientific Technique Project of Xian City (No.GG06049).-9-The paper studies remote argon plasma sterilization on medical Poly(tetrafluoroethylene)(PTFE) film contaminated by E.coli and characterized surface structure, performances of sterilized PTFE film in order to obtain both the optimal inactivation of E. coli and surface performances for use in medicine.2. Experiments2.1 MaterialsMedical PTFE film(20 mm thickness, made by Blood Center of Shanghai) was cut into pieces of dimensions 5025mm. Prior to the experiment, the film was degrease, washed with distilled water, disinfected and dried naturally at room temperature7.2.2 Microbial Strains and PreparationE.coli (ATCC 8099, Biosafety level 1) which were obtained from our microbial lab were incubated on nutritional agar substrate for 2 days at 370C prior to experimention. Then selected the representative strain to inoculate on the incline at 370C for 24h. Cells were subsequently transferred to 50ml of phosphate buffer solution (PBS, PH 7.0) under sterile surroundings. The cell suspension was diluted serially to the required concentration range (106108cfu/ml), and then, 0.01ml of it was droped onto presterilized PTFE film surface and spreaded uniformity by inoculative ring. The PTFE films were dried naturally at room temperature7.2.3 Remote Plasma SterilizationA self-designed ideal tube-reactor was used. In Figure 1, we show the Remote plasma reactive system. The reactor includes four parts: gas inlet, reaction chamber, gas exhaust, power supply (SY-500W 13.56MHz) and matching network (SP-matcher) which are made by the Science Academy of China. Thereaction chamber is Pyrex glass tube (length 1000mm, diameter 45mm), where inductance-coupling discharge is applied.Figure 1: Schematic Structure of Remote Plasma Reactor: (1) Gas bottle; (2) Valve; (3) Mass flow meter; (4) Inductance coil; (5) Reaction chamber; (6) Sample; (7)Vacuum gauge; (8) Electromagnetism valve; (9) Vacuum pump; (10) RF generator; (11) Grounding protection; (12) Matching system.The PTFE films, which contaminated by E.coli 8099, were positioned on the carrier to be sterilized by remote argon plasma. The purity of argon was more than 99.999%. The conditions of the sterilization were at an Radio Frequency (RF) power of 20100W, treatment time of 10120s, and argon flux of 20100 cm3/min. After the plasma treatment, bacterium were transferred from the films to Petri dishes containing nutritional agar and incubated at 370C for 2 day prior to determining the resulting number of colonyforming units (cfu) 7-9 . The Germicidal Effect (GE) can be calculated by the following equation7:GE = log N0 log NtWhere N0 and Nt are the number of colony forming units (cfu) of control and sterilized, respectively.(1)The plasma-sterilized PTFE flims were washed with distilled water for 2 min, dried naturally at room temperature and to be used for measuring surface properties.2.4 Contact Angle MeasurementThe measurements were carried out immediately after plasma sterilization. The contact angle was measured by a contact angle meter (JY-82) made in Chengde, China. To lessen the effect of gravity, the volume of each drop was regulated to about 0.2 cc by a micro syringe. The measurement was carried out at200C and humidity of 45% R.H. The average value of the angles of the both sides of each drop wascounted as one measurement. Each contact angle was determined from an average of 10 measurements with the mean standard deviation (n = 10).2.5 Changes in MassMasses of PTFE films were obtained by electronic analytical balance (Mettler AE240). Three plasma-sterilized PTFE films of each sample position were weighted and mass loss was calculated by comparing the mass of unterilized PTFE film (M0) with that of plasma-sterilized PTFE film(Mt). The results of which are presented as the mean standard deviation (n =2.5), as calculated by10:( M 0 M t )Mass loss =100%M 0(2)2.6 Scanning Electron MicroscopyThe surface morphology of the PTFE film was examined using scanning electron microscopy (SEM) in aJEOL instrument (Model JSM5800, Japan) after vacuum coating the specimens with gold.2.7 Platelet Adhesion MeasurementA drop blood plasma with rich platelet was dropped on PTFE film surface after sterilization, immerged in PBS solution to strip down the loose platelet after 5 min, then fixated for 30 min in Glutaraldehyde solution (25% Glutaraldehyde 0.4L, 0.2mol/L PBS solution 5L and distilled water 4.6L). Finally, grads dehydration was carried out in turn in ethanol-water solution with 40%, 50%, 60%, 70%, 80%, 90% and 100% for each time 1530min. Platelet adhesion states on PTFE film surface was examined by SEM after drying naturally at room temperature under super clean surroundings.2.8 X-ray Photoelectron SpectroscopyThe X-ray photoelectron spectroscopy (XPS, PHI1600ESCA System Perkin-Elmer, USA) with a nonmonochromatic Mg K photon source was used to analyze chemical compositions of surface layer of the PTFE film at a pressure lower than 510-8 Pa. The anode voltage was 10 kV and the anode current 30 mA.The size of the X ray spot was 2 mm diameters and the take-off angle of photoelectrons was 150 withrespect to the sample surface. O/C and F/C atomic ratios in the PTFE film surface were estimated from the relative intensity of the O1s core level against the C1s core within an experimental error of 0.01.3. Results and Discussion3.1 Germicidal Effect on Medical PTFE Film Surface by Argon PlasmaThe PTFE films contaminated by E.coli were positioned at a constant distance of 0, 20, 40, 60, and 80 cm from the center of the RF coil in the Pyrex glass tube of the reactor and were exposed to the argon plasma at RF power levels of 20, 40, 60, 80 and 100 W. The surface sterilization of the PTFE film by the argonplasma was investigated from the GE as a function of the RF power, the treatment time, the argon flux and the sample position. The GE varies strongly with these conditions. Figures 24 show the effects of the RF power, the treatment time, and the argon flux on the GE at any sample position and Figure 5 shows the typical effect of the sample position on the GE.The GE values change undulately with the RF power increasing at first, as shown in Figure 2, the changes are small, and subsequently, a large increase in GE values shows between 80W and 100W regardless of the sample position, the peak GE values reach 4.633. The effects of plasma treatment time were also observed after 30s (Figure 3), especially at sample positions of 0, 20 and 40cm: GE values at 30s are 1.517, 1.676,1.328 and 4.633, 4.192, 3.769 at 120s, respectively. On the other hand, at sample positions of 60 and 80cm, the GE values increase small correspondingly, which are 1.252, 1.167 at 30s and 2.579, 2.042 at 120s, respectively. These results indicate that GE for PTFE film surface are affected more strongly by changing treatment time at sample position of 040cm than of 6080cm. However, the reducing GE before 30s is not well understood, but it could be as follows.In RF argon plasma, Ar+ ions and quick atoms which have high mass and low velocity of flow needundergoing 2530 RF periods to achieve stable states while electrons with lesser mass vary fluctuantly during the course. So at the start of discharge, all particles are non- stable states11. Here, either mutual collision of ions, atoms and electrons with non-stable states or action on the walls so as to increasing the losses of the energy, as well as some higher energy electrons ionizing metastable states causing a reduction of electron temperature, which slows down the rate of diffusion12 lead to interact between these particlesand cells stochastic with lower efficiency.5.04.54.03.5G E3.02.52.01.51.00.50.020 40 60 80100RF power (W)Figure 2: Effect of RF Power on Germicidal Effect at Sample Position of ()0cm; () 20cm; ()40cm; ()60cm; and()80cm (treatment time: 120s; argon flux: 20cm3/min).5.04.54.0G E3.53.02.52.01.51.0020406080100120Plasma treatment time (sec)Figure 3: Effect of Plasma Treatment Time on Germicidal Effect at Sample Position of ()0cm; ()20cm; ()40cm; ()60cm; and ()80cm (power: 100W; argon flux: 20cm3/min).5.04.54.0G E3.53.02.52.01.51.020406080100Argon flux (cm3/min)Figure 4: Effect of Argon Flux on Germicidal Effect at Sample Position of ()0cm; () 20cm; ()40cm; ()60cm; and()80cm (power: 100W; treatment time: 120s).The argon flux effects are distinguished from the plasma RF power and treatment time effects. Figure 4 shows GE for the PTFE film surface at 100W for 120s as a function of the argon flux. GE values reduce continually in spite of the sample position. All these values in Figures 24 indicate that the optimal sterilization conditions of RF argon plasma for PTFE film surface contaminated by E.coli are the plasmaRF power 100W, the treatment time 120s and argon flux 20 cm3/min.5.04.54.0G E3.53.02.52.01.5020406080Sample position from RF coil (cm)Figure 5: Effect of Sample Position on Germicidal Effect(power: 100W; treatment time: 120s; argon flux: 20cm3/min).Figure 5 shows GE for the PTFE film surface under the optimal sterilization conditions as a function of the sample position, GE values at sample positions of 0, 20, 40, 60, 80cm are 4.633, 4.192, 3.769, 2.579, 2.042, respectively. These values indicate that the argon plasma sterilization can be carried out at sample positions of 040cm. Here we call the argon plasma sterilization at a sample position of 40cm the remote argon plasma sterilization to distinguish it from conventional argon plasma sterilization, which is done at a sample position of 0cm. We also call conventional argon plasma sterilization direct argon plasma sterilization. From these results, we conclude the following: (1) Both direct argon plasma (at a sample position of 0cm) and remote argon plasma (at a sample position of 40cm) can realize the surface sterilization of PTFE film contaminated by E.coli; (2) the GE depends on the plasma RF power as well as the treatment time and the argon flux at any sample position.3.2 Changes in Surface Properties of Medical PTFE Film after Argon PlasmaSterilization3.2.1 Contact Angle of Water on the Plasma-sterilized PTFE FilmThe water contact angle on the surface of plasma-sterilized PTFE film as a function of the sample position under the optimal sterilization conditions was investigated. The contact angle measurements were carried out immediately after finishing the plasma sterilization experiments to minimize changes in the surface properties. The hydrophilicity is evaluated by the contact angle of water (). The PTFE films sterilized by the argon plasma showed smaller contact angles than the unsterilized PTFE film ( = 1080 ) and their surfaces became hydrophilic. Figure 6 shows for the PTFE film sterilized under the optimal sterilization conditions as a function of the sample position, at sample positions of 040cm decreases from 70.50 to58.50 and on the other hand, at sample positions of farther than 40cm rises up by increasing the sampleposition. This result indicates that the argon plasma exposure at a sample position of 40cm is the best way for hydrophilic modification. So remote argon plasma sterilization can obtain optimal hydrophilicity ofPTFE film surface.120110Unsterilized10090(O)80706050400 20 40 6080Sample position from RF coil (cm)Figure 6: Effect of Sample Position on the Contact Angle to Water of PTFE Film (power: 100W; treatment time: 120s;argon flux: 20cm3/min) (n=10, mean standard deviations are reported).3.2.2 Mass Loss of the Plasma-sterilized PTFE FilmFigure 7 shows effect of the sample position on the mass loss of the PTFE film under the optimal sterilization conditions. With the farther the sample position from RF coil, as shown in Figure 7, the mass loss decreases rapidly from 37% to 5.9%. But in view of GE is worse at sample position of 60 and 80cm, sothe argon plasma sterilization at a sample position of 40cm, that is, remote argon plasma sterilization makesthe minimal degradation of PTFE film surface.40Mass loss ( % )30201000 20 40 6080Sample position from RF coil (cm)Figure 7: Effect of Sample Position on Mass Loss of PTFE Film (power: 100W; treatment time: 120s; argon flux:20cm3/min) (n=2.5, mean standard deviations are reported).3.2.3 Surface Morphology of Plasma-sterilized PTFE FilmThe contact angle and mass loss measurements show that the remote argon plasma sterilization leads to higher hydrophilic and lower degradation of PTFE film than direct one. A main difference between the remote and direct argon plasma is a relative concentration between argon radicals and charged species such as argon ions and electrons. The argon radicals in the remote zone are in higher concentration than the charged species. On the other hand, both charged species and argon radicals in the discharged zone are in high concentration. Therefore, sterilization with remote argon plasma will escape from surface degradation of the PTFE film initiated by heavy collision of the charged species. Surface morphology of the PTFE film sterilized by argon plasma was examined with scanning electron microscopy (SEM) in order to investigate how the PTFE film surface was damaged by the plasma exposure. The PTFE films, which were sterilizedwith the remote and direct argon plasma at RF power 100 W and argon flux 20 cm3/min for 120s were usedas specimens for SEM measurements and Figure 8 shows these SEM pictures. By increasing the sample position, the damages of plasma-sterilized PTFE film surface are mitigated (Figure 8 (b)(d) and in remote zone, the plasma-sterilized PTFE film surface is as smooth as the unsterilized PTFE film (Figure 8 (d), but the direct argon plasma-sterilized PTFE film shows a rougher surface and there are numerous lumps all over it. This comparison shows that the remote argon plasma sterilization caused little change in PTFE film surface, but reverse of the direct one. From Figures 68, we can conclude that going with complete sterilization, the remote argon plasma does not initiate remarkable degradation and damage on the PTFE film surface with hydrophilic modification while the direct argon plasma initiates heavy degradation and damage.(a) (b)(c) (d)Figure 8: Scanning Electron Micrograph (8000) of (a) Unsterilized PTFE Film; and Plasma-sterilied PTFE Film atSample Position of (b) 0cm; (c) 20cm; (d) 40cm (power: 100W; treatment time: 120s; argon flux: 20cm3/min).3.2.4 Surface Antithrombogenicity of Plasma-sterilized PTFE FilmSurface antithrombogenicity of the PTFE film sterilized by argon plasma was examined with SEM. The PTFE films, which were sterilized by the remote and direct argon plasma at RF power 100 W and argon flux 20 cm3/min for 120s were used as specimens for SEM measurements. On direct argon plasma-sterilized PTFE film surface, as shown in Figure 9(a), more platelet adhere to it, assembling and distorting severely, but these become unconspicuous by increasing the sample position (Figure 9 (b)(d), till the remote zone, adhesion amount drop off