Unearthed Chinese silk fabrics, especially cultural silk relics, carry precious cultural heritage and are essential to the study of traditional cultures, particularly the ancient aesthetic features.^[1–3] Exploring the preserved relic pieces made of the unearthed silk fabrics can facilitate the understanding of our history, the appreciation of the creative power of human beings, and the revealing of the development of human society,^[4–6] all of which promote the development of contemporary and future society. However, mycete cultured on these unearthed silk fabrics are known to significantly shorten the life-spans of such fabrics, which have endured a harsh environment under the ground for a long period of time. Therefore, it is of practical importance to establish protocols for sterilizing mycete while preserving the quality of the unearthed silk fabrics. The conventional methods used for sterilizing the unearthed silk fabrics are typically based on either thermal or chemical treatment, or exposure to ionizing radiation.^[7] However, these conventional processes easily cause thermal damage or chemical corrosion to the sterilized pieces. Moreover, the chemical reagents are not environmentally friendly and very effective when fungus contamination is concerned.^[8] Therefore, a more effective sterilization protocol for the treatment of the unearthed silk fabrics is worthwhile exploring.

Recently, the development of the plasma based sterilization technology has made tremendous progress and it has been widely used in different industries, such as medical, biological, food-processing, and agricultural applications.^[9–14] For example, Natthaporn Khamsen et al. designed a system to produce atmospheric hybrid cold-discharge plasma (HCP) for inactivating microorganisms that commonly attach to the rice seed husk, and the cold HCP completely inactivated the pathogenic fungi and other microorganisms, enhancing the germination percentage and seedling quality.^[15] Sakai et al. generated oxygen plasma using a narrow gap radio-frequency (RF) discharge to sterilize dental instruments.^[16] The atmospheric pressure plasma jet (APPJ), which has the characteristics of high sterilization efficiency, short treatment time, no pollution, no residue, and no damage to the materials,^[17,18] may be an ideal candidate for the sterilization/inactivation of mycete.

The sterilization performance of the APPJ treatment depends heavily on the active species (e.g., highly energized atoms, charged particles, electric fields, and UV radiation)^[19–21] that it generates and such active species are impacted strongly by the electrode configuration, the gas flow rate, and the applied voltage.^[22–24] When helium/argon (He/Ar) mixed with oxygen act as the reactive gases in the APPJ system, the active radicals are generated by bombarding with energetic electrons, which has also been identified as an important factor to determine the bacteria sterilization outcomes.^[25,26]

While various APPJ systems have been reported for many different sterilization applications, to the best knowledge of the authors of this paper, no such report on the sterilization treatment of the unearthed silk fabrics has been published. Herein, an Ar/He–O₂ APPJ system was employed to sterilize the mycete cultured on the simulated unearthed silk fabrics to study the feasibility for treating the authentic unearthed silk fabrics. The effects of the APPJ treatment time, the discharge power, and the gas type of the APPJ system on the inactivation of mycete attached to the simulated unearthed silk fabrics were investigated. The experimental data obtained in this work was used to determine the optimized sterilization parameters and the protocol employing the Ar/O₂ APPJ system was shown to be a promising candidate for the heritage protection of the authentic unearthed silk fabrics.

Figure 2 shows the optical emission spectra from 200 nm to 800 nm of Ar and He plasmas. The Ar lines mainly dominated the region of 700–800 nm and the peaks at 548 nm and 620 nm were also detected (Fig. 2(a)), and the He lines at 550 nm, 616 nm, 667 nm, and 706 nm were detected (Fig. 2(b)). Both optical emission spectra revealed the presence of excited species in the plasma jets, such as N₂, OH, H, and . The excited observed at 427 nm and O emission resulting from the 3⁵S–3⁵P transition at 740 nm were also observed in the OES. It is well known that some of these species (e.g., O and OH) are very active in such a way that they play a very important role in sterilization of mycete. This may be due to the fact that a significant fraction of the plasma electrons used to produce O-containing species from the O₂ molecules are present in the plasma-forming gas.^[28] The probable formation paths of in He plasma include both direct electron-impact ionization ) and Penning ionization ).^[29,30] Since N₂ represents 70% of the ambient air, its emission is likely to be significant with respect to the intensity of O and OH emission. The presence of N₂ (265 nm, 315 nm, 337 nm, 357 nm, and 380 nm) and (378 nm and 470 nm) lines in Fig. 2 clearly revealed the presence of ambient air in the plasma, which was also reported by other researchers.^[31–34] However, comparing Fig. 2(a) with Fig. 2(b), it can be found that the intensity of these species in Ar and He is obviously different, and the peak intensities of some functional species in Ar are higher than those occurred in He APPJ, which may be more effective on the mycete sterilization.

Fig. 2.

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	Fig. 2. Optical emission spectra of the APPJ with the silk fabrics, an Ar or He flow rate of , an O2 flow rate of , and an input power of 50 W: (a) Ar, (b) He.

Figure 3 shows the photographs of the AN funguses attached on the silk fabrics treated different times by Ar or Ar/O₂ APPJ. As shown in Fig. 3(a₀), the number of AN colonies cultured in the PDA medium was about 110, indicating a CFU quantity of AN about 2.2×10⁵ on the original untreated silk fabric. When the samples were treated by the Ar APPJ for 1 min, 2 min, 3 min, 4 min, and 5 min (Figs. 3(a₁)–3(a₅) respectively, the AN number decreased noticeably as the plasma treatment time increased. The AN sterilization percentage could reach 99.0% if a treatment time of 5 min was used with an Ar APPJ plasma. However, the CFU number of AN on the silk fabric was still about 2×10³, which exceeded the maximum limit when protecting the unearthed silk fabrics was concerned. Figures 3(b₀)–3(b₅) show the effect of treatment time on the AN sterilization when the Ar/O₂ APPJ was used, and the AN quantities decreased sharply as the plasma treatment time increased. Under the same discharge power as the Ar plasma, the AN attached on the untreated silk fabric (CFU: 1.36×10⁵) were completely inactivated when the silk fabric was treated by the Ar/O₂ APPJ for 4.0 min (Fig. 3(b₄)), indicating that when the sterilization of mycete on the silk fabrics is concerned, the Ar/O₂ plasma will be more effective than the Ar plasma.

Fig. 3.

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	Fig. 3. (color online) The photographic illustration of the AN samples: (a0), (b0) the untreated silk fabric; the silk fabrics treated by the (a1)–(a5) Ar or the (b1)–(b5) Ar/O2 APPJ plasma for 1.0 min, 2.0 min, 3.0 min, 4.0 min, and 5.0 min, respectively.

Figure 4 presents the sterilization rate of the AN attached on the silk fabrics by the Ar or Ar/O₂ plasma. The experimental data depicted by Fig. 4 showed that the sterilization rate of the silk fabrics steeped with the AN was augmented noticeably as the treatment time increased for both the Ar and the Ar/O₂ APPJ plasmas. For the Ar plasma, the sterilization rate of AN was about 77% with a treating of 1.0 min, whereas nearly 90% AN was killed by the Ar/O₂ plasma within 1.0 min with the same plasma treatment parameters. Although the exact role of the O₂ contained in the Ar/O₂ plasma for sterilization is still controversial,^[35] the active species of OH and O generated in the Ar/O₂ plasma may play a crucial role for inactivating the microorganisms.^[23]

Fig. 4.

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	Fig. 4. (color online) The sterilization rate of the AN fungus injected onto silk fabrics treated by the Ar or the Ar/O2 APPJ plasma with different treatment time.

It is well known that the reactive species of the APPJ system depend closely on the plasma characteristics and the discharge parameters. The AN counts on the original simulated unearthed silk fabrics (Fig. 5(a)) were about 4.2×10⁵, and it decreased to 1.84×10⁵ when the sample was treated by the Ar plasma with a discharge power of 30.0 W for 2.0 min (a 65.2% reduction in the AN population) (Fig. 5(c₁). When the discharge power increased (40.0 W, 50.0 W, and 60.0 W), as shown in Figs. 5(c₂)–5(c₄), the AN counts decreased drastically (down to 2.0×10⁴, a 95.2% reduction when 60.0 W was used) under the same treatment time. Such significant increase in the sterilization rate suggests that the AN can be efficiently inactivated by increasing the plasma discharge power (which understandably results in increased particle densities). In addition, the relationship between the sterilization efficiency and the discharge gas type was also investigated, and it can be seen that the Ar plasma (Fig. 5(d₂), CFU: 2.2×10⁵) is more effective than the He plasma (Fig. 5(d₁), CFU: 3.8×10⁵). It should be noted that the sterilization efficiencies of the He/O₂ (Fig. 5(d₃), CFU: 3.6×10⁴) and the Ar/O₂ (Fig. 5(d₄), CFU: 1.8×10⁴) plasmas are higher than those obtained by pure inert gases, suggesting that the presence of O₂ in the plasma jet is of essential importance.

Fig. 5.

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	Fig. 5. (color online) The photographic illustration of the AN samples on (a) the untreated silk fabrics, and the silk fabrics treated by Ar APPJ with discharge powers of (c1) 30.0 W, (c2) 40.0 W, (c3) 50.0 W, and (c4) 60.0 W; the silk fabrics treated by (d1) He, (d2) Ar, (d3) He/O2, and (d4) Ar/O2 APPJ plasmas.

In order to further explore the relationship between the sterilization rate and the discharge parameters, the effect of the discharge power and the gas types on the AN inactivation was also analyzed, as shown in Fig. 6. Figure 6(a) shows the AN reduction performance as a function of the discharge power when the Ar APPJ plasma was used. The experimental data depicted in Fig. 6(a) showed that the sterilization rate of AN increased from 56.2% (30.0 W) to 95.2% (60.0 W). Under the same discharge power of 50.0 W, the sterilization efficiency of the Ar plasma (91.0%) was nearly twice that achieved by the He plasma (47.6%). It should be particularly noted that the He/O₂ plasma could achieve almost the same sterilization performance (91.4%), suggesting once again that the presence of O₂ in the plasma jet may be crucial to the AN sterilization performance. Based on the results discussed above, the Ar/O₂ plasma is better suited for the microorganism inactivation applications than the He or He/O₂ plasma.

Fig. 6.

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	Fig. 6. (color online) The sterilization rate of the simulated unearthed silk fabrics steeped with AN treated by the APPJ plasmas with (a) different discharge powers, and (b) different gases.

In order to better illustrate the morphological evolution of AN before and after plasmatic inactivation, the series digital microscope was used. Figure 7(a) shows the microscope image of the AN spores attached on the simulated unearthed silk fabrics, and the average diameter of the spore was about (the inset of Fig. 7(a)). When the AN attached silk fabrics was treated by the Ar/O₂ plasma with a discharge power of 50.0 W for 4.0 min, as shown in Fig. 7(b), all the AN spores were deformed, and the average nominal diameter of a residue of spore decreased to (the inset of Fig. 7(b)). Moreover, the AN hyphas were completely destructed under the function of the Ar/O₂ plasma, resulting in permanent loss in the meristem ability. Figures 7(c) and 7(d) exhibit the three-dimensional images of the AN spores having undergone treatments by the Ar/O₂ plasma, and that the spore wall was breached by the reactive species generated in the Ar/O₂ APPJ system, as indicated by the tissue fluid that has flown out from the AN spore. From the captured images of the AN spores, the inactivation processes and mechanism of spores by the APPJ plasma may be described as follows. (i) The active species (e.g. Ar⁺, O, OH, O₃, UV light) generated in the Ar/O₂ plasma firstly etch or oxidize the spore walls and hyphas.^[36–38] (ii) The tissue fluids flow out from the holes on the breached spore walls while the spores are continuously oxidized by the active species.^[39–41] (iii) Finally, the spores die without getting enough alimentation, and the AN lose the function of reproduction and the ability of self-replication.^[38,42–45]

Fig. 7.

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	Fig. 7. (color online) The digital microscope images of AN spores: (a) and (c) untreated, (b) and (d) treated by Ar/O2 APPJ.

Figure 8 shows the SEM images of the AN attached silk fabrics treated by the Ar/O₂ (volumetric ratio: 5/1) APPJ with different time. Compared with the untreated silk fabrics (Fig. 8(a)), there is no observable change in the surface morphology of the sample treated with the Ar/O₂ plasma for 2.0 min (Fig. 8(b)). Under the same discharge power (50.0 W), the silk fabric profiles were similarly very smooth, independent of the treatment time (3.0 min in Fig. 8(c) and 4.0 min in Fig. 8(d)). Furthermore, the effect of the plasmatic treatment time on the silk fabrics’ morphology was also investigated using the high resolution SEM images (insets of Fig. 8), and the Ar/O₂ plasma left the silk fibre almost intact, as opposed to what it did to the AN fungi. Such a noticeable mitigation of the adverse destructive effects of the Ar ions showed that the Ar ions pushed by a high electric field bombard the substrate with very high velocities was attributed to the nonexistence of a strong electric field between the inner electrode and the substrate.^[46] Consequently, the velocity of the Ar ions was similar to the gas flow rate, and the etching effect of the Ar ions on the silk fibre was negligible.

Fig. 8.

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	Fig. 8. The SEM micrographs of silk fabrics injected by fungus with (a) untreated; and different plasma-treated time for (b) 2.0 min, (c) 3.0 min, and (d) 4.0 min, respectively.

In order to enhance the storability of the plasmatic sterilized unearthed silk fabrics, it is of vital importance that such plasmatic treatment should have minimal impact on the physical/chemical/mechanical properties of the treated silk fabrics. Figure 9 presents the mechanical property of the silk fabrics treated by the Ar/O₂ APPJ with different time, and the elongation at break and tensile strength of the samples were tested using an electronic universal testing machine controlled by a microcomputer. As the black line shows in Fig. 9, there was an upward trend for the elongation at the initial stage, and the elongation values at break changed marginally after a critical treatment time of about 40 s. Compared with the original sample, the elongation at break increased by about 10.0% when 100% sterilization of the AN on the silk fabrics was achieved by the Ar/O₂ plasma in 4.0 min. Such experimental results showed that contrary to reducing the mechanical strength of the silk fabrics, the Ar/O₂ plasma treatment performed in this work actually enhanced it. Such an increase in mechanical strength was attributed to the surface modification during the plasma treatment process. It should be noted, however, that the tensile strength did decline slightly in the initial plasma process (60 s). Beyond this point, on the other hand, the further change in the tensile strength of the plasma treated silk fabrics tended to be negligible. Such a marginal intensity decrease indicates that the physical damage of the Ar/O₂ plasma on the silk fibre can be safely neglected.

Fig. 9.

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	Fig. 9. (color online) The mechanical properties of the silk fabrics treated by the Ar/O2 APPJ for different time.

Since the major components of both the silk fabrics and the AN are proteins and saccharides, it is important to confirm the selectivity of the active species sterilizing the silk fabrics towards the AN. The FTIR spectroscopy in ATR mode was used to investigate the compositional changes of the plasma treated silk fabrics. Figure 10 exhibits the FTIR spectra of the silk fabrics treated with different gases, and the significant peaks in line d₀ indicate that the original silk fabrics injected with the AN were covered with proteins and a small amount of sugar and chitin.^[21] Compared with the original untreated silk fabric sample, no obvious change of peak positions, especially peaks of –NH at 3300 cm⁻¹ and C=O at 1625 cm⁻¹, occurred in the FTIR curves for the samples treated by the He (line d₁) and the He/O₂ (line d₃) plasmas for 2.0 min. Meanwhile, similar phenomena were also observed in the samples treated by the Ar (line d₂) and the Ar/O₂ (line d₄) plasmas. It should be noted that the peak intensities of the treated samples at 1625 cm⁻¹ for C=O and 1520 cm⁻¹ for C–C increased noticeably. The results indicated that the active species generated by the APPJ system do not alter the chemical structure of the silk fibre, but only the DNA and cytoderm of the AN.

Fig. 10.

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	Fig. 10. (color online) FTIR absorption spectra of the silk fabrics treated by different gases with a discharge power of 50.0 W: (d0) original sample injected with AN, (d1) He, (d2) Ar, (d3) He/O2 (Vol: 5/1), and (d4) Ar/O2 (Vol: 5/1).

In order to further explore the possible changes in the chemical composition of the samples before and after the plasmatic sterilization, the silk fabrics treated by the APPJ system employing different gases were characterized by an x-ray photoelectron spectrometer. Figure 11 shows the XPS survey and the high resolution C 1s of the silk fabrics surface treated by the Ar (O₂) or the He (O₂) APPJ plasma. As shown in Figs. 11(a) and 11(b), the elements on the original fabric surface injected AN were C, N, and O, and the chemical assignment for the deconvoluted carbon peaks was based on the binding energy from the literature:^[47,48] C–C/C–H (284.6 eV), C–OH/C–N (285.9 eV), C=O (287.9 eV), and C–C–OH/C–C–OR (288.7 eV). Compared with the carbon peak of the original sample (Fig. 11(b)), the chemical bonds of the silk fabrics treated by the He (Fig. 11(c)), Ar (Fig. 11(d)), He/O₂ (Fig. 11(e)), and Ar/O₂ (Fig. 11(f)) APPJ plasmas differed noticeably, especially the change of the C=O bond. The C=O bonds mainly existed in the protein of the silk fabrics and chitin of the cell wall, and a small part of them may come from the unsaturated fatty acid and protein in the mycete cells. The peak intensity of the C=O bond is relatively weak in Fig. 11(c), and the possible reason is that the mycetes covered the surface protein of the silk fabrics due to the lower sterilization efficiency of the He plasma (47.6%). Meanwhile, some of the charged particles were gathered on the surface of the cell wall, destroying the chitin structure. However, the peak intensity of the C=O bond is relatively strengthened in Figs. 11(d)–11(f), and it may be due to the destruction of the AN by the active species (e.g. Ar⁺, O, OH, O₃), which is also indicated by the fact that the carbon contents of all treated samples decreased (Table 2). On the other hand, the average diameter of spores became smaller with the increased sterilization efficiency of Ar, He/O₂ and Ar/O₂ plasmas, therefore, the exposed area of the silk fabrics increased. Moreover, the oxidation species of O, OH, and O₃ destroyed the cell membrane, resulting in the outflow of protein containing C=O bonds. It should be noted that the oxygen contents of all the samples treated by the APPJ plasmas employing different gases also increased obviously, mainly attributed to the grafted O and OH radicals, which can destroy unsaturated fatty acids and exert strong oxidative effects on the outer structure of the mycete cells.^[38,42]

Fig. 11.

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	Fig. 11. (color online) (a) The XPS survey and high resolution carbon of original silk fabrics, and C 1s peaks of silk fabrics (b) untreated, and treated with different gases: (c) He, (d) Ar, (e) He/O2, and (f) Ar/O2.

Table 2.

Table 2.

Table 2. The surface element composition ratio of the samples treated by APPJ with different gases. . Element C 1s N 1s O 1s Control sample 65.1 14.19 20.71 He atmosphere 46.59 10 43.42 Ar atmosphere 53.75 16.14 30.11 He/O2 atmosphere 35.88 18.61 45.52 Ar/O2 atmosphere 35.07 16.73 48.2

Table 2. The surface element composition ratio of the samples treated by APPJ with different gases. .

In summary, a novel sterilization method employing an APPJ system is successfully implemented to sterilize mycete attached on simulated unearthed silk fabrics. The sterilization performance of AN attached on the silk fabrics depends closely on the discharge parameters of the APPJ plasma, and a longer treating time (up to 4 min) and a sufficiently higher discharge power (up to 60 W) are more beneficial when destroying the AN spores is concerned. Amongst the various plasma jets tested in this work, the Ar/O₂ APPJ plasma has been proven to be the most effective. Typically, the AN injected on silk fabrics were completely inactivated by the Ar/O₂ APPJ plasma with a treatment time of 4 min and the elongation at break strengths of the treated silk fibre increased about 10%. The experimental results showed that the Ar/O₂ APPJ plasma could successfully inactivate the AN attached on the silk fabrics without any significant damage to the integrity of the silk fibre. Therefore, the APPJ system presented in this work should be considered as a viable, minimum-destructive, and high-performance protocol for sterilizing unearthed silk fabrics. The methodology presented in this work is highly recommended for archaeological applications.