MATERIAL WITH SUPERCAPACITANCE MODIFIED SURFACE AND PREPARATION METHOD AND APPLICATION THEREOF

Abstract

Disclosed are a material with supercapacitance modified surface and a preparation method and application thereof. Specifically, the present disclosure introduces a material having a controllably supercapacitive surface. The surface is chargeable, the full-charged modified surface can interact with bacteria disturbing the electron transfer of respiratory chain of bacteria and inhibiting the growth and reproduction of bacteria in a short-term. The antibacterial rate can be improved by cyclically charging-discharging without losing capacitance, and prevent formation of biofilm of bacteria. The antibacterial system can quantitatively control the antibacterial process without affecting the biocompatibility of the material, and has the advantages of environmental protection and controllability.

Claims

1. A material with supercapacitance modified surface, comprising: a material body; and a supercapacitance layer on surface; wherein the material body is selected from a metal material or other conductors, the supercapacitance layer refers to a functional layer having a surface capacitance greater than 10 mF.Math.cm.sup.−2.

2. The material according to claim 1, wherein the metal material is selected from the group consisting of titanium or an alloy thereof, aluminum or an alloy thereof, stainless steel, nickel or an alloy thereof, manganese or an alloy thereof, tungsten or an alloy thereof, zinc or an alloy thereof; the other conductors are selected from the group consisting of conductive polymers, including polypyrrole, polyacetylene, polythiophene, polyaniline; the surface capacitance of the supercapacitance layer is greater than 50 mF.Math.cm.sup.−2, preferably greater than 100 mF.Math.cm.sup.−2.

3. The material according to claim 2, wherein the metal material is selected from the group consisting of a titanium alloy, an aluminum alloy, a stainless steel, a nickel alloy, and a zinc alloy; and the supercapacitance layer is selected from the group consisting of a titanium dioxide nanotube array layer, a zinc oxide nanorod layer, or a reduced graphene oxide.

4. The material according to claim 3, wherein the titanium dioxide nanotubes or zinc oxide nanorods have a diameter of 10 nm to 1000 nm, preferably 20 to 800 nm, most preferably 50 to 500 nm; and a pipe diameter of 500 nm to 10 μm.

5. The material according to claim 4, wherein the titanium dioxide nanotube array layer further comprises deposited carbon; and the zinc oxide nanorod layer is doped with silver, gold, copper or platinum nanoparticles.

6. A preparation method of material with supercapacitance modified surface according to claim 1, comprising: anodizing the surface of metal material, the electrolyte used to anodize is a mixed solution of an ammonium salt, a lower alcohol, water, and a polyol.

7. The preparation method according to claim 6, wherein the ammonium salt is selected from an ammonium halide, preferably ammonium fluoride; the lower alcohol is selected from methanol or ethanol; and the polyol is selected from ethylene glycol; the voltage of anodization is 10-100 V and the time of anodization is 20-1000 min.

8. The preparation method according to claim 6, wherein the array of nanotubes obtained from anodization is placed in a vacuum tube furnace annealing in vacuum to achieve carbon deposition, so as to enhance the capacitive characteristics; and the temperature of vacuum anneal is 500-800° C., the annealing time is 1 to 5 h, and the heating rate is 1 to 20° C. min.sup.−1.

9. A preparation method of material with supercapacitance modified surface according to claim 1, comprising: growing zinc oxide nanorods on the surface of the metal material by a hydrothermal method and sputtering the doped silver, gold, copper or platinum nanoparticles by magnetron sputtering, the specific steps are as follows: (1) preparation of zinc oxide seed crystal: dissolving zinc acetate and a strong base in a lower alcohol, quickly spin-coating on the surface of the metal material to obtain a wet film, heating to volatilize the solvent and pyrolyzation to obtain a metal material with a crystal seed layer; (2) growth of zinc oxide nanorods: placing the sample from (1) into a reactor, adding a mixed aqueous solution of a zinc salt and a base, sealing and heating the reactor; sputtering silver, gold, copper or platinum nanoparticles by magnetron sputtering; the base is preferably hexamethylenetetramine, sodium hydroxide, potassium hydroxide, calcium hydroxide, and aqueous ammonia.

10. A preparation method of the material with supercapacitance modified surface according to claim 1, comprising: using metal material as working electrode to electrodeposition; adding graphene oxide into alcoholic aqueous solution as eletro-deposited solution and connecting it with a reference electrode and a counter electrode, electrodeposition with DC to obtain a layer of graphene oxide; hydrothermal treatment of the obtained sample in hydrazine solution to obtain a reduced graphene oxide-metal composite.

11. A sterilization method, comprising using the material with supercapacitance modified surface according to claim 1.

12. The sterilization method according to claim 11, further comprising: charging the material to DC or an AC circuit and interacting with the bacterial cultural solution; the electric charge of charging process is preferably positive charge.

13. The sterilization method according to claim 12, wherein the voltage of the circuit is set referring to the response interval of capacitance, the charging time is 5-180 min, and the time of interaction with the bacterial cultural solution is more than one minute.

14. The sterilization method according to claim 11, wherein the charging sterilization process is carried out for several times, preferably two or more times.

15. The sterilization method according to claim 14, wherein the cyclical sterilization is achieved by converting the mechanical energy from body movement to the electrical energy repeatedly to charging-discharging material.

16. A sterilization method, comprising using the material with supercapacitance modified surface according to claim 2.

17. A sterilization method, comprising using the material with supercapacitance modified surface according to claim 3.

18. A sterilization method, comprising using the material with supercapacitance modified surface according to claim 4.

19. The sterilization method according to claim 12, wherein the charging sterilization process is carried out for several times, preferably two or more times.

20. The sterilization method according to claim 13, wherein the charging sterilization process is carried out for several times, preferably two or more times.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1a shows the SEM (scanning electron microscope) image of titanium dioxide nanotube array (TNT-C-15) with the insets showing the corresponding enlarged and cross-sectional images (scale bar=500 nm), after vacuum annealing at heating rate of 15° C. min.sup.−1 following anodization for 60 min under a scanning electron microscope; and, TNT represents an array of titanium dioxide nanotubes annealed in air.

[0055] FIG. 1b AFM (atomic force microscopy) image of shows the surface and cross section morphology of TNT-C-15 (anodization for 60 min and heating rate of vacuum annealing is 15° C. min.sup.−1).

[0056] FIG. 1c shows the analysis of TNT-C-15 via STEM-EELS (scanning transmission electron microscope electron energy loss spectroscopy) maps (scale bar=50 mn)(anodization for 60 min and heating rate of vacuum annealing is 15° C. min.sup.−1).

[0057] FIG. 1d shows the comparison of XRD patterns of TNT-C-15 and TNT (anodization for 60 min and heating rate of vacuum or air annealing is 15° C. min.sup.−1).

[0058] FIG. 1e shows the high-resolution carbon electron spectra of samples from the surface (heating rate in air is 15° C. min.sup.−1 (TNT), the heating rate of vacuum annealing is 5, 10, 15 and 20° C. min.sup.−1, corresponding to TNT-C-5, TNT-C-10, TNT-C-15 and TNT-C-20, respectively).

[0059] FIG. 1f shows the high-resolution carbon X-ray photoelectron spectra of samples from the surface after sputtering for 6 min with Ar.sup.+ at a sputtering speed of 21 nm min.sup.−1.

[0060] FIG. 2a shows the cyclic voltammetry (CV) curve of the samples annealed at different heating rates (heating rate in air is 15° C. min.sup.−1, and 5, 10, 15 and 20° C. min.sup.−1 in vacuum).

[0061] FIG. 2b shows the curve of the sample at galvanostatic charging-discharging (GCD) plots, annealed at different heating rates (heating rate in air is 15° C. min.sup.−1, and 5, 10, 15 and 20° C. min.sup.−1 in vacuum).

[0062] FIG. 3 shows the SEM image of hydrothermally synthesized zinc oxide nanorods magnetically sputtering with gold for 2 min.

[0063] FIG. 4a shows the CV curve of the capacitive properties of reduced graphene oxide prepared by electrodeposition in combination with hydrothermal method.

[0064] FIG. 4b shows the curves of capacitive properties—GCD of reduced graphene oxide prepared by electrodeposition in combination with hydrothermal method.

[0065] FIG. 5 shows a schematic diagram of charging a TNT-C sample.

[0066] FIG. 6a shows the post-charging antimicrobial rates of charged sample within 20 min (heating rate in air of 15° C. min.sup.−1, and 5, 10, 15 and 20°C. min.sup.−1 in vacuum); wherein P denotes direct current positive charging and N denotes direct current negative charging.

[0067] FIG. 6b shows the post-charging antimicrobial rates of charged sample with 180 min (heating rate in air of 15° C. min.sup.−1, and 5, 10, 15 and 20° C. min.sup.−1 in vacuum).

[0068] FIG. 7 shows the antibacterial rates of TNT-C against Staphylococcus epidermidis and Pseudomonas aeruginosa after charging for 20 min.

[0069] FIG. 8 shows the antibacterial rates of TNT-C-15 which is DC charging-discharging for three times (anodization for 60 min and heating rate of vacuum annealing is 15° C. min.sup.−1).

[0070] FIG. 9 shows the 3D morphology of the fluorescent stained biofilm of TNT-C-15 after DC charging-discharging for 8 times (anodization for 60 min and heating rate of vacuum annealing is 15° C. min.sup.−1); where DC represents direct current.

[0071] FIG. 10 shows the post-charging antimicrobial rates of TNT-C-15 against Escherichia coli and Staphylococcus aureus at AC (anodization for 60 min and heating rate of vacuum annealing is 15° C. min.sup.−1).

[0072] FIG. 11 shows the post-charging antimicrobial rates of sample at 20 min, at different power supply and different charged times (the bacterium used is Escherichia coli); wherein AC represents alternating current; On 0.5 min, On 5 min and On 15 min represent charging for 0.5, 5 and 15 min, respectively.

[0073] FIG. 12 shows the post-charging antimicrobial rates of different ZnO samples against bacteria at 20 min, the time of gold sputtered on the surface of zinc oxide is 0, 2, 4, and 6 min (corresponding to ZnO, ZnO—Au-2, ZnO—Au-4, and ZnO—Au-6, respectively).

[0074] FIG. 13 shows the post-charging antimicrobial rates of the reduced graphene oxide-titanium alloy composite against bacteria at different time points.

DETAILED DESCRIPTION

[0075] Pretreatment of Titanium Alloy and Supercapacitive Modification of Surface

[0076] The titanium alloy is cut to dimension of 30×30×0.5 mm, polished and ground, and ultrasonically cleaned in acetone, ethanol, and water in series for 10 min, and dried with nitrogen for future use.

[0077] The capacitive surface of the material can be designed by anodizing the surface of the titanium alloy to form titanium dioxide nanotube array of diameter of 10 nm to 500 nm; wherein the electrolyte used in anodization is ammonium fluoride (1-10%), methanol (1-10%), deionized water (1-10%) and ethylene glycol (70-95%); the voltage of anodization is 10-100 V; the reaction time is 20-1000 min; the sample is rinsed in 5 mL deionized water for 2 min and dried in nitrogen. The anodized nanotube array is placed into vacuum tube furnace annealing to acquire carbon concentrations (named as TNT-C) which enhances the capacitive properties, the temperature of anneal is 500-800° C., the time is 1-5 h, and the heating rate is 0.1-20° C. min.sup.−1. The capacitance of the material can be quantitatively controlled by heating rate and temperature.

[0078] The capacitive surface of material can also be manufactured by hydrothermal method that growing zinc oxide nanorods doped with gold nanoparticles on the surface of titanium alloy. The specific operations are as follows: (1) Preparation of zinc oxide seed crystal: Weighing zinc acetate, sodium hydroxide and methanol and fully mixing to a 0.001-1 M solution at 50-70° C. for 1-10 h. The above solution is spin-coated on the treated titanium foil at a speed of 500-3000 r/min for 5-30 s to form a wet film, heating at 250 degrees for 5-20 min to volatilize the solvent and pyrolyzation the procedure is repeated 3-5 times, and cooling to acquire titanium foil with crystal seed layer. (2) Growth of zinc oxide nanorods: placing the sample from (1) in reactor of volume of 10-1000 mL, adding 0.001-1 M of 8-800 mL mixed solution of zinc nitrate and hexamethylenetetramine to reactor, sealing the reactor and heating in a muffle furnace at 90-120° C. for 8-48 h. The titanium foil growing with zinc oxide nanorods on the surface obtained from prior step will be ultrasonically cleaned. The gold particles with a particle size of 1-100 nm are sputtered by magnetron sputtering method to obtain the sample having supercapacitive characteristics.

[0079] In addition, the reduced graphene oxide is used as supercapacitive material to modify the titanium alloy. The pretreated titanium foil is sequentially immersed in 10%-30% nitric acid and 1-10 M sodium hydroxide solution for 5 min, washed with deionized water and dried at room temperature used as working electrode for electrodeposition. Contacting a 0.01-1 mg/mL electrodeposition solution of graphene oxide and aqueous ethanol solution (concentration of 10%-80%) with a reference electrode and a counter electrode to electrodeposition at 1-20 V direct current voltage at 40-50° C. for 1-60 min to acquire a graphene oxide layer. It was placed in 4% hydrazine solution and hydrothermally treated at 95° C. for 1 h to obtain a reduced graphene oxide-titanium alloy composite.

[0080] Charging and Sterilization Application of a Supercapacitive Material

[0081] Connecting the carbon-deposited titanium dioxide nanotube-modified titanium alloy above to an electrochemical workstation to test the capacitive property response voltage interval. Then the material is connected to DC or AC (peak-to-peak value of 2-40, frequency of 1 Hz-1 MHz) circuit, the voltage is set with reference to the responsive interval of capacitance (0.1-50 V), charging the capacitor for 5-180 min. Spreading the bacterial culture solution of 10-10.sup.6 CFU mL.sup.−1 on the surface of post-charging material. After waiting for 1-180 min, the bacteria is cultured on agar plate and the physiological activity is measured, to analyze the antimicrobial effect.

Embodiment 1

[0082] The titanium foil having a dimension of 30×30×0.5 mm is polished, grounded, and ultrasonically cleaned in acetone, alcohol, and deionized water in series. Connecting sample to positive electrode of the DC power supply to anodization, the electrolyte of anodization comprises ammonium fluoride (5.5%), methanol (5%), deionized water (5%) and ethylene glycol (70-90%), the voltage of anodization is 60 V, the time of anodization is 60 min, and the obtained sample is rinsed in 5 mL water for 2 min, dried in nitrogen. The anodized nanotube array is placed in a vacuum tube furnace annealing to obtain deposited carbon, which improves the rate of electron transfer of semiconductor titanium dioxide and reduces the rate of neutralization of positive and negative charges, increasing the specific surface area to enhance the capacitive properties, the temperature of anneal is 500° C., the time is 3 h, and the heating rate is 15° C. min.sup.−1, the sample annealed in air under the same conditions is conducted as control group with no deposited carbon. The SEM image of the microscopic morphology of sample surface is shown in FIG. 1a. As shown in FIG. 1a, the anodized titanium dioxide nanotubes has an outer diameter of 160 nm, wall thickness of 25 nm, and nanotubes length of 10 μm. Similar results are shown in AFM image (FIG. 1b). Comparing with titanium dioxide nanotubes annealed in air, the titanium dioxide nanotubes annealed in argon do not cause a significant change in morphology. It can be concluded that deposited carbon did not cause a significant change in the microscopic morphology to titanium dioxide nanotube array.

Embodiment 2

[0083] The surface of the sample obtained from Embodiment 1 is tested by elemental content analysis. The STEM-EELS maps indicate carbon was evenly precipited on the wall of titanium dioxide nanotube (shown in FIG. 1c). The main peak of anatase crystalline titanium dioxide (2θ=25.3° (101), 48.0° (200), and 70.3° (220)) is shown in XRD patterns (FIG. 1d). Further researches of X-ray photoelectron spectra (XPS) revealed that the distribution pattern of carbon elements on the surface of the sample is dominated by C-C bond (FIG. 1e), but the C—Ti bond turns to the dominant way after sputtering for 6 min (FIG. 1f), which can be explained by that the carbon partially substitutes oxygen in titanium dioxide and precipitating evenly. The elemental analysis results above indicate the titanium dioxide nanotube array with uniformly distributed carbon was prepared.

Embodiment 3

[0084] Capacitance analysis of the prepared sample is carried out by electrochemical workstation. It can be detected obviously under 15° C. min.sup.−1 annealing condition that the sample has electric double layer capacitive properties(FIG. 2a), and the sample can accumulate more charges under 15° C. min.sup.−1 annealing condition (FIG. 2b), indicating that there can have more electron transfer when antibacterial was carried out thereafter.

Embodiment 4

[0085] Titanium foil having a dimension of 30×30×0.5 mm is polished, grounded, and ultrasonically cleaned in acetone, alcohol, and deionized water in series. 0.219 g of zinc acetate, 0.12 g of sodium hydroxide and 100 mL of methanol are weighed to formulate a mixed solution, which is stirred at 60° C. for 2 h to mix well. The mixed solution is spin-coated on the treated titanium foil at a speed of 3000 r/min for 20 s to obtain a wet film, heating it at 250 degrees for 5 min to volatilize the solvent and pyrolyzation, this procedure repeats 3 times, cooling to acquire titanium foil with a crystal seed layer. Placing sample into a reactor of 20 mL volume, prepare 10 mL of mixed solution of zinc nitrate and hexamethylenetetramine with a concentration of 100 μM, add the mixed solution to the reactor. Sealing the reactor and place it into a muffle furnace at 90° C. for 10 h. The sample is taken out and the obtained titanium foil growing with zinc oxide nanorods is ultrasonically cleaned for 10 s. The gold particles are sputtered on surface of sample by magnetron sputtering method for 2 min to acquire a capacitive characteristic. the SEM image of sample is shown in FIG. 3.

Embodiment 5

[0086] The ground and cleaned titanium foil is sequentially immersed in 20% nitric acid and 5 M sodium hydroxide solution for 5 min, washed in deionized water and dried at room temperature used as a working electrode to electrodeposition. Adding graphene oxide to 30% aqueous ethanol solution to prepare a concentration of 0.3 mg/mL of electrodeposition solution, connecting with a reference electrode and a counter electrode to electrodeposition at direct current voltage of 10 V at 40° C. for 20 min to obtain a graphene oxide layer. Putting it into 4% hydrazine solution and hydrothermally treating at 95° C. for 1 h to obtain a reduced graphene oxide-titanium alloy composite which are connected to electrochemical workstation to analyze its capacitive characteristics, the figures of CV curve and curve of GCD are shown in FIGS. 4a and 4b.

Embodiment 6

[0087] The sample obtained in Embodiment 1 is subjected to DC charging, the charging voltage is 2 V, and the charging time is 20 min. The schematic diagram of charging is shown in FIG. 5.

Embodiment 7

[0088] The fully charged sample in Embodiment 6 is taken out and applied to the antibacterial (Staphylococcus aureus and Escherichia coli) test. The antimicrobial effect is evaluated by the coated plate counting method and the results are shown in FIG. 6. For samples with larger capacitance, Higher sterilization rate can be achieved after fully charged. For example, about 80% and about 70% of sterilization rates to Escherichia coli and Staphylococcus aureus can be achieved at 15° C. min.sup.−1 when interacted with bacteria for 20 minutes after fully charged (FIG. 6a). Extending the interaction time of the material to the bacteria for up to 180 min did not significantly increase the antibacterial effect (FIG. 6b), indicating that the antibacterial process occurred at early stage of the contact. In addition, the sterilizing efficiency of the positively charged surface of the sample is significantly higher than that of the negatively charged ones.

Embodiment 8

[0089] The antibacterial operation in Embodiment 7 is applied to two other bacteria (Pseudomonas aeruginosa and Staphylococcus epidermidis) to further verify its antibacterial effect, and the results showed that TNT-C-15 could realize about 75% and about 45% of antibacterial effects against Pseudomonas aeruginosa and Staphylococcus epidermidis within 20 min after being positively charged (FIG. 7). Compared with the antibacterial results in Embodiment 7, it can be seen that the antibacterial system based on the supercapacitor material has a significantly higher antibacterial effect against Gram-negative bacteria than Gram-positive bacteria.

Embodiment 9

[0090] In order to improve the antibacterial efficiency, the bacteria are collected after sterilizing for 20 minutes in Embodiment 7, and the sample is recharged (positively charged), and then the collected bacteria are added to the surface of the material for secondary sterilization, and the antibacterial results are shown in FIG. 8. The results showed that the sterilization rate of the four bacteria can be increased to about 90% in the second cycle charging process, and the antibacterial rate of greater than 90% can be achieved after three cycles of charging.

Embodiment 10

[0091] The bacteria on the material after sterilization for 20 min in Embodiment 7 are cultured in a bacterial culture medium at 37° C., the material is charged every 6 h, and co-cultured to 48 h. The formation of biofilm is observed by fluorescent staining method, as shown in FIG. 9. A strong biofilm is formed on the uncharged titanium dioxide nanotubes. The biofilm is also formed on the DC-charged titanium sheets but the thickness is significantly lower than that of the uncharged titanium dioxide group. Died bacteria could be obviously detected on the charged and discharged titanium dioxide and carbon deposited titanium dioxide and no continuous biofilm is formed thereon, these results proved that the titanium alloy based on supercapacitor material can effectively inhibit the formation of biofilm during charge and discharge, and the suppression effect is positively correlated with the capacitance.

[0092] It is confirmed by experiments that the titanium dioxide nanotube array with diameter of 160 nm is prepared by Redox method , which is annealed in argon (annealing temperature of 500° C., annealing time of 3 h, and heating rate of 15° C. min.sup.−1) to obtain the carbon deposited titanium dioxide nanotube array, which has supercapacitor characteristics. It is charged with a DC power source (2 V) for 15 min, and a sterilization rate of more than 80% can be achieved within 20 min, and more than 90% of sterilization rate can be achieved after three cycles of charging and formation of biofilm can be effectively inhibited. in the body, after the bacteria breed, it is often easy to form a biofilm with extracellular polymer matrix, specific structure and stronger resistance, which can cause serious postoperative infection, the anti-biofilm efficacy of the present disclosure can significantly reduce the risk of postoperative infection.

Embodiment 11

[0093] The TNT-C-15 sample in Embodiment 1 is subjected to AC charging with a voltage peak-to-peak value of 2 V, a frequency of 50 Hz, and a charging time of 15 min. The fully charged sample is taken out and applied to the antibacterial (Staphylococcus aureus and Escherichia coli) test. The antibacterial effect is evaluated by the coated plate counting method, and the results are shown in FIG. 10. The results showed that about 80% and 60% of antibacterial rate against E. coli and S. aureus can be achieved during the 15 min charging period. After de-energized, more than 40% of antibacterial rate could still be achieved within 20 min and 180 min when contacting with the bacteria. It is indicated that the alternating current can charge the material in the present disclosure to achieve a sterilization effect by using its capacitance.

Embodiment 12

[0094] The sample in Embodiment 1 is charged (the AC-DC parameters are the same as above) for different periods of time to obtain samples carrying different charge densities, and then the sample is contacted with E. coli (its concentration is the same as above), and the sterilization effect within 20 min is judged by the coated plate counting method. The results are as shown in FIG. 11. For samples treated with AC and DC positively, the sample can achieve a higher sterilization rate within 20 min as the charging time is extended. This result indicated that the longer the charging time of the material with capacitive properties, the more charges are accumulated on the surface, and the higher is the sterilization efficiency

Embodiment 13

[0095] The ZnO sample obtained in Embodiment 4 is subjected to DC charging, the charging voltage is 2 V, and the charging time is 20 min. The schematic diagram of charging is shown in FIG. 5 (the TNT-C sample is replaced with a ZnO sample). The fully charged sample is taken out and applied to the antibacterial (Staphylococcus aureus and Escherichia coli) test. The antibacterial effect is evaluated by the coated plate counting method, and the results are shown in FIG. 12. For samples with larger capacitance, higher sterilization rate can be achieved after fully charged. For example, ZnO—Au-6, about 90% and about 80% of sterilization rates to Escherichia coli and Staphylococcus aureus can be achieved when interacted with bacteria for 20 minutes after fully charged (FIG. 12).

Embodiment 14

[0096] The sample obtained in Embodiment 5 is subjected to DC charging, the charging voltage is 1.5 V, and the charging time is 20 min. The schematic diagram of charging is shown in FIG. 5 (the TNT-C sample is replaced with a reduced graphene oxide-titanium alloy composite sample). The fully charged sample is taken out and applied to the antibacterial (Staphylococcus aureus and Escherichia coli) test. The antibacterial effect is evaluated by the coated plate counting method. The results are shown in FIG. 13. The bactericidal effect is gradually increased during the first 20 min of the interaction of the sample with the bacteria after charging, and the bactericidal effect is slowly increased within the treatment time of 20-360 min, and finally more than 90% of the sterilization rate can be achieved.