CN110151994A - A kind of bacteriophage and its application in the light power preparation for preparing inactivation of bacterial - Google Patents
A kind of bacteriophage and its application in the light power preparation for preparing inactivation of bacterial Download PDFInfo
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- CN110151994A CN110151994A CN201910480938.0A CN201910480938A CN110151994A CN 110151994 A CN110151994 A CN 110151994A CN 201910480938 A CN201910480938 A CN 201910480938A CN 110151994 A CN110151994 A CN 110151994A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
- C12N2795/10121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
- C12N2795/10132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/14011—Details ssDNA Bacteriophages
- C12N2795/14111—Inoviridae
- C12N2795/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/14011—Details ssDNA Bacteriophages
- C12N2795/14111—Inoviridae
- C12N2795/14132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
Abstract
The present invention discloses a kind of bacteriophage and its application in the light power preparation for preparing inactivation of bacterial, is related to a nanometer new material technology field.The surface of the bacteriophage is connected with photosensitizer by weak interaction, the weak interaction can interact for electrostatic interaction or hydrophobe, pass through the specificity interaction between bacteriophage and bacterium, allow certain selectively targeted bacterium of photosensitizer, the invention also discloses the bacteriophages of photosensitizer attachment can be applied to prepare in the light power preparation of inactivation of bacterial.
Description
Technical field
The present invention relates to nanometer new material technology fields.It is inactivated more particularly, to a kind of bacteriophage and its in preparation thin
Application in the light power preparation of bacterium.
Background technique
Bacterium infection poses a serious threat human health, and the generation of bacterial drug resistance caused by the abuse of antibiotic makes this
One problem becomes more serious.Due to Noninvasive, high space-time is selective the features such as, light dynamic pasteurization causes extensive pass
Note.Light dynamic pasteurization is made of photosensitizer, light source and oxygen three parts, these three components are all nontoxic under individualism.
Photosensitizer can be by energy or electron transmission to oxygen, so as to generate reactive oxygen species, such as singlet under light source irradiation
Oxygen (1O2), superoxide anion (O2 ·-) and hydroxyl radical free radical (OH) etc..Reactive oxygen species have the oxidisability of height, can
With to many important biomolecule, such as phospholipid bilayer, albumen, DNA etc. forms irreversible damage.Therefore light power
Treatment can effectively kill bacterium, including drug-fast bacteria.At the same time, light dynamic pasteurization is also not easy that bacterium is made to generate drug resistance.
But photosensitizer usually lacks targeting to bacterium, this can cause serious side effect to normal mammalian cell.Compared to leather
Lan Shi positive bacteria, the presence of Gram-negative bacteria outer membrane weaken light dynamic pasteurization effect significantly.
Make photosensitizer specificity targeted bacteria, is a critical issue in light dynamic pasteurization.Usual way includes: with receiving
Such as, polymer or MOF etc. wrap up photosensitizer to meter Zai Ti, so that it is discharged (Mao D, et in specific site by stimulating responsive
al.Metal–Organic-Framework-assisted in vivo bacterial metabolic labeling and
precise antibacterial therapy.[J]Adv Mater 2018,2018,1706831.).The load that this method uses
Body may be such that its biological safety declines due to non-degradable etc..Another method is by the segment of targeted bacteria, such as
Sugar, polypeptide etc. carry out covalent cross-linking with photosensitizer, make its targeted bacteria (Xiao F, et al.Pathogen-specific
polymeric antimicrobials with significant membrane disruption and enhanced
photodynamic damage to inhibit highly opportunistic bacteria.[J]ACS Nano
2019,13,1511.).This method preparation step is relative complex, and the introducing of organic solvent also will increase its life in chemical synthesis
Object toxicity.
Bacteriophage (phage) is the virus for invading bacterium, and main component is protein and nucleic acid, has bio-safety
Property, it is nontoxic to humans and animals.Nature pnagus medius exists extensively, is easy to obtain.Due to the osculum and receptor of bacteriophage
Bacterium surface receptor molecule has complementarity, and bacteriophage has stringent host specificity.In this patent, we pass through weak phase interaction
With the phage surface that photosensitizer is adsorbed in targeting Gram-negative bacteria, such as M13.Pass through the spy between bacteriophage and bacterium
Opposite sex interaction, allows certain selectively targeted bacterium of photosensitizer.Compared to the method reported before, this method targeting
By force, simple and easy, biological safety is high.
Summary of the invention
The purpose of the present invention is to provide a kind of bacteriophages that bacterium is killed by photodynamic therapy.
In order to achieve the above objectives, the present invention adopts the following technical solutions:
A kind of bacteriophage of photosensitizer attachment, the surface of the bacteriophage is connected with photosensitizer by weak interaction.
Furthermore it is preferred that scheme be that the weak interaction is that electrostatic interaction or hydrophobe interact.
Furthermore it is preferred that scheme be, the bacteriophage be target Gram-negative bacteria bacteriophage；
Furthermore it is preferred that scheme be that the bacteriophage is M13 bacteriophage, any one in T1-T7 bacteriophage.
Furthermore it is preferred that scheme be, the bacteriophage be target E. coli bacteriophage.
Furthermore it is preferred that scheme be, the photosensitizer be Porphyrin-Based Sensitizer, phthalocyanines photosensitizer, porphin photosensitizer
With any one in aggregation-induced emission property photosensitizer；
Furthermore it is preferred that scheme be, the aggregation-induced emission property photosensitizer be TPE base photosensitizer；
Furthermore it is preferred that scheme be, the Porphyrin-Based Sensitizer be haematoporphyrin, Photofrin or Photogem；
Furthermore it is preferred that scheme be, the phthalocyanines photosensitizer be ZnPc, Photosens, Pc4 or CGP55847；
Furthermore it is preferred that scheme be, the porphin photosensitizer be Ce6, Foscan, Aptocine or Laserphyrin.
Furthermore it is preferred that scheme be, when the photosensitizer is positively charged photosensitizer, the phagocytosis of photosensitizer attachment
The preparation method of body the following steps are included:
Bacteriophage and positively charged photosensitizer are mixed, outer surface must be arrived by electrostatic interaction and be adsorbed with photosensitizer
Bacteriophage.
Furthermore it is preferred that scheme be, when the photosensitizer is hydrophobic photosensitizer, the phagocytosis of photosensitizer attachment
The preparation method of body the following steps are included:
Thermo-sensitive hydrophobic molecule is modified in phage surface；
The bacteriophage for being modified with Thermo-sensitive hydrophobic molecule and hydrophobic photosensitizer are mixed, Thermo-sensitive is adjusted the temperature to and dredges
The phase transition temperature of hydrone makes Thermo-sensitive hydrophobic molecule be changed into hydrophobic by hydrophilic, wraps up the photosensitizer.
Furthermore it is preferred that scheme be, the Thermo-sensitive hydrophobic molecule be class elastin laminin ELP；Bacteriophage and photosensitizer phase
When mixing, 37 DEG C are adjusted the temperature to.
Furthermore it is preferred that scheme be the ratio that the bacteriophage and photosensitizer are 1:0.5-1:50 according to the ratio between amount of substance
Example mixing.
The present invention also provides application of the bacteriophage as described above in the light power preparation for preparing inactivation of bacterial.
Beneficial effects of the present invention are as follows:
This patent by weak interaction by photosensitizer be adsorbed in can targeted bacteria phage surface, by bacteriophage with
Specificity interaction between bacterium, allows certain selectively targeted bacterium of photosensitizer, so by photodynamic therapy into
Row efficient sterilizing.Compared with prior art, the targeting of this method is strong, simple and easy, and biological safety is high, and sterilization effect is good.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 shows the TEM electron microscope of 1 pnagus medius M13 of the embodiment of the present invention.
Fig. 2 shows E.coli in test example 1 of the present invention by TPE-Pt-MC@M13 treated SEM electron microscope.
Specific embodiment
Since photosensitizer lacks targeting in the light dynamic pasteurization of the prior art, normal mammalian cell will cause sternly
The side effect of weight；And compared to gram-positive bacteria, the presence of Gram-negative bacteria outer membrane makes the effect of light dynamic pasteurization significantly
It reduces.It, should if in the carrier by photosensitizer package, discharging photosensitizer by stimulating responsive specific site such as polymer or MOF
The carrier organism safety that method uses is low, not degradable；If in the segment of photosensitizer surface grafting targeted bacteria, such as sugar or more
Peptide etc., for enhancing photosensitizer to the targeting of bacterium, this method step is complicated, and introducing organic solvent can make during chemical synthesis
Biological safety reduces.
Based on problems of the prior art, the present invention provides a kind of bacteriophage of photosensitizer attachment, the bacteriophage
Surface photosensitizer is connected with by weak interaction, using specificity interaction strong between bacteriophage and bacterium, make photosensitive
The ability of agent acquisition targeted bacteria.
In the present invention preferably, the weak interaction is that electrostatic interaction or hydrophobe interact.Both are weak
The forming process of interaction is simple and easy, and photosensitizer can be effectively attached to the surface of bacteriophage, makes bacteriophage can be with
Bacterium is effectively killed by photodynamic therapy, in this self assembling process, it is organic molten that additional chemical synthesis can't be introduced
Agent will not generate bio-toxicity.Those skilled in the art in the actual operation process, also can use design provided by the invention
Thinking, according to the concrete type of photosensitizer and bacteriophage, using metal coordination, host-guest interaction, receptors ligand phase
The type of other weak interactions such as interaction.
Compared to gram-positive bacteria, the presence of Gram-negative bacteria outer membrane substantially reduces the effect of light dynamic pasteurization.
In the present invention preferably, the bacteriophage is the bacteriophage for targeting Gram-negative bacteria, and further, the bacteriophage is targeting
The bacteriophage of E. coli；The bacteriophage is M13 bacteriophage, any one in T1-T7 bacteriophage.
In the preferred embodiment of the present invention, the photosensitizer is Porphyrin-Based Sensitizer, phthalocyanines photosensitizer, porphines class light
Any one in quick dose and aggregation-induced emission property photosensitizer.TPE-Pt-MC in aggregation-induced emission property photosensitizer
Per se with positive charge, the surface of bacteriophage can be attached to by electrostatic interaction, this method step is simple, without additional
The addition of organic solvent will not generate side effect.
Preferably, the aggregation-induced emission property photosensitizer is TPE base photosensitizer, such as TPE-Pt-MC；The porphyrin
Photosensitizer is haematoporphyrin, Photofrin or Photogem；The phthalocyanines photosensitizer be ZnPc, Photosens, Pc4 or
CGP55847；The porphin photosensitizer is Ce6, Foscan, Aptocine or Laserphyrin.
In a preferred embodiment of the invention, when the photosensitizer is positively charged photosensitizer (such as TPE-Pt- itself
When MC), the weak interaction between the photosensitizer and bacteriophage can be electrostatic interaction, at this point, photosensitizer attachment
The preparation method of bacteriophage passes through electrostatic the following steps are included: bacteriophage and aggregation-induced emission property photosensitizer are mixed
Interact to be adsorbed with to outer surface the bacteriophage of photosensitizer.It is further preferred that the bacteriophage and photosensitizer are according to object
The ratio that the ratio between amount of matter is 1:0.5-1:50 mixes.
When the weak interaction between photosensitizer and bacteriophage is hydrophobe interaction, the photosensitizer is hydrophobic light
When quick dose (such as ZnPc, Ce6, haematoporphyrin), the preparation method of the bacteriophage of photosensitizer attachment the following steps are included:
Thermo-sensitive hydrophobic molecule is modified in phage surface；The bacteriophage of Thermo-sensitive hydrophobic molecule and hydrophobic will be modified with
Photosensitizer mixes, and adjusts the temperature to the phase transition temperature of Thermo-sensitive hydrophobic molecule, makes Thermo-sensitive hydrophobic molecule by hydrophilic transformation
Be it is hydrophobic, wrap up the photosensitizer.It is further preferred that the bacteriophage and photosensitizer are 1:0.5- according to the ratio between amount of substance
The ratio of 1:50 mixes；The actual temp of adjusting is determined according to the difference of Thermo-sensitive hydrophobic molecule type, for example, working as
When M13 phage surface modifies class elastin laminin (ELP), temperature-adjustable is to 37 DEG C.
The present invention also provides application of the bacteriophage as described above in the light power preparation for preparing inactivation of bacterial, Ke Yitong
It crosses and selects different bacteriophage types, kill different types of bacterium using photodynamic therapy.
In order to illustrate more clearly of the present invention, below with reference to preferred embodiment, the present invention is described further.Ability
Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, this should not be limited with this
The protection scope of invention.
Embodiment 1
M13 bacteriophage is obtained by the method for the prior art, TEM photo is as shown in Fig. 1.It is by the ratio between the amount of substance
The M13 bacteriophage of 1:2 and TPE base photosensitizer (TPE-Pt-MC) with aggregation-induced emission property mix, and pass through electrostatic phase
Interaction obtains the M13 bacteriophage of outer surface absorption photosensitizer TPE-Pt-MC, i.e. TPE-Pt-MC@M13.By TPE-Pt-MC@
Bacterium M13 selectively targeted for M13, carries out antibacterial experiment.
Embodiment 2
T2 bacteriophage is obtained by the method for the prior art, the ratio between amount of substance for the T2 bacteriophage of 1:1 and is had poly-
The TPE base photosensitizer (TPE-Pt-MC) of collection induced luminescence property mixes, and obtains outer surface by electrostatic interaction and adsorbs light
The T2 bacteriophage of quick dose of TPE-Pt-MC, i.e. TPE-Pt-MC@T2.TPE-Pt-MC@T2 is used for the selectively targeted bacterium of T2,
Carry out antibacterial experiment.
Embodiment 3
M13 bacteriophage is obtained by the method for the prior art, class elastin laminin (ELP) is modified in M13 phage surface, obtains
To ELP-M13.The polypeptide has Thermo-sensitive, i.e., as temperature raising can occur by hydrophilic to hydrophobic transformation.By substance
The ratio between amount mixes at 37 DEG C with photosensitizer ZnPc for the ELP-M13 bacteriophage of 1:50, interacts to obtain by hydrophobe
Adsorb the M13 bacteriophage of photosensitizer ZnPc, i.e. ZnPc@M13 in outer surface.ZnPc@M13 is used for the selectively targeted bacterium of M13,
Carry out antibacterial experiment.
Test example 1
The TPE-Pt-MC@M13 that embodiment 1 is prepared is used for Escherichia coli, carries out antibacterial experiment.
Antibacterial experiment step:
(1) single E.coli bacterium colony is chosen into 50mL pancreas medium and soy protein medium, is trained overnight in 37 DEG C of 200rpm shaking tables
It supports.
(2) by the bacterium solution of overnight incubation 10mM pH 7.4K2HPO4-KH2PO4Buffer solution dilution certain multiple obtains
105The bacterium solution of CFU/mL.
(3) take the bacteriophage (TPE-Pt-MC@M13) of absorption photosensitizer (concentration is 20 μM of TPE-Pt-MC+10 μM of M13)
With 105The bacterium solution of CFU/mL mixes, and interact 15min in dark.
(4)20mW/cm2After 420nm light source (such as LED light, laser etc.) irradiation interacts with TPE-Pt-MC@M13
Bacterium solution, time 10-20min.Suitable illumination wavelength is selected according to the UV absorption of photosensitizer, such as TPE-Pt-MC@M13
Illumination wavelength be 420nm.
(5) bacterium solution after 100 μ L illumination is drawn, is placed on MH solid medium, after even spread, back-off is placed on 37 DEG C
In incubator, counted after staying overnight.
Treated that SEM picture is as shown in Fig. 2 by TPE-Pt-MC@M13 by E.coli, and bacterial membrane rupture can be observed.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention may be used also on the basis of the above description for those of ordinary skill in the art
To make other variations or changes in different ways, all embodiments can not be exhaustive here, it is all to belong to this hair
The obvious changes or variations that bright technical solution is extended out are still in the scope of protection of the present invention.
Claims (10)
1. a kind of bacteriophage of photosensitizer attachment, which is characterized in that the surface of the bacteriophage is connected with by weak interaction
Photosensitizer.
2. bacteriophage according to claim 1, which is characterized in that the weak interaction is electrostatic interaction or close and distant
Water phase interaction.
3. bacteriophage according to claim 1, which is characterized in that the bacteriophage is the phagocytosis for targeting Gram-negative bacteria
Body；Preferably, the bacteriophage is M13 bacteriophage, any one in T1-T7 bacteriophage.
4. bacteriophage according to claim 1, which is characterized in that the bacteriophage is to target biting for E. coli
Thallus.
5. bacteriophage according to claim 1, which is characterized in that the photosensitizer is Porphyrin-Based Sensitizer, phthalocyanines light
Any one in quick dose, porphin photosensitizer and aggregation-induced emission property photosensitizer；
Preferably, the aggregation-induced emission property photosensitizer is TPE base photosensitizer；
Preferably, the Porphyrin-Based Sensitizer is haematoporphyrin, Photofrin or Photogem；
Preferably, the phthalocyanines photosensitizer is ZnPc, Photosens, Pc4 or CGP55847；
Preferably, the porphin photosensitizer is Ce6, Foscan, Aptocine or Laserphyrin.
6. bacteriophage according to claim 1, which is characterized in that when the photosensitizer is positively charged photosensitizer, institute
State photosensitizer attachment bacteriophage preparation method the following steps are included:
Bacteriophage and positively charged photosensitizer are mixed, outer surface must be arrived by electrostatic interaction and be adsorbed with biting for photosensitizer
Thallus.
7. bacteriophage according to claim 1, which is characterized in that when the photosensitizer is hydrophobic photosensitizer, institute
State photosensitizer attachment bacteriophage preparation method the following steps are included:
Thermo-sensitive hydrophobic molecule is modified in phage surface；
The bacteriophage for being modified with Thermo-sensitive hydrophobic molecule and hydrophobic photosensitizer are mixed, adjust the temperature to hydrophobic point of Thermo-sensitive
The phase transition temperature of son makes Thermo-sensitive hydrophobic molecule be changed into hydrophobic by hydrophilic, wraps up the photosensitizer.
8. bacteriophage according to claim 7, which is characterized in that the Thermo-sensitive hydrophobic molecule is class elastin laminin ELP.
9. the described in any item bacteriophages of according to claim 6 or 7, which is characterized in that the bacteriophage and photosensitizer are according to object
The ratio that the ratio between amount of matter is 1:0.5-1:50 mixes.
10. application of the bacteriophage as described in claim 1 in the light power preparation for preparing inactivation of bacterial.
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| CN113171470A (en) * | 2020-01-24 | 2021-07-27 | 香港科技大学 | Aggregation-induced emission molecule-phage bioconjugate |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113171470A (en) * | 2020-01-24 | 2021-07-27 | 香港科技大学 | Aggregation-induced emission molecule-phage bioconjugate |
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| CN111956817A (en) * | 2020-08-10 | 2020-11-20 | 武娟 | Indoor environment photodynamic broad spectrum sterilization and disinfection method |
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| CN110151994B (en) | 2021-07-27 |
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