CN108753154A - 利用羧基修饰的碳纳米管抑制聚二甲基硅氧烷表面生物膜中真核生物附着生长的方法 - Google Patents
利用羧基修饰的碳纳米管抑制聚二甲基硅氧烷表面生物膜中真核生物附着生长的方法 Download PDFInfo
- Publication number
- CN108753154A CN108753154A CN201810485784.XA CN201810485784A CN108753154A CN 108753154 A CN108753154 A CN 108753154A CN 201810485784 A CN201810485784 A CN 201810485784A CN 108753154 A CN108753154 A CN 108753154A
- Authority
- CN
- China
- Prior art keywords
- cmwnts
- pdms
- eucaryote
- carboxyl modified
- stained
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D183/00—Coating compositions based on macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon, with or without sulfur, nitrogen, oxygen, or carbon only; Coating compositions based on derivatives of such polymers
- C09D183/04—Polysiloxanes
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
- C09D5/16—Antifouling paints; Underwater paints
- C09D5/1656—Antifouling paints; Underwater paints characterised by the film-forming substance
- C09D5/1662—Synthetic film-forming substance
- C09D5/1675—Polyorganosiloxane-containing compositions
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
- C09D5/16—Antifouling paints; Underwater paints
- C09D5/1687—Use of special additives
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K2201/00—Specific properties of additives
- C08K2201/002—Physical properties
- C08K2201/006—Additives being defined by their surface area
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Materials Engineering (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Paints Or Removers (AREA)
Abstract
本发明属于生物技术领域,涉及一种利用羧基修饰的多壁碳纳米管(carboxyl modified multiwall carbon nanotubes,cMWNTs)作为纳米添加剂抑制聚二甲基硅氧烷(Polydimethylsiloxane,PDMS)表面生物膜中附着污损真核生物生长的方法。通过将不同种类cMWNTs引入PDMS成膜体系,制备不同种类cMWNTs‑PDMS复合涂层,利用cMWNTs添加剂有效降低和改变PDMS表面附着污损真核生物群落的多样性水平及丰度,抑制污损真核生物的生长。
Description
技术领域
本发明涉及一种利用不同碳纳米管(carbon nanotubes,CNTs)抑制PDMS表面真核生物生长的方法。应用菌群结构或群落结构的测定方法检测污损真核生物群落结构,并从分子生态学角度从群落整体水平上评价不同CNTs-PDMS复合涂层表面污损真核生物群落结构动态变化。本发明属于生物技术领域。
背景技术
海洋生物污损给全世界海洋产业的发展带来严重危害,极大程度上限制了人类对于海洋资源的开发和利用。据统计,全世界每年由海洋生物污损造成的船体自重增加会使燃油消耗增加40%,整体航运成本增加77%。每年由海洋生物污损造成的经济损失高达300-500亿美元。研究表明,如果没有采取任何防污策略,半年内船体上附着的污损生物即可以达到100-150kg/m2。除海洋工程领域外,生物污损也大量存在于其他领域。
聚二甲基硅氧烷(Polydimethylsiloxane,PDMS)是目前颇具吸引力的低表面能高分子聚合物之一。向PDMS及其衍生物的成膜体系中加入不同种类纳米粒子是增强其防污性能和机械性能的有效方法。鉴于CNTs材料的独特的分子结构特征,良好的化学稳定性,优良的机械性能和热力学性能,是PDMS基涂层理想的纳米添加剂之一。
很多研究表明,海洋生物膜与后期大型海洋污损生物幼虫的附着存在密切联系,生物膜的形成是后期大型生物幼虫附着的前提条件。从这一角度来说,生物膜因此被认为是大型污损生物的附着关键的介质。几乎所有的海洋生物污损的核心问题都与生物膜的动力过程有关。因此抑制海洋生物污损的有效途径之一就是控制涂层表面生物膜的组成及动态变化过程。海洋真核(微) 生物群落对于污损后期大型污损生物幼虫的定性附着具有重要影响。因此,研究不同材料表面海洋真核(微)生物群落结构和动态变化非常重要。可以通过涂层表面生物膜中分子生态学参数变化对其中真核(微)生物群落的变化特征进行表征。
群落生态学参数测定依赖于各种菌群结构或群落结构的解析技术。传统的微生物分析测定技术,如选择性培养基计数、纯种分离等,由于方法本身的局限性,很难真实准确地反映出复杂微生物种群中细菌的种类和数量,近年发展起来的分子生物学技术已被用于这一问题的解决,相关的技术利用不同微生物核糖体RNA基因遗传信息或特殊功能基因来进行微生物种类的鉴定和定量分析。在上世纪九十年代以来,基因克隆文库、聚合酶链式反应(Polymerase Chain Reaction, PCR)、梯度凝胶电泳(Denaturing/TemperatureGradient Gel Electrophoresis,DGGE/TGGE)、荧光原位杂交(Fluorescence in SituHybridization,FISH)、Real-Time PCR定量测定技术、单链构象多态性技术(SingleStrand Comformation Polymorphism,SSCP)等分子生物学手段,尤其是高通量DNA测序技术都可以应用于微生物菌群结构或包含微生物和真核生物的生物群落的组成测定。Denovo sequencing技术直接把群落中的所有宏基因组进行提取和后续操作,可以直接测定所有生物的基因组序列,进而对群落中真核原核等不同生物统一进行分子生态学的测定和表征。
群落生物多样性是包括物种多样性、遗传(基因)多样性、生态系统多样性以及功能多样性。它不仅反映了生物多样性水平和分布稳定性,而且是监测对环境变化和胁迫的反应的重要指标。生物种群结构分子生态学参数主要Shannon diversity index,Speciesrichness,Total abundance等。对生物多样性的分析能够从群落整体水平进一步揭示不同生存条件下生物数量和功能上的差异以及生物群落动态变化规律。
发明内容
本发明的目的在于应用不同CNTs抑制PDMS表面真核生物(不限于真核微生物)群落生长。利用菌群结构或群落结构测定方法检测不同CNTs-PDMS复合涂层表面附着真核(微)生物群落结构。本发明不依赖传统的分离培养,利用真核(微)生物ITS(Internaltranscribed spacer) 等通用引物扩增核糖体RNA编码基因,分析不同CNTs-PDMS复合涂层表面污损样品中海洋污损真核(微)生物群落结构。也可以使用其他方法如高通量测序来解析真核生物群落结构。利用生物群落分子生态学参数从群落整体水平揭示不同CNTs-PDMS复合涂层表面真核(微)生物群落结构动态变化。
本发明的技术方案包括:不同CNTs-PDMS复合涂层的制备;定期采集污损生物膜样品;生物膜生物群落结构测定,比如(不限于)利用PCR(polymerase chain reaction)试剂盒扩增真核 (微)生物核糖体RNA基因,继而检测不同取样时间不同CNTs-PDMS复合涂层表面附着污损真核(微)生物群落结构;依据群落结构测定结果对不同表面污损真核(微)生物群落进行结构分析。
本发明的目的在于应用不同CNTs抑制PDMS涂层表面真核(微)生物群落生长,对于不同 CNTs-PDMS复合涂层抑制生物污损之分子机制的探索提供新的视角和应用基础。
附图说明
图1CNTs-PDMS涂层表面污损真核微生物SSCP指纹。
M11指的是M1-PDMS涂层的第1次取样(2d),M12指的是M1-PDMS涂层的第2次取样(5d),M13 指的是M1-PDMS涂层的第3次取样(8d),M15指的是M1-PDMS涂层的第5次取样(14d),以此类推。C1~C5指的是不添加纳米材料的PDMS对照样本。
图2不同CNTs-PDMS涂层表面污损真核微生物Shannon diversity index均值变化。
注:误差线代表标准差。*代表显著水平(P<0.05),**代表极显著水平(P<0.01)
图3不同CNTs-PDMS涂层表面污损真核微生物Species richness均值变化。
注:误差线代表标准差。*代表显著水平(P<0.05),**代表极显著水平(P<0.01)
图4不同CNTs-PDMS涂层表面污损真核微生物Total abundance均值变化。
注:误差线代表标准差。*代表显著水平(P<0.05),**代表极显著水平(P<0.01)
具体实施方式
实施案例1.不同种类CNTs-PDMS复合涂层的制备:
PDMS涂层的制备方法为将Sylgard184体系组分A和组分B按照10:1的比例(重量比)充分混合后静置30min脱气,105℃固化35min。CNTs-PDMS复合涂层的制备方法为优选CNTs与组分A共混后充分混匀,之后再添加组分B充分混匀静置30min脱气(A、B组分重量比10:1), 105℃固化35min。
实施案例2.不同CNTs-PDMS复合涂层表面污损微生物样品的采集:
所有涂层浸没于相同海域,挂板深度1.5m。从挂板后第2天起每隔3天定期取样,取样时间2 周。取样方法为:(1)取样前将毛刷浸没在无菌ddH2O中保证毛刷充分润湿。(2)取样时先用无菌ddH2O反复冲洗钢板表面2~3次。(3)用量程1000μL微量移液器在钢板表面滴加1mL 无菌ddH2O,用毛刷用力刷洗钢板表面,再用量程200μL微量移液器在吸取毛刷上的菌液至15mL EP管中。(4)重复上述操作一次。小钢板的另一面样品的采集采用相同方法处理,两面共取样4mL。取样时注意将15mL EP管始终置于冰水中。(5)将样品均匀分装至4个1.5mL EP 管中,7000rpm离心3min。(6)弃上清,标记好样品名称,然后将菌体置于-80℃超低温冰箱中保存备用。
实施案例3.海洋污损真核微生物群落SSCP分析:
利用本实验室自主研发的纳米PCR试剂盒(NPK02,山东大正)扩增海洋真核微生物ITS-2区序列。所采用的通用引物为:ITS 3(5'-GCA TCG ATG AAG AAC GCA GC-3')和ITS4(5'-TCC TCC GCT TAT TGA TAT GC-3')。PCR程序为:94℃预变性3min,94℃预变性30s,56℃退火40s,72℃延伸40s,共35个循环,最后72℃延伸5min,4℃保存。12μL PCR体系包括2×buffer(NPK02) 6.0μL,TaqDNA聚合酶0.2μL(5U/μL),上下游引物1.6μL,裂解菌液4.2μL。PCR产物经1.2%琼脂糖凝胶电泳检测后,PCR产物加入等体积的变形缓冲液后经98℃经变性处理10min后, 15min复性,可直接用于SSCP分离。SSCP分离条件主要为:8%丙烯酰胺凝胶,分离电压90V, 4℃电泳20h。PCR产物与变性缓冲液比例为1:1,上样量4uL,电极缓冲液为1×TBE。SSCP 凝胶所采用的银染方法为:银染液为0.1%AgNO3;显影液主要为含0.2gNa2CO3的2%NaOH 500mL,使用前加4mL质量分数为37%的甲醛。银染步骤为:电泳后的凝胶仔细从玻板上剥离, ddH2O水洗15s;染色液染胶8min;去离子水水洗15s;显影液显色3-4min;终止显影。银染结果用数码相机拍照记录记录。SSCP分析结果如图1所示。
实施案例4.SSCP指纹图谱的识别与数据处理:
海洋污损微生物群落SSCP指纹图谱的识别采用Quantity one 4.6.2软件(Bio-rad)进行。将条带的光密度值输入到Biodap软件,根据条带的位置和强度所形成二元矩阵,并应用Biodap软件进行群落分子生态学参数的计算。应用Biodap软件计算群落分子生态学参数。主要分子生态学参数包括:Shannon diversity index,Total abundace,Speciesrichness等。应用Graphpad6.0 软件和SPSS19.0软件完成对于后续数据的处理相关表格的制作分析。
实施案例5.海洋污损真核微生物群落结果动态变化分析:
主要对比了将6种MWNTs、6种hMWNTs、6种cMWNTs引入PDMS体系前后PDMS表面附着污损真核微生物分子生态学参数的变化。CNTs的基本信息如表1所示。其结果如图2-4所示。从图2-4可以看出,同PDMS涂层相比较,3类CNTs均能够不同程度地降低PDMS涂层表面污损真核微生物群落的多样性水平和丰度,抑制生物膜中污损真核生物的生长,但是羧基修饰的多壁碳纳米管效果最好。
表1 CNTs基本信息
Claims (3)
1.一种利用羧基修饰的多壁碳纳米管抑制PDMS表面生物膜中真核生物附着生长的方法,该方法主要包括:不同种类羧基修饰的多壁碳纳米管cMWNTs-聚二甲基硅氧烷PDMS复合涂层的制备,污损生物样品的采集与制备,不同cMWNTs-PDMS复合涂层表面污损真核生物群落结构的测定,检测真核生物群落结构信息并进行分子生态学评价。
2.根据权利要求1所述的方法,其特征在于所采用的cMWNTs主要为羧基修饰的碳纳米管,其羧基含量在0.5-5%之间,粒径8-100纳米,长度10-50微米,纯度不低于95%,比表面积40-600m2/g。
3.根据权利要求1所述的方法,其特征在于所采用的有机硅树脂为美国道康宁公司生产的Sylgard 184弹体(含有A,B两个成分)。cMWNTs-PDMS复合涂层的制备方法为cMWNTs与组分A共混后充分混匀,之后再添加组分B充分混匀(A、B组分重量比10:1),105℃固化35-60min。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810485784.XA CN108753154A (zh) | 2018-05-21 | 2018-05-21 | 利用羧基修饰的碳纳米管抑制聚二甲基硅氧烷表面生物膜中真核生物附着生长的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810485784.XA CN108753154A (zh) | 2018-05-21 | 2018-05-21 | 利用羧基修饰的碳纳米管抑制聚二甲基硅氧烷表面生物膜中真核生物附着生长的方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN108753154A true CN108753154A (zh) | 2018-11-06 |
Family
ID=64007181
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810485784.XA Pending CN108753154A (zh) | 2018-05-21 | 2018-05-21 | 利用羧基修饰的碳纳米管抑制聚二甲基硅氧烷表面生物膜中真核生物附着生长的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108753154A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112226155A (zh) * | 2020-09-24 | 2021-01-15 | 常熟理工学院 | 一种仿生污损释放型有机硅海洋防污涂料及其制备方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101528867A (zh) * | 2006-10-18 | 2009-09-09 | 纳诺塞尔股份有限公司 | 海洋防生物污损和污损物脱除涂料组合物的用途 |
CN102782058A (zh) * | 2009-12-22 | 2012-11-14 | 纳诺赛尔股份有限公司 | 用于制备抗生物污损涂层的组合物 |
CN104129776A (zh) * | 2014-08-15 | 2014-11-05 | 北京万源工业有限公司 | 羧基功能化碳纳米管的可控制备方法 |
-
2018
- 2018-05-21 CN CN201810485784.XA patent/CN108753154A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101528867A (zh) * | 2006-10-18 | 2009-09-09 | 纳诺塞尔股份有限公司 | 海洋防生物污损和污损物脱除涂料组合物的用途 |
CN102782058A (zh) * | 2009-12-22 | 2012-11-14 | 纳诺赛尔股份有限公司 | 用于制备抗生物污损涂层的组合物 |
CN104129776A (zh) * | 2014-08-15 | 2014-11-05 | 北京万源工业有限公司 | 羧基功能化碳纳米管的可控制备方法 |
Non-Patent Citations (1)
Title |
---|
孙源: "CNTs/PDMS复合涂层对污损早期微生物群落影响研究", 《万方数据》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112226155A (zh) * | 2020-09-24 | 2021-01-15 | 常熟理工学院 | 一种仿生污损释放型有机硅海洋防污涂料及其制备方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Koch et al. | Cytometric fingerprinting for analyzing microbial intracommunity structure variation and identifying subcommunity function | |
Kirk et al. | Methods of studying soil microbial diversity | |
Wilderer et al. | Modern scientific methods and their potential in wastewater science and technology | |
Amann et al. | Modern methods in subsurface microbiology: in situ identification of microorganisms with nucleic acid probes | |
Siqueira Jr et al. | Exploiting molecular methods to explore endodontic infections: part 1—current molecular technologies for microbiological diagnosis | |
Kozdroj et al. | Structural diversity of microorganisms in chemically perturbed soil assessed by molecular and cytochemical approaches | |
Jin et al. | Bacterial communities and potential waterborne pathogens within the typical urban surface waters | |
Fakruddin et al. | Methods for analyzing diversity of microbial communities in natural environments | |
Dorigo et al. | Molecular approaches to the assessment of biodiversity in aquatic microbial communities | |
Gilbride et al. | Molecular techniques in wastewater: understanding microbial communities, detecting pathogens, and real-time process control | |
Hirsch et al. | Culture-independent molecular techniques for soil microbial ecology | |
Stults et al. | Application of the 5′ fluorogenic exonuclease assay (TaqMan) for quantitative ribosomal DNA and rRNA analysis in sediments | |
Dubey et al. | Exploration of soil bacterial communities for their potential as bioresource | |
Christian et al. | Key issues concerning Biolog use for aerobic and anaerobic freshwater bacterial community‐level physiological profiling | |
Shafi et al. | Aquatic bacterial diversity: Magnitude, dynamics, and controlling factors | |
Collins et al. | Accessing the black box of microbial diversity and ecophysiology: recent advances through polyphasic experiments | |
Logue et al. | Progress in the ecological genetics and biodiversity of freshwater bacteria | |
Preston et al. | Near real‐time, autonomous detection of marine bacterioplankton on a coastal mooring in Monterey Bay, California, using rRNA‐targeted DNA probes | |
CA2887416A1 (en) | Field-based qpcr microbial monitoring | |
Ogram et al. | Methods of soil microbial community analysis | |
Urrea-Valencia et al. | Molecular techniques to study microbial wastewater communities | |
CN108753154A (zh) | 利用羧基修饰的碳纳米管抑制聚二甲基硅氧烷表面生物膜中真核生物附着生长的方法 | |
Kim et al. | Rapid fingerprinting of methanogenic communities by high-resolution melting analysis | |
Jiang et al. | A microbial analysis primer for biogeochemists | |
Bakal et al. | Suitability and setup of next-generation sequencing-based method for taxonomic characterization of aquatic microbial biofilm |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WD01 | Invention patent application deemed withdrawn after publication | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20181106 |