CN111670913A - 负载Ag纳米颗粒的介孔氧化铁单晶、制备方法及其在抗菌抗病毒领域的应用 - Google Patents
负载Ag纳米颗粒的介孔氧化铁单晶、制备方法及其在抗菌抗病毒领域的应用 Download PDFInfo
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Abstract
本发明公开了负载Ag纳米颗粒的介孔氧化铁单晶、制备方法及其在抗菌抗病毒领域的应用。本发明采用硼氢化钠还原法将Ag纳米颗粒负载到介孔氧化铁单晶上,Ag纳米颗粒尺寸在10~30纳米,并且Ag颗粒分布在介孔氧化铁单晶的表面和孔洞内。这种材料可应用在光催化抑菌以及杀灭病毒方面,在光照条件下,Ag‑Fe2O3对金黄色葡萄球菌及大肠杆菌具有优异的光催化抑菌性能,明显优于未负载Ag纳米颗粒的介孔氧化铁单晶;另外,Ag‑Fe2O3对基孔肯雅病毒(CHIKV)ROSS株有良好的病毒杀伤效果。该方法操作简便,工艺设备简单,原料易得,制备成本较低,反应周期短,可重复性高。
Description
技术领域
本发明属于一种负载Ag纳米颗粒的介孔氧化铁单晶材料,该材料在光催化抑菌以及杀伤病毒方面具备优异性能,在其他能源开发和环境保护领域也具备潜在应用性能。
背景技术
α-Fe2O3为n型半导体,其禁带宽度较窄(1.9-2.2eV),在可见光区域有很强的光吸收能力,纳米α-Fe2O3的制备成本低廉且无毒,在催化,光致变色以及光化学器件中都有广泛的应用。但是,α-Fe2O3平带电位较正,同时量子效率也比较低。并且,对于纳米α-Fe2O3单晶而言,当尺寸超过微米级时,相对较低的比表面积限制了材料的应用性能。因此,在单晶半导体氧化物中引入具有纳米尺度的孔洞近年来受到关注,具有介孔结构的半导体氧化物单晶不仅可以提供高表面积,而且可以在整个晶体框架内快速转移电荷。此外,将少量贵金属沉积到介孔α-Fe2O3表面及孔洞内,可减少光电子和空穴复合,从而提高α-Fe2O3光催化活性。近年来,人们对银纳米颗粒(Ag NPs)的制备和研究产生了越来越大的兴趣,适量的Ag可以增强与抗菌活性相关的光催化活性。与纯α-Fe2O3相比,负载Ag NPs的α-Fe2O3表现出增强的抗菌活性以及对病毒的杀伤作用。因此,制备一种同时具备介孔氧化铁单晶材料和金属助催化剂优势的复合材料在光催化抗菌抗病毒性能上有着潜在的应用。
发明内容
鉴于以上问题,本发明的目的是提供一种负载Ag纳米颗粒的介孔氧化铁单晶材料的制备方法及在抗菌抗病毒方面的应用。制备方法简单,成本低。所制备的纳米材料具有优异的光催化抑菌性能和杀伤病毒作用。合成过程中不需要复杂的仪器、操作简单,有利于大规模的生产。
为实现上述目的,本发明采用如下技术方案:
负载Ag纳米颗粒的介孔氧化铁单晶材料,所述Ag纳米颗粒尺寸为10~30纳米,其中Ag颗粒分布在介孔氧化铁单晶的孔洞中。
负载Ag纳米颗粒的介孔氧化铁单晶材料的制备方法,包括如下步骤:
(1)步骤一、介孔氧化铁单晶的制备
在反应釜的聚四氟乙烯内衬,将0.3~1.0g六水氯化铁,0.03~1.00g氟化钠,0.2~1.2mL氧化硅胶体溶液加入20~60mL去离子水,在温度为150~220℃烘箱中反应12~72个小时后取出。用去离子水洗涤3次,再用无水乙醇洗涤1次。洗涤完成后,将样品放置入氟化氢铵溶液(0.5~2.0M),蚀刻12~24小时后取出样品用去离子水洗涤三次后,80℃烘干3小时,获得介孔氧化铁单晶α-Fe2O3。
(2)步骤二、负载Ag纳米颗粒的介孔氧化铁单晶的制备
将20~40mg介孔氧化铁单晶粉末分散在40mL去离子水中,超声1h。然后,向该溶液中加入500μL硝酸银溶液(0.2~1.0M),再搅拌1h。接着,加入5mL柠檬酸三钠溶液(0.3~0.6M)和2.4~4.8mL双氧水(H2O2),并在常温下搅拌20分钟。之后,将2.5mL硼氢化钠(NaBH4,0.4~0.8M)快速注入。该混合物常温下搅拌4h。通过离心收集所得产物,并用去离子水洗涤三遍,在真空烘箱中于60℃干燥12h,得到负载Ag纳米颗粒的介孔氧化铁单晶粉末。所述Ag纳米颗粒尺寸为10~30纳米,其中Ag颗粒分布在介孔氧化铁单晶的孔洞中。
本发明还提供了上述负载Ag纳米颗粒的介孔氧化铁单晶的应用,是用于光催化抑菌和杀伤病毒方面。
上述应用方法如下:
(一)光催化抑菌:在全波段光(300W Xe Lamp)下,负载Ag纳米颗粒的介孔氧化铁单晶材料对金黄色葡萄球菌及大肠杆菌光催化的抑菌性能。在超净台的平板上均匀放入灭菌滤纸片(直径5.6mm),分别在滤纸片上滴加10μL材料悬液及空白对照液体(卡那霉素和无菌水)。在光照组给予光照5min,不光照组直接放入培养箱培养,将平板放入培养箱培养,37℃,16-18h后,读取结果。然后用游标卡尺测量抑菌环的直径,抑菌环的边缘以肉眼见不到细菌明显生长为限,记录光照与不光照处理后样品对金黄色葡萄球菌及大肠杆菌抑菌环的直径。
(二)病毒灭活:取对数生长期人肝癌细胞系Huh7细胞铺入96孔板,静置培养16h。使用减血清培养基(Opti-MEM)将负载Ag纳米颗粒的介孔氧化铁单晶材料配制成为0.5mg/mL,与自身带有增强绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)的CHIKV病毒(MOI=1)等份混匀,室温下孵育30min后,12000rpm,离心2min,加入到细胞密度为80-90%的Huh7细胞培养板中,每孔加入200μL,37℃恒温细胞培养箱中培养2h,然后将培养基换成10%FBS的DMEM培养基,继续培养16h。利用荧光显微镜检测病毒对Huh7细胞的感染滴度。
上述负载Ag纳米颗粒的介孔氧化铁单晶材料的应用,用于光催化抑菌性能和杀伤病毒方面。评判抑菌性能优劣的方法如下:在对样品分别进行光照和不光照处理后,通过对比两种处理方式下材料对金黄色葡萄球菌及大肠杆菌出现抑菌环直径的大小,来判断材料抑菌性能的优劣。评判对病毒杀伤作用的方法如下:利用荧光显微镜检测病毒对Huh7细胞的感染滴度,来验证负载Ag纳米颗粒的介孔氧化铁单晶材料的病毒杀伤效果。
实验结果:在全波段光照条件下,负载Ag纳米颗粒的介孔氧化铁单晶材料出现的抑菌环直径明显大于未光照处理的样品,表现出较优异的抑菌性能;通过检测病毒对Huh7细胞的感染滴度,证实负载Ag纳米颗粒的介孔氧化铁单晶材料有良好的病毒杀伤效果。
本发明的有益效果在于:
(1)采用简单的一步水热法以及硼氢化钠还原法合成负载Ag纳米颗粒的介孔氧化铁单晶材料,Ag纳米颗粒尺寸为10~30纳米,其中Ag颗粒均匀分布在介孔氧化铁单晶的孔洞中。合成方法简单,操作简便,条件温和,目标产物纯度高,安全无毒,可以大批量合成;
(2)将负载Ag纳米颗粒的介孔氧化铁单晶材料用于光催化抑菌和杀伤病毒应用,结果表明其具有较好的光催化抑菌性能以及病毒杀伤作用。在光催化抑菌性能方面,全波段光照射5min后对金黄色葡萄球菌及大肠杆菌均出现抑菌环,且抑菌环直径明显大于未光照处理的样品;另外,在病毒灭活应用中,加入Ag-Fe2O3样品的培养基中,病毒对Huh7细胞的感染滴度明显低于对照组。
(3)制备过程中,所有试剂均为商业产品,不需要进一步处理;
(4)合成方法简单,得到的材料易于应用。
附图说明
图1是实施例1所制备的负载Ag纳米颗粒的介孔氧化铁单晶材料分别在光照与未光照处理条件下对(A)大肠杆菌和(B)金黄色葡萄球菌抑菌环电子照片,(C)对大肠杆菌及金黄色葡萄球菌抑菌环直径柱状图以及(D)大肠杆菌及金黄色葡萄球菌最小抑菌浓度柱状图;
图2是实施例1所制备的负载Ag纳米颗粒的介孔氧化铁单晶材料对基孔肯雅病毒(CHIKV)ROSS株杀伤效果的电子照片;
图3是实施例1所制备的介孔氧化铁单晶材料的X射线衍射图谱;
图4是实施例1所制备的负载Ag纳米颗粒的介孔氧化铁单晶材料的透射电镜图;
图5是实施例1所制备的负载Ag纳米颗粒的介孔氧化铁单晶材料的高分辨透射电镜图;
图6是实施例1所制备的负载Ag纳米颗粒的介孔氧化铁单晶材料的扫描电镜图。
具体实施方式
下面,结合附图和实施例,对本发明的具体实施方式做进一步详细的说明,但不应以此限制本发明的保护范围。
本文所公开的“范围”以下限和上限的形式。可以分别为一个或多个下限,和一个或多个上限。给定范围是通过选定一个下限和一个上限进行限定的。选定的下限和上限限定了特别范围的边界。所有可以这种方式进行限定的范围是包含和可组合的,即任何下限可以与任何上限组合形成一个范围。例如,针对特定参数列出了60-120和80-110的范围,理解为60-110和80-120的范围也是预料到的。此外,如果列出的最小范围值1和2,和如果列出了最大范围3,4和5,则下面的范围可全部预料到:1-2、1-4、1-5、2-3、2-4和2-5。
本发明中,除非有其他说明,数值范围“a-b”表示a到b之间的任意实数组合的缩略表示,其中a和b都是实数。例如数值范围“0-5”表示本文中已经全部列出了“0-5”之间的全部实数,“0-5”只是这些数值组合的缩略表示。
在本发明中,如果没有特别的说明,本文所提到的所有实施方式以及优选实施方式可以互相组合形成新的技术方案。
在本发明中,如果没有特别的说明,本文所提到的所有技术特征以及优选特征可以互相组合形成新的技术方案。
下面将结合具体实施例来具体阐述本发明的优选实施方法,但是应当理解,本领域技术人员可以在不背离权利要求书限定的范围的前提下,对这些实施例进行合理的变化、改良和相互组合,从而获得新的具体实施方法,这些通过变化、改良和相互组合获得的新的具体实施方式也都包括在本发明的保护范围之内。
实施例1
一、负载Ag纳米颗粒的介孔氧化铁单晶材料的制备
(1)步骤一、介孔氧化铁单晶的制备
在反应釜的聚四氟乙烯内衬,将0.5g六水氯化铁,0.03g氟化钠,0.4mL氧化硅胶体溶液加入40mL去离子水,在温度为220℃的烘箱中反应48个小时后取出。用去离子水洗涤3次,再用无水乙醇洗涤1次。洗涤完成后,将样品放置入氟化氢铵溶液(1.0M),蚀刻12小时后取出样品用去离子水洗涤三次后,80℃烘干3小时。获得介孔氧化铁单晶α-Fe2O3。
(2)步骤二、负载Ag纳米颗粒的介孔氧化铁单晶的制备
将介孔氧化铁单晶粉末30mg分散在40mL去离子水中,超声1h。然后,向该溶液中加入500μL硝酸银溶液0.5M,再搅拌1h。接着,加入5mL柠檬酸三钠溶液(0.5M)和3mL双氧水(H2O2),并在常温下搅拌20分钟。之后,将2.5mL硼氢化钠(NaBH4,0.5M)快速注入该混合物常温下搅拌4小时。通过离心收集所得产物,并用去离子水洗涤三遍,在60℃的真空烘箱中干燥12小时,得到负载Ag纳米颗粒的介孔氧化铁单晶粉末。
二、抑菌性能测试
(一)培养基
LB培养基:大肠杆菌、金黄色葡萄球菌。
(二)药敏纸片
抗菌药物纸片直径约为5.6mm,厚度约1mm,每片的吸水量约20μL。
(三)质控菌株
K-B法质量控制菌株常规用:金黄色葡萄球菌ATCC25923、大肠杆菌ATCC25922。
三、试验方法与结果
1、菌种复苏:取-80℃保种于ep管中的菌液,在琼脂平板上划线,置37℃温箱孵育12h,放置于4℃冰箱中可保存一周,作为接种平板。
2、制备接种菌液:挑选琼脂平板上形态相同的单个菌落,移种于10-12mL含有LB液体培养基(3-5mL)的具塞试管中,置37℃温箱孵育12h,作为接种菌液。
3、接种平板:(两层铺板法)底层铺空白的LB培养基(10mL),冷凝;在LB温度降到40℃左右,加入1%菌液(v/v)的LB培养基(10mL),冷凝,作为上层含菌培养基。
4、贴纸片:待平板上的水分被琼脂完全吸收后(约15min),用无菌镊子取纸片(剪好或购买的纸片,放置在平板中,报纸包裹,灭菌、烘干,无菌条件下使用)贴在琼脂平板表面,并用镊尖轻压一下,使其贴平。每张纸片间距不少于24mm,纸片中心距平皿边缘不少于15mm。
5、加药:每纸片上加10μL测试液,设阳性对照(抗生素)和阴性对照(空白溶剂无菌水),挥干,待溶剂被琼脂完全吸收。
6、每种菌的3块板,分为光照组,不光照组以及不加样平板。全波段光照时间均为5min。光照组和不光照组所加测试样品分别为:负载Ag纳米颗粒的介孔氧化铁单晶材料,介孔氧化铁单晶材料,卡那霉素(阳性对照)和无菌水(阴性独照)(50mg/mL)各10μL,分别等距加于平板上。
7、孵育:把贴好药敏纸片的平皿放进37℃温箱培养,最好单独摆放,不超过2个叠在一起,孵育16-18h后,读取结果。
8、判定结果:培养后取出平板,用游标卡尺测量抑菌环的直径,抑菌环的边缘以肉眼见不到细菌明显生长为限,然后根据抑菌环直径大小加以记录。
9、记录结果:
ATCC25923板:
(1)光照组:卡那霉素抑菌环直径22.7mm,负载Ag纳米颗粒的介孔氧化铁单晶材料出现抑菌环,抑菌环直径17.3mm,介孔氧化铁单晶材料未出现抑菌环。
(2)不光照组:卡那霉素抑菌环直径22.2mm,负载Ag纳米颗粒的介孔氧化铁单晶材料出现抑菌环,抑菌环直径13.94mm,介孔氧化铁单晶材料未出现抑菌环。
ATCC25922板:
(1)光照组:卡那霉素抑菌环直径14.5mm,负载Ag纳米颗粒的介孔氧化铁单晶材料出现抑菌环,抑菌环直径11.8mm,介孔氧化铁单晶材料未出现抑菌环。
(2)不光照组:卡那霉素抑菌环直径14.8mm,负载Ag纳米颗粒的介孔氧化铁单晶材料出现抑菌环,抑菌环直径10.6mm,介孔氧化铁单晶材料未出现抑菌环。
三、基孔肯雅病毒(CHIKV)灭活性能测试
实验步骤:取对数生长期人肝癌细胞系Huh7细胞铺入96孔板,静置培养16h。使用减血清培养基(Opti-MEM)将Ag-Fe2O3配制成为0.5mg/mL,与自身带有增强绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)的CHIKV病毒(MOI=1)等份混匀,室温下孵育30min后,12000rpm,离心2min,加入到细胞密度为80-90%的Huh7细胞培养板中,每孔加入200μL,37℃恒温细胞培养箱中培养2h,然后将培养基换成10%FBS的DMEM培养基,继续培养16h。利用荧光显微镜检测病毒对Huh7细胞的感染滴度。
实验结果:加入负载Ag纳米颗粒的介孔氧化铁单晶材料的培养基在荧光显微镜检测下发现病毒感染滴度明显低于对照组。
图1是实施例1所制备的负载Ag纳米颗粒的介孔氧化铁单晶材料分别在光照与未光照处理条件下对(A)大肠杆菌和(B)金黄色葡萄球菌抑菌环电子照片,(C)其对大肠杆菌及金黄色葡萄球菌抑菌环直径柱状图以及(D)大肠杆菌及金黄色葡萄球菌最小抑菌浓度柱状图。可以看出光照处理和无光照处理后所制备材料对大肠杆菌、金黄色葡萄球菌抑菌环直径有明显变化,光照处理后抑菌环直径明显增加,抑菌性能提高,并且,所需的最小抑菌浓度也明显降低。
图2是实施例1所制备的负载Ag纳米颗粒的介孔氧化铁单晶材料对基孔肯雅病毒(CHIKV)ROSS株杀伤效果的电子照片,可以看出加入Ag-Fe2O3样品的培养基在荧光显微镜检测下发现病毒感染滴度明显低于对照组,表明Ag-Fe2O3材料有良好的病毒杀伤效果。
图3是实施例1所制备介孔氧化铁单晶材料的X射线衍射图谱,通过谱图可以清晰地观察到氧化铁的衍射峰。曲线是所制备的材料在扫描速度为3°/min,扫描范围为10°-80°的X射线衍射图谱。
图4是实施例1所制备的负载Ag纳米颗粒的介孔氧化铁单晶材料的透射电镜图,通过观察样品形貌,Ag纳米颗粒尺寸约为10~30纳米,分布在介孔氧化铁单晶的孔洞内。
图5是实施例1所制备的负载Ag纳米颗粒的介孔氧化铁单晶材料高分辨透射电镜图,通过观察,Ag纳米颗粒晶面间距为0.235nm,对应(111)晶面,Fe2O3晶面间距为0.220nm,对应(113)晶面。
图6是实施例1所制备的负载Ag纳米颗粒的介孔氧化铁单晶材料的扫描电镜图,通过观察,介孔氧化铁单晶表面分布着20纳米左右的孔洞。
实施例2
(1)步骤一、介孔氧化铁单晶的制备
在反应釜的聚四氟乙烯内衬,将0.8g六水氯化铁,0.06g氟化钠,0.6mL氧化硅胶体溶液加入20~60mL去离子水,在温度为220℃的烘箱中反应48个小时后取出。用去离子水洗涤3次,再用无水乙醇洗涤1次。洗涤完成后,将样品放置入氟化氢铵溶液(1.2M),蚀刻12小时后取出样品用去离子水洗涤三次后,80℃烘干3小时。获得介孔氧化铁单晶α-Fe2O3。
(2)步骤二、负载Ag纳米颗粒的介孔氧化铁单晶的制备
将40mg介孔氧化铁单晶粉末分散在40mL去离子水中,超声1h。然后,向该溶液中加入500μL硝酸银溶液(0.6M),再搅拌1h。接着,加入5mL柠檬酸三钠溶液(0.5M)和3.6mL双氧水(H2O2),并在常温下搅拌20分钟。之后,将2.5mL硼氢化钠(NaBH4,0.6M)快速注入该混合物中,常温下搅拌4小时。通过离心收集所得产物,并用去离子水洗涤三遍,在真空烘箱中于60℃干燥12小时,得到负载Ag纳米颗粒的介孔氧化铁单晶粉末。
(3)步骤三、抑菌测试以及杀伤病毒特征和性能与实施例1类似。
实施例3
(1)步骤一、介孔氧化铁单晶的制备
在反应釜的聚四氟乙烯内衬,将1.0g六水氯化铁,0.05g氟化钠,0.8mL氧化硅胶体溶液加入40mL去离子水,在温度220℃的烘箱中反应48个小时后取出。用去离子水洗涤3次,再用无水乙醇洗涤1次。洗涤完成后,将样品放置入氟化氢铵溶液(1.5M),蚀刻12小时后取出样品用去离子水洗涤三次后,80℃烘干3小时。获得介孔氧化铁单晶α-Fe2O3。
(2)步骤二、负载Ag纳米颗粒的介孔氧化铁单晶的制备
将50mg介孔氧化铁单晶粉末分散在40mL去离子水中,超声1h。然后,向该溶液中加入500μL硝酸银溶液(1.0M),再搅拌1h。接着,加入5mL柠檬酸三钠溶液(0.8M)和4.8mL双氧水(H2O2),并在常温下搅拌20分钟。之后,将2.5mL硼氢化钠(NaBH4,1.0M)快速注入该混合物常温下搅拌4小时。通过离心收集所得产物,并用去离子水洗涤三遍,在真空烘箱中于60℃干燥12h,得到负载Ag纳米颗粒的介孔氧化铁单晶粉末。
(3)步骤三、抑菌测试以及杀伤病毒特征和性能与实施例1类似。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (5)
1.负载Ag纳米颗粒的介孔氧化铁单晶,其特征在于,所述Ag纳米颗粒为10~30纳米,并且分散在介孔氧化铁单晶的孔洞中。
2.根据权利要求1所述的负载Ag纳米颗粒的介孔氧化铁单晶,其特征在于,介孔氧化铁单晶表面分布着20纳米左右的孔洞。
3.一种如权利要求1所述负载Ag纳米颗粒的介孔氧化铁单晶的制备方法,包括如下步骤:
(1)步骤一、介孔氧化铁单晶的制备
在反应釜的聚四氟乙烯内衬中,将0.3~1.0g六水氯化铁,0.03~1.00g氟化钠,0.2~1.2mL氧化硅胶体溶液加入20~60mL去离子水,在温度为150~220℃的烘箱中反应12~72个小时后取出,经水洗、无水乙醇洗涤后,经氟化氢铵溶液蚀刻12~24小时后,经水洗后烘干后获得介孔氧化铁单晶α-Fe2O3;
(2)步骤二、负载Ag纳米颗粒的介孔氧化铁单晶的制备
取经步骤一制得的20~40mg介孔氧化铁单晶粉末分散在40mL去离子水中,经超声处理,向该溶液中加入0.2~1.0M硝酸银溶液500μL,再搅拌均匀后加入0.3~0.6M柠檬酸三钠溶液5mL和2.4~4.8mL双氧水,搅拌均匀;将0.4~0.8M硼氢化钠2.5mL注入上述混合物,搅拌均匀;离心收集所得产物,水洗干燥后得到负载Ag纳米颗粒的介孔氧化铁单晶粉末。
4.一种如权利要求1所述负载Ag纳米颗粒的介孔氧化铁单晶的应用,用于光催化抑菌或病毒灭活的应用。
5.如权利要求4所述负载Ag纳米颗粒的介孔氧化铁单晶的应用,其特征在于,将负载Ag纳米颗粒的介孔氧化铁单晶材料用于对金黄色葡萄球菌和大肠杆菌细菌光催化的抑菌应用或用于对病毒的杀伤作用。
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