CN115254068A - 一种含植酸的磁性纳米捕菌剂及其制备方法和应用 - Google Patents
一种含植酸的磁性纳米捕菌剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种含植酸的磁性纳米捕菌剂及其制备方法和应用,属于微生物检测以及磁性材料的制备和应用技术领域。该磁性纳米捕菌剂是以纳米Fe3O4为磁性核,用植酸对Fe3O4表面进行修饰得到。该磁性纳米捕菌剂对革兰氏阳性菌具有很强的特异性吸附的能力,可以用于快速吸附和分离样品的中革兰氏阳性菌,不需要进行复杂的样品前处理;用于食品卫生领域,可以避免食品风味损失和品质破坏。
Description
技术领域
本发明属于微生物检测以及磁性材料的制备和应用技术领域,具体涉及一种含植酸的磁性纳米捕菌剂及其制备方法和应用。
背景技术
Fe3O4纳米粒子具有许多优良的性质,在外磁场下能够定向移动,并且在外加交变电磁场作用下能产生热量,其化学性能稳定,用途广泛。但是,Fe3O4纳米粒子在生产、应用过程中却受到很多的限制。首先Fe3O4本身具有磁性,容易团聚;另一方面由于纳米颗粒具有很高的比表面积,处于高能状态,为不稳定体系,因此具有强烈的聚集倾向;其次,纳米颗粒表面原子存在许多不饱和键,极易与其他原子相结合而趋于稳定;再次,纳米颗粒表面出现非化学平衡、非整数配位的化学价。因而,需要对Fe3O4纳米粒子进行表面修饰,通过表面修饰能够降低纳米颗粒的表面能,一方面减小纳米粒子间的相互作用,达到稳定纳米粒子不使其团聚的效果,另一方面使粒子表面产生新的物理和化学功能。目前,对Fe3O4磁性粒子表面进行修饰得到具有新功能的磁性材料的技术已广泛应用于生物医学材料制备、有机合成、环境污染治理、食品安全和卫生等技术领域。
近年来食源性致病菌引发的食品安全事件对人类健康构成了严重威胁。在全球范围内,由食源性致病菌介导的食源性疾病受到人们的高度重视。食源性细菌暴发是全世界疾病和死亡的主要原因之一,严重威胁着我国的社会公共卫生安全,给医疗保健和社会经济造成沉重的经济负担。有效预防食源性疫情的爆发和控制食源性疾病的扩散的一个关键在于病原微生物的快速筛查,通常包括样品采集、样品前处理和微生物检测等步骤。然而,食品样品成分比较复杂,通常需要进行有效的样品前处理才能得到准确可靠的检测结果。样品前处理的目的就是浓缩被测物质、消除基质干扰、保护仪器、提高方法的准确性、精密度、选择性和灵敏度。在整个食品安全性的检测分析中,70%~80%甚至更多的时间用在样品的前处理上,而给实验带来的误差有60%以上来自样品的前处理。而传统的微生物分离方法如过滤和离心等,分别是基于微生物大小和重量,均没有特异性;传统的热杀菌方法包括巴氏杀菌、高温短时间处理和超高温处理,对食品的营养成分、感官品质、理化性质和风味都有不利影响;非热处理方法如光动力灭活、高压处理和低压等离子处理,则需要复杂的设备,且还存在裂解后留下残留微生物细胞的缺点。
食品生产中所使用的原料来源于日益多样化,基础的样品富集方法如离心过滤对于富集处理高度加工、脂类、酸类、高或低DNA含量的食品样品,常对检测灵敏度产生干扰,导致结果的不可靠。另一方面对于复杂的食品基质和低水平的目标,特别是对于痕量化合物的检测,样品制备程序尤为重要。适当的样品制备方法可以有效分离和富集目标分析物,减少样品基质和其他杂质的干扰,从而提高分析结果的灵敏度、准确性和可靠性。因此,迫切需要一种有效的策略来快速、灵敏、可靠和简单地分离病原微生物或早期检测它们的存在。具有代表性的磁分离方法以其用户友好、快速和成本效益高的模式而受到越来越多的关注,并成为一种强大的预处理工具。
植酸(Phytic acid,PA)是从植物种子中提取的一种有机磷酸类化合物,在自然界中普遍存在。植酸因其有12个可电离质子的独特的结构,不仅对钙、锌和铁等多价金属离子有很强的亲和力,可相互作用形成金属配合物,还能与蛋白质形成配合物。同时植酸已被证明能破坏细菌细胞壁细胞膜,从而产生很强的抑菌作用。利用金属离子与PA的螯合作用形成稳定的配位化合物,实现对磁性材料有效的表面包覆改性,具有简单、天然、安全和环保等优点。
目前的微生物样品前处理存在较大的局限性,通过功能化改性磁性纳米材料开发出能特异性吸附食源性致病菌的捕菌剂和其使用方法,不仅能够高效准确地检测致病菌,还能避免食品风味损失和品质破坏,同时简化细菌检测方法,对于食品药品安全和环境卫生领域的细菌检测具有重要意义。
发明内容
为了解决上述问题,本发明提供了一种含植酸的磁性纳米捕菌剂(Fe3O4-PA),该磁性纳米捕菌剂是以磁性纳米Fe3O4为核,用植酸对磁性纳米Fe3O4表面进行修饰得到;它能够特异性吸附革兰氏阳性菌,然后通过磁性分离将吸附的革兰氏阳性菌从样品中分离出来。
本发明还提供了该磁性纳米捕菌剂的制备方法,包括以下步骤:将磁性纳米Fe3O4在乙醇中超声分散,得到悬浮液A;制备含植酸的乙醇溶液B;将悬浮液A与乙醇溶液B混合,振荡反应,通过磁分离得到反应产物,用去离子水清洗后得到Fe3O4-PA磁性纳米捕菌剂;所述磁性纳米Fe3O4与所述植酸的摩尔比为(81.4:1)~(4.1~3);所述植酸是指纯植酸。
优选的,步骤S2中,制备所述乙醇溶液B时,使用的是70wt%植酸水溶液。
优选的,步骤S2中,超声分散的时间为5~30min。
优选的,步骤S2中,振荡反应的条件为在室温下,以200~400rpm的速度振荡反应6小时。
优选的,所述磁性纳米Fe3O4的制备方法包括以下步骤:将可溶于水的三价铁盐和无水醋酸钠加入乙二醇中溶解,再加入聚丙烯酸,氮气保护下搅拌均匀,得到溶液C;溶液C在聚四氟乙烯内衬的高压釜中,在110~130℃下反应2h,继续加热至190~210℃反应10h;待反应体系冷却至室温,所得黑色产品用无水乙醇洗涤至上清无色透明,真空干燥,即得磁性纳米Fe3O4。
进一步优选的,所述可溶于水的三价铁盐、无水醋酸钠、聚丙烯酸的摩尔比为72.5:381:1。
进一步优选的,所述可溶于水的三价铁盐为六水合氯化铁。
按照上述方法制备的Fe3O4-PA磁性纳米捕菌剂可用于捕获和分离样品中的革兰氏阳性菌;尤其是样品中的单增李斯特菌、金黄色葡萄球菌或耐甲氧西林金黄色葡萄球菌。
革兰氏阳性菌细胞壁主要由肽聚糖和磷壁酸组成。与革兰氏阳性菌不同,革兰氏阴性菌的细胞壁具有疏水性外膜双层,并且更复杂,包括脂多糖、磷脂和外膜蛋白。与具有更多肽聚糖的革兰氏阳性菌相比,革兰氏阴性菌的多糖外膜可能会阻碍PA结合。
本发明中将该Fe3O4-PA磁性纳米捕菌剂用于捕获和分离样品中的革兰氏阳性菌的方法包括以下步骤:若被污染样品为液体,则直接取被污染样品,加入所述磁性纳米捕菌剂,振荡8~16min,通过磁分离将已吸附革兰氏阳性菌的磁性纳米捕菌剂分离出来;若被污染样品为非液体状态,则将被污染样品制成液态匀浆,再加入所述磁性纳米捕菌剂,振荡8~16min,通过磁分离将已吸附革兰氏阳性菌的磁性纳米捕菌剂分离出来;所述磁性纳米捕菌剂中纳米Fe3O4与植酸的摩尔比为(81.4:1)~(4.1:3);所述磁性纳米捕菌剂的用量根据反应体系中革兰氏阳性菌的浓度确定。实际应用中,先测定样品中革兰氏阳性菌的浓度,再根据革兰氏阳性菌的浓度确定磁性纳米捕菌剂的浓度。当被污染样品中革兰氏阳性菌的浓度较低时,相应地使用浓度较低的磁性纳米捕菌剂,例如,一般情况下,食品中革兰氏阳性菌的浓度不会超过104CFU/mL,因此使用的磁性纳米捕菌剂在反应体系中的终浓度为0.05mg/mL左右。
进一步优选的,所述磁性纳米捕菌剂中磁性纳米Fe3O4与植酸的摩尔比为(20.4:1)~(4.1:3)。
更进一步优选的,所述磁性纳米捕菌剂中磁性纳米Fe3O4与植酸的摩尔比为12.2:1。
优选的,振荡时间为8min。
与现有技术相比,本发明的有益效果是:
(1)本发明中使用植酸对磁性纳米四氧化三铁表面进行修饰,制备出Fe3O4-PA磁性纳米捕菌剂;该Fe3O4-PA磁性纳米捕菌剂对革兰氏阳性菌具有高度特异性,能够吸附样品中的革兰氏阳性菌,然后在外加磁场作用下,能够将被吸附的细菌从样品中分离出来。
(2)使用该Fe3O4-PA磁性纳米捕菌剂对样品中的革兰氏阳性菌进行吸附时,吸附效果不受样品中细菌浓度影响,只需根据样品中细菌浓度调整磁性纳米捕菌剂的用量即可;例如当样品中细菌浓度为108CFU/mL时,反应体系中Fe3O4-PA磁性纳米捕菌剂的浓度可以选择0.3mg/mL;当样品中细菌浓度为103~104CFU/mL时,反应体系中Fe3O4-PA磁性纳米捕菌剂的浓度可以选择0.05mg/mL;当样品中浓度低于103CFU/mL时,可以调整反应体系中Fe3O4-PA磁性纳米捕菌剂的浓度低于0.05mg/mL。
(3)将本发明中的细菌富集和分离方法应用于食品药品检测领域,能够提高检测速度和灵敏度,减少样品本身复杂成分所引起的基质干扰,不需要进行复杂的样品前处理,同时还能避免热杀菌导致的食品风味损失和品质破坏,或者避免其他非热处理的杀菌方式所需的复杂设备和导致的细胞残留问题。
附图说明
图1中a图为本发明制备的磁性纳米Fe3O4的扫描电镜图,b图为本发明制备的Fe3O4-PA磁性纳米捕菌剂的扫描电镜图;
图2为纳米Fe3O4与PA在不同用量比例下对MRSA的捕获效率图;
图3为不同浓度的Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率图;
图4为Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率随时间变化的曲线图;
图5为Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率随温度变化的结果图;
图6为Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率随pH变化的结果图;
图7为Fe3O4-PA磁性纳米捕菌剂对不同细菌的捕获效率图;
图8为Fe3O4-PA磁性纳米捕菌剂用于分离被污染样品中革兰氏阳性菌的平板对比图;
图9为Fe3O4-PA磁性纳米捕菌剂用于捕获、分离革兰氏阳性菌的流程图。
具体实施方式
以下结合实施例对本发明技术方案进行清楚、完整的描述,显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施例,都属于本发明所保护的范围。本领域技术人员依据以下实施方式所作的任何等效变换或替代,均属于本发明的保护范围之内。
以下实施例中PA或附图中的PA都是指市售的70wt%植酸水溶液。本发明中使用70wt%植酸水溶液仅仅是因为购买的是这种规格的植酸溶液,而不是用于限制反应体系中植酸的浓度,本领域技术人员根据浓度换算也可以采用其他规格的植酸溶液,例如使用50wt%植酸水溶液。本发明的实施例中使用的磁性纳米Fe3O4可以为市售产品,也可以按照实施例中的方法制备,还可以按照其他方法制备,只要能成功用于制备Fe3O4-PA磁性纳米捕菌剂即可。
实施例1
本实施例提供了一种含植酸的磁性纳米捕菌剂的制备方法、不同条件下对细菌的捕获效率和应用于被污染样品中细菌分离的方法。
1、制备含植酸修饰的磁性纳米捕菌剂
S1、通过溶剂热反应法合成磁性纳米Fe3O4
将1.36g六水合氯化铁和3.6g醋酸钠加入40mL乙二醇中充分溶解,再加入0.125g聚丙烯酸(分子量为1800),磁力搅拌下冲入氮气2小时得到溶液C;将溶液C转移至聚四氟乙烯内衬的不锈钢高压釜中,加热至120℃并保持2小时;然后继续加热至200℃反应10小时。让反应釜自然冷却至室温,所得黑色产品用无水乙醇洗涤至上清无色透明,并在60℃下真空干燥,即可制得磁性纳米Fe3O4。
S2、制备含植酸的磁性纳米捕菌剂
取30mg磁性纳米Fe3O4于30mL乙醇中超声15min以均匀分散,得到悬浮液A;将10mg70%植酸水溶液于30mL乙醇中均匀溶解得到含植酸的乙醇溶液B;将悬浮液A与乙醇溶液B混合,在室温下以350rpm速度振荡反应6小时,通过磁分离得到固体的反应产物,弃去上清,固体用去离子水洗涤3~5次,即得到含植酸的磁性纳米捕菌剂(Fe3O4-PA磁性纳米捕菌剂)。
分别配制磁性纳米Fe3O4和Fe3O4-PA磁性纳米捕菌剂的悬浊液,用移液枪取样品超声后的悬浊液,滴一滴于硅片上,自然风干,将风干后的硅片用导电胶粘在样品台上,并使用溅射镀膜仪喷金处理,随后使用扫描电子显微镜拍摄样品形貌、能谱mapping图等进行测试,通过扫描电镜能谱点扫在点范围获得样品的元素半定量信息,分析结果见表1。
表1磁性纳米粒子的重量百分比(wt%)及原子百分比(at%)
图1中a图为步骤S1中制备的磁性纳米Fe3O4的扫描电镜图,从图中可以看出,磁性纳米Fe3O4本身表面较为光滑;b图为步骤S2中制备的Fe3O4-PA磁性纳米捕菌剂的扫描电镜图;从图中可以看出,磁性纳米Fe3O4表面被植酸修饰后其结构没有受到影响,只是表面变得更为粗糙。
2、Fe3O4-PA磁性纳米捕菌剂对细菌的富集效果
2-1、细菌培养
采用平板划线法将细菌在LB琼脂平板上划线,放入37℃的培养箱过夜培养,然后在培养好的平板上挑出单个菌落,接种在30mL LB肉汤中,并于37℃振荡温育12小时;从培养好的细菌的肉汤中移取8mL至无菌离心管中,在6000rpm、4℃条件下离心5min,弃去上清的肉汤,加入0.9%生理盐水(pH=6)后重悬,再次离心,最后用0.9%生理盐水(pH=6)稀释已经洗涤的菌悬液,使得其在OD600nm处的吸光值为0.5,将制备好的菌悬液存放至4℃环境,备用。
上述步骤中所使用的LB琼脂平板的培养基的成分包括:10g/L胰蛋白胨,5g/L酵母粉,10g/L NaCl,15g/L琼脂,pH为7.1±0.2;LB肉汤培养基的成分包括:10g/L胰蛋白胨,5g/L酵母粉,10g/L NaCl,pH为7.0±0.2。
按照上述方法,分别制备阪崎肠杆菌、沙门氏菌、大肠杆菌、金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌和单增李斯特菌的菌悬液。
2-2、Fe3O4-PA磁性纳米捕菌剂对细菌的富集效果
将耐甲氧西林金黄色葡萄球菌(MRSA)作为模型细菌,以评估Fe3O4-PA磁性纳米捕菌剂对细菌的吸附性能。
(1)测定磁性纳米Fe3O4与植酸用量比例不同时Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率
按照步骤S2中的方法制备Fe3O4-PA磁性纳米捕菌剂,不同之处在于分别将0.6mg、1.5mg、3mg、6mg、15mg、30mg、60mg、90mg 70%植酸水溶液于30mL乙醇中均匀溶解得到含植酸的乙醇溶液B。
将制备出的磁性纳米Fe3O4与植酸用量比例不同的Fe3O4-PA磁性纳米捕菌剂分别超声分散于水中,均制备成浓度为20mg/mL的悬浮液,每份该悬浮液取25μL分别加入到8份2475μL耐甲氧西林金黄色葡萄球菌菌悬液(步骤2-1中制备)中得到8份混合液,每份混合液均在室温下振荡10分钟,通过磁分离将Fe3O4-PA磁性纳米捕菌剂分离出来,测定磁分离后的溶液在600nm处的吸光度值,计算细菌的捕获效率,结果如图2所示。从图2中可以看出,当磁性纳米Fe3O4与70%植酸水溶液的质量比为(10:1)~(1:3)时,Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率为69.87%~84.31%;当磁性纳米Fe3O4与70%植酸水溶液的质量比为(5:1)~(1:3)时,Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率为82.92%~84.31%;当磁性纳米Fe3O4与70%植酸水溶液的质量比为3:1时,Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率最高,为85.95%。
(2)测定不同浓度的Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率
将步骤S2中制备的Fe3O4-PA磁性纳米捕菌剂超声分散于水中,制备成浓度为10~40mg/mL的悬浮液;取25μL该悬浮液加入到2475μL耐甲氧西林金黄色葡萄球菌菌悬液(步骤2-1中制备)中,在室温下振荡10分钟,通过磁分离将Fe3O4-PA磁性纳米捕菌剂分离出来,测定磁分离后的溶液在600nm处的吸光度值,计算细菌的捕获效率,结果如图3所示。从图3中可知,反应体系中Fe3O4-PA磁性纳米捕菌剂的浓度为0.1~0.4mg/mL时,Fe3O4-PA磁性纳米捕菌剂对耐甲氧西林金黄色葡萄球菌(MRSA)的捕获效率为72.76%~97.57%;反应体系中Fe3O4-PA磁性纳米捕菌剂的浓度低于0.3mg/mL时,随着浓度增加,Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率增加;达到0.3mg/mL以后,随着浓度增加捕获效率增长趋势趋于平缓;当反应体系中Fe3O4-PA磁性纳米捕菌剂的浓度为0.3mg/mL时,Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率为96.85%。
(3)测定不同反应时间下Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率
将步骤S2中制备的Fe3O4-PA磁性纳米捕菌剂超声分散于水中,制备成浓度为30mg/mL的悬浮液;取25μL该悬浮液加入到2475μL耐甲氧西林金黄色葡萄球菌菌悬液(步骤2-1中制备)中,制备10份相同的反应体系;在室温下,依次测定10个反应体系在振荡第0min、2min、4min、6min、8min、10min、12min、14min、15min、16min后进行磁分离后的溶液在600nm处的吸光度值,计算细菌的捕获效率,结果如图4所示。从图4中可知,在8min之前,随着反应时间延长,Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率增大;到8min后,捕获效率的增长趋势变得平缓,即反应体系至少需要8min中的反应时间才能达到较高的捕获效率。
(4)测定不同温度下Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率
将步骤S2中制备的Fe3O4-PA磁性纳米捕菌剂超声分散于水中,制备成浓度为30mg/mL的悬浮液;取25μL该悬浮液加入到2475μL耐甲氧西林金黄色葡萄球菌菌悬液(步骤2-1中制备)中,相同的反应体系制备6份,分别在4℃、10℃、20℃、30℃、37℃、45℃下振荡8分钟,通过磁分离将Fe3O4-PA磁性纳米捕菌剂分离出来,测定磁分离后的溶液在600nm处的吸光度值,计算细菌的捕获效率,结果如图5所示。从图5中可知,不同反应温度下,Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率均超过90%,这说明温度对Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率的影响可以忽略不计。
(5)测定不同pH条件下Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率
按照步骤2-1中的方法制备耐甲氧西林金黄色葡萄球菌的菌悬液,不同之处是加入相同体积的pH分别为3、4、5、6、7、8、9的0.9%生理盐水后重悬,再次离心,最后用对应pH值的0.9%生理盐水稀释已经洗涤的菌悬液,使得所有菌悬液在OD600nm处的吸光值为0.5,即得到pH值不同的MRSA菌悬液;不同pH值的0.9%生理盐水是通过盐酸和氢氧化钠溶液调节0.9%生理盐水的pH到所需的值得到。
将步骤S2中制备的Fe3O4-PA磁性纳米捕菌剂超声分散于水中,制备成浓度为30mg/mL的悬浮液;取pH分别为3、4、5、6、7、8、9的MRSA菌悬液各2475μL,每份中分别加入该悬浮液25μL,得到的7份混合液在室温下振荡8分钟,通过磁分离将Fe3O4-PA磁性纳米捕菌剂分离出来,测定磁分离后的溶液在600nm处的吸光度值,计算细菌的捕获效率,结果如图6所示。从图6中可以看出,在pH=3~9的范围内,Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率均超过90%,这说明pH的不同对Fe3O4-PA磁性纳米捕菌剂对MRSA捕获效率的影响可以忽略不计。
(6)测定Fe3O4-PA磁性纳米捕菌剂对不同细菌的捕获效率
将步骤S2中制备的Fe3O4-PA磁性纳米捕菌剂超声分散于水中,制备成浓度为30mg/mL的悬浮液;分别取步骤2-1中制备的每种菌悬液2475μL,向每种菌悬液中各加入25μL该悬浮液,得到的混合液在室温下振荡8分钟,通过磁分离将Fe3O4-PA磁性纳米捕菌剂分离出来,测定磁分离后的溶液在600nm处的吸光度值,计算细菌的捕获效率,结果如图7所示。从图7中可以看出,Fe3O4-PA磁性纳米捕菌剂对单增李斯特菌的捕获效率为72.09%,对金黄色葡萄球菌的捕获效率为95.59%,对耐甲氧西林金黄色葡萄球菌的捕获效率为98.76%;而对阪崎肠杆菌不捕获,对大肠杆菌、沙门氏菌的捕获效率低于20%。这说明,Fe3O4-PA磁性纳米捕菌剂对革兰氏阳性菌具有显著的特异性捕获的能力。
3、Fe3O4-PA磁性纳米捕菌剂用于捕获和分离被污染样品中的革兰氏阳性菌
取市售的汇源100%苹果汁两份,分别添加金黄色葡萄球菌和单增李斯特菌,使细菌浓度均为103~104CFU/mL,再加入Fe3O4-PA磁性纳米捕菌剂使反应体系中Fe3O4-PA的浓度为0.05mg/mL;反应体系在室温下振荡8分钟,通过磁分离将Fe3O4-PA磁性纳米捕菌剂分离出来,通过平板菌落计数法测定分离前反应体系和分离后溶液中的菌落数,结果如图8所示。从图8中可以看出,磁分离后溶液中的金黄色葡萄球菌和单增李斯特菌均显著减少,尤其是金黄色葡萄球菌几乎不存在与磁分离后的溶液中。
步骤2-2第(2)条中测定不同浓度的Fe3O4-PA磁性纳米捕菌剂对MRSA的捕获效率时,细菌浓度为108CFU/mL,因此反应体系中的Fe3O4-PA磁性纳米捕菌剂浓度相对较高;而实际情况下,被细菌污染的食品药品中革兰氏阳性菌的浓度一般不会超过104CFU/mL,因此从被污染的苹果汁中分离革兰氏阳性菌时,反应体系中Fe3O4-PA磁性纳米捕菌剂的终浓度可以根据细菌浓度进行调整,降低至0.05mg/mL,而不必使用0.3mg/mL以免浪费材料。
实施例2
本实施中提供了一种含植酸的磁性纳米捕菌剂的制备方法,该制备方法与实施例1中第1项基本相同,不同之处在于,步骤S1中将溶液C转移至聚四氟乙烯内衬的不锈钢高压釜中,加热至110℃并保持2小时;然后继续加热至210℃反应10小时;步骤S2中超声30min得到悬浮液A,将悬浮液A与乙醇溶液B混合,在室温下以200rpm速度振荡反应6小时。
实施例3
本实施中提供了一种含植酸的磁性纳米捕菌剂的制备方法,该制备方法与实施例1中第1项基本相同,不同之处在于,步骤S1中将溶液C转移至聚四氟乙烯内衬的不锈钢高压釜中,加热至130℃并保持2小时;然后继续加热至190℃反应10小时;步骤S2中超声5min得到悬浮液A,将悬浮液A与乙醇溶液B混合,在室温下以400rpm速度振荡反应6小时。
如图9所示,本发明中以磁性纳米Fe3O4为核,用植酸对Fe3O4表面进行修饰制备了Fe3O4-PA磁性纳米捕菌剂,该Fe3O4-PA磁性纳米捕菌剂可以用于快速分离被致病菌污染的样品中的致病菌。实施例2和实施例3中Fe3O4-PA磁性纳米捕菌剂对细菌的富集效果以及应用的实验方法均与实施例1中相同,效果也与实施例1中的结论一致,因此,这里不再赘述。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明的保护范围。对于任何熟悉本领域的技术人员来说,本发明可以有各种更改和变化。任何依据本发明申请保护范围及说明书内容所作的简单的等效变化和修饰,均应包含在本发明的保护范围之内。
Claims (9)
1.一种含植酸的磁性纳米捕菌剂,其特征在于,所述磁性纳米捕菌剂是以磁性纳米Fe3O4为核,用植酸对所述磁性纳米Fe3O4表面进行修饰得到。
2.权利要求1所述的磁性纳米捕菌剂的制备方法,其特征在于,包括以下步骤:
将磁性纳米Fe3O4在乙醇中超声分散,得到悬浮液A;制备含植酸的乙醇溶液B;将悬浮液A与乙醇溶液B混合,振荡反应,通过磁分离得到反应产物,用去离子水清洗后得到Fe3O4-PA磁性纳米捕菌剂;所述磁性纳米Fe3O4与所述植酸的摩尔比为(81.4:1)~(4.1~3)。
3.根据权利要求2所述的磁性纳米捕菌剂的制备方法,其特征在于,所述磁性纳米Fe3O4的制备方法包括以下步骤:将可溶于水的三价铁盐和无水醋酸钠加入乙二醇中溶解,再加入聚丙烯酸,氮气保护下搅拌均匀,得到溶液C;溶液C在聚四氟乙烯内衬的高压釜中,在110~130℃下反应2h,继续加热至190~210℃反应10h;待反应体系冷却至室温,所得黑色产品用无水乙醇洗涤至上清无色透明,真空干燥,即得磁性纳米Fe3O4。
4.根据权利要求3所述的磁性纳米捕菌剂的制备方法,其特征在于,所述可溶于水的三价铁盐、无水醋酸钠、聚丙烯酸的摩尔比为72.5:381:1。
5.根据权利要求3所述的磁性纳米捕菌剂的制备方法,其特征在于,所述可溶于水的三价铁盐为六水合氯化铁。
6.权利要求1所述的磁性纳米捕菌剂的应用,其特征在于,将所述磁性纳米捕菌剂用于捕获和分离样品中的革兰氏阳性菌。
7.根据权利要求6所述的磁性纳米捕菌剂的应用,其特征在于,所述革兰氏阳性菌为单增李斯特菌、金黄色葡萄球菌或耐甲氧西林金黄色葡萄球菌中的一种或几种。
8.根据权利要求6所述的磁性纳米捕菌剂的应用,其特征在于,将所述磁性纳米捕菌剂用于捕获和分离样品中的革兰氏阳性菌的方法包括以下步骤:若被污染样品为液体,则直接取被污染样品,加入所述磁性纳米捕菌剂,振荡8~16min,通过磁分离将已吸附革兰氏阳性菌的磁性纳米捕菌剂分离出来;若被污染样品为非液体状态,则将被污染样品制成液态匀浆,再加入所述磁性纳米捕菌剂,振荡8~16min,通过磁分离将已吸附革兰氏阳性菌的磁性纳米捕菌剂分离出来;所述磁性纳米捕菌剂中磁性纳米Fe3O4与植酸的摩尔比为(81.4:1)~(4.1:3);所述磁性纳米捕菌剂的用量根据反应体系中革兰氏阳性菌的浓度确定。
9.根据权利要求8所述的磁性纳米捕菌剂的应用,其特征在于,所述磁性纳米捕菌剂中磁性纳米Fe3O4与植酸的摩尔比为(20.4:1)~(4.1:3)。
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