CN113522229A - 一种高效吸附环境中胞外dna的磁珠及其制备方法 - Google Patents
一种高效吸附环境中胞外dna的磁珠及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种高效吸附环境中胞外DNA的磁珠及其制备方法,该磁珠为纳米球状双层结构,粒径在15‑30nm,比表面积在65‑210m2/g,其中内层为具有磁性的纳米四氧化三铁(nFe3O4),外层为负载介孔的硅烷涂覆层,并含有羟基官能团;首先将纳米四氧化三铁(nFe3O4)磁珠用二氧化硅包裹,随后利用表面活性剂再次形成涂覆层,将表面活性剂洗脱后得到高效吸附环境中胞外DNA的磁珠,该磁珠具有粒径小和比表面积大的特征,能够在有污染物存在的情况下高选择性吸附环境中胞外DNA,吸附时间短,吸附容量大,可用于环境中胞外DNA的高效提取。
Description
技术领域
本发明涉及一种高效吸附环境中胞外DNA的磁珠及其制备方法,该磁珠具有粒径小和比表面积大的特征,能够在有污染物存在的情况下高选择性吸附环境中胞外DNA,吸附时间短,吸附容量大,可用于环境中胞外DNA的高效提取。
背景技术
胞外DNA是环境中DNA的重要组成部分,能够独立于细胞长期存在,蕴含着丰富的遗传信息,如考古学中常通过胞外DNA来发掘远古生物信息,另外胞外DNA还被用于探索植物与传粉媒介之间的相互联系,对食物链进行分析检测以及判别是否有外来物种入侵,预测新型污染物,甚至评估空气质量及其对人类健康的影响等。此外,胞外DNA对生物的生长进化也具有重要作用。因此建立一种高效的胞外DNA的提取方法至关重要。
目前常用的胞外DNA提取方法有醇沉淀法、CTAB(十六烷基三甲基溴化铵)法等。醇沉淀法主要是利用乙醇、戊二醇等洗去核酸表面的水化层,使核酸中带负电磷酸基团暴露出来,然后利用水中带正电的离子在静电力作用下吸附下沉。如专利“一种高效提取沉积物中胞外DNA的方法”(申请号:CN201610841848.6)公开了一种高效提取沉积物中胞外DNA的方法,该方法采用苯酚、氯仿和异戊醇等有机溶液体系进行DNA的提取。但该方法对操作复杂,且对短链和低浓度DNA的提取效率较低。CTAB法是利用CTAB作为一种阳离子物质,能够与富含阴离子的DNA相结合。如专利“同时提取污水生物处理水样中微生物胞内和胞外DNA的方法”(申请号:CN201510344497.3)公开了一种同时提取污水生物处理水样中微生物胞内和胞外DNA的方法,采用含有CTAB的DNA提取缓冲液来提取胞外DNA。但该方法的缺点是CTAB不能与DNA形成特异性结合,因此提取的DNA质量较差。因此,发展新的胞外DNA提取方法势在必行。
磁珠法主要是依靠Fe3O4纳米粒子具有强磁性、高比表面积以及优异生物容性等特征,通过修饰后对DNA具有较好的吸附能力。但当前制备的磁珠提取DNA多需要在高盐体系下进行,吸附特异性受负载的物质的影响较大,对低浓度DNA的提取效率也较低。如专利“一种磁珠法提取游离核酸的方法及其试剂盒”(申请号:CN201510939670.4)公开了一种磁珠法提取游离核酸的方法,该专利未涉及磁珠的制备方法,且需要在多种盐溶液下进行DNA的吸附。专利“一种磁珠法核酸提取液及其制备方法”(申请号:CN202011571423.0)公开了一种磁珠法核酸提取液及其制备方法,制备了改性的壳聚糖包覆的磁珠。该磁珠对DNA的吸附特异性不够高。因此发展新的磁珠制备方法势在必行。
发明内容
本发明的目的是为了改进现有技术不足而提供一种能够高效吸附环境中胞外DNA的磁珠,本发明的另一目的是提供上述高效吸附环境中胞外DNA的磁珠的制备方法,该磁珠能够在无盐条件下高效吸附DNA,且不易受污染物的干扰,具有较好的吸附特异性可用于环境中胞外DNA的提取。
本发明的技术方案为:一种高效吸附环境中胞外DNA的磁珠,其特征在于该磁珠为纳米球状双层结构,粒径在15-30nm,比表面积在65-210m2/g,其中内层为具有磁性的纳米四氧化三铁(nFe3O4),外层为负载介孔的硅烷涂覆层,并含有羟基官能团;该磁珠对胞外DNA的饱和吸附量达365-537mg/g,吸附平衡时间为1-5分钟;该磁珠对胞外DNA呈特异性吸附,有竞争污染物存在时磁珠对DNA的吸附量在没有污染物时的85%以上。
本发明还提供了制备上述的高效吸附环境中胞外DNA的磁珠的方法,首先将纳米四氧化三铁(nFe3O4)磁珠用二氧化硅包裹,随后利用表面活性剂再次形成涂覆层,将表面活性剂洗脱后得到高效吸附环境中胞外DNA的磁珠,其具体步骤如下:
1)将纳米四氧化三铁颗粒nFe3O4溶于水后加入正硅酸乙酯(TEOS)和甘油,用弱酸调节混合液pH在3-5;然后将混合液在70-95℃温度下搅拌加热1-3小时;反应完成后,将产物冷却后通过磁铁从溶液中分离;将分离的固体洗涤,得到负载SIO2的nFe3O4纳米颗粒SiO2@nFe3O4;其中TEOS和nFe3O4纳米颗粒的质量比为1-5;甘油和nFe3O4纳米颗粒的质量比为10-40;
2)将上述步骤制得的SiO2@n Fe3O4分散于醇溶液中,加入表面活性剂超声溶解,在60-90℃下连续搅拌回流6-12h;用磁铁回收固体物质并洗涤;接着将样品分散在含有硝酸铵的醇溶液中并在60-90℃下回流12-36小时;用磁铁回收固体物质洗涤后得到高效吸附胞外DNA的磁珠;其中表面活性剂与SiO2@Fe3O4的质量比为0.5-2;硝酸铵与SiO2@nFe3O4的质量比为0.5-2。
优选步骤1)中nFe3O4的粒径为为15-30nm;所述的弱酸为冰醋酸或硼酸。优选步骤1)中所述的洗涤均为分别用水和乙醇洗涤。
优选步骤2)中所述的醇溶液为甲醇或乙醇;上述硝酸铵的醇溶液中醇的加入量只要溶解硝酸铵即可,醇溶液为甲醇或乙醇。表面活性剂优选为十六烷基三甲基溴化铵(CTAB)或聚氧乙烯-聚氧丙烯-聚氧乙烯(P123)。
优选SiO2@n Fe3O4在醇溶液中的质量浓度为5-10g/L。
有益效果:
该磁珠对胞外DNA具有高吸附量,饱和吸附量达385-537mg/g,比表面积在65-210m2/g;该磁珠对胞外DNA具有极快的吸附速率,在1-5分钟之内可以达到吸附平衡;该磁珠胞外DNA的吸附具有高特异性,有竞争污染物存在时磁珠对DNA的吸附量在没有污染物时的85%以上。
附图说明
图1:实施例1制备的磁珠的透射电镜图;
图2:实施例1制备的磁珠的红外光谱图;
图3:实施例1制备的磁珠对胞外DNA的吸附等温线拟合图;
图4:实施例1制备的磁珠对胞外DNA的吸附动力学拟合图。
具体实施方式
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前提下所获得的所以其他实施例,都属于本发明保护的范围。
实施例1
1)取4g nFe3O4(粒径为15nm)溶于80mL水后加入8g正硅酸乙酯(TEOS)和75g甘油,用冰醋酸调节混合液pH在4.2。然后将混合液在90℃温度下搅拌加热2小时。反应完成后,将产物冷却后通过磁铁从溶液中分离。将分离的固体分别用水和乙醇洗涤,得到干净的负载SiO2的Fe3O4纳米颗粒(SiO2@nFe3O4)。
2)将1g上述步骤制得的SiO2@nFe3O4分散于200mL乙醇溶液中,加入1g CTAB,在80℃下连续搅拌回流12h。用磁铁回收固体物质并分别用水和乙醇洗涤。接着将样品分散在100mL含有1g硝酸铵的乙醇溶液中并在80℃下回流24小时。用磁铁回收固体物质并分别用水和乙醇洗涤得到高效吸附胞外DNA的磁珠。
制备的高效吸附胞外DNA的磁珠的透射电镜图见图1,可以发现该磁珠的平均粒径在15nm;该磁珠的红外光谱图见图2,可以发现其携带羟基官能团;该磁珠的比表面积为210m2/g;该磁珠对胞外DNA的吸附等温线拟合图见图3,最大吸附量为537mg/g;该磁珠对胞外DNA的吸附动力学拟合图见图4,吸附平衡时间为1min;模拟污染物葡萄糖对该DNA选择性吸附剂吸附DNA的影响不超过9%,模拟污染物蛋白质对该DNA选择性吸附剂吸附DNA的影响不超过9%,模拟污染物腐殖酸对该DNA选择性吸附剂吸附DNA的影响不超过12%。
实施例2
1)取2g nFe3O4(粒径为30nm)溶于100mL水后加入2g正硅酸乙酯(TEOS)和20g甘油,用冰硼酸调节混合液pH在5.0。然后将混合液在70℃温度下搅拌加热1小时。反应完成后,将产物冷却后通过磁铁从溶液中分离。将分离的固体分别用水和乙醇洗涤,得到干净的负载SiO2的Fe3O4纳米颗粒(SiO2@nFe3O4)。
2)将2g上述步骤制得的SiO2@nFe3O4分散于200mL甲醇溶液中,加入1g P123,在60℃下连续搅拌回流6h。用磁铁回收固体物质并分别用水和乙醇洗涤。接着将样品分散在100mL含有1g硝酸铵的甲醇中并在60℃下回流12小时。用磁铁回收固体物质并分别用水和乙醇洗涤得到高效吸附胞外DNA的磁珠。
制备的高效吸附胞外DNA的磁珠的平均粒径在30nm,红外光谱图显示该磁珠携带羟基官能团,该磁珠的比表面积为65m2/g;该磁珠对胞外DNA最大吸附量为365mg/g,吸附平衡时间为5min;模拟污染物葡萄糖对该DNA选择性吸附剂吸附DNA的影响不超过7%,模拟污染物蛋白质对该DNA选择性吸附剂吸附DNA的影响不超过11%,模拟污染物腐殖酸对该DNA选择性吸附剂吸附DNA的影响不超过13%。
实施例3
1)取5g nFe3O4(粒径为25nm)溶于200mL水后加入20g正硅酸乙酯(TEOS)和200g甘油,用冰醋酸调节混合液pH在3.0。然后将混合液在95℃温度下搅拌加热3小时。反应完成后,将产物冷却后通过磁铁从溶液中分离。将分离的固体分别用水和乙醇洗涤,得到干净的负载SiO2的Fe3O4纳米颗粒(SiO2@nFe3O4)。
2)将3g上述步骤制得的SiO2@nFe3O4分散于300mL乙醇溶液中,加入6g CTAB,在90℃下连续搅拌回流12h。用磁铁回收固体物质并分别用水和乙醇洗涤。接着将样品分散在100mL含有6g硝酸铵的乙醇中并在90℃下回流36小时。用磁铁回收固体物质并分别用水和乙醇洗涤得到高效吸附胞外DNA的磁珠。
制备的高效吸附胞外DNA的磁珠的平均粒径为25nm,红外光谱图显示该磁珠携带羟基官能团,该磁珠的比表面积为128m2/g;该磁珠对胞外DNA最大吸附量为479mg/g,吸附平衡时间为4min;模拟污染物葡萄糖对该DNA选择性吸附剂吸附DNA的影响不超过8%,模拟污染物蛋白质对该DNA选择性吸附剂吸附DNA的影响不超过10%,模拟污染物腐殖酸对该DNA选择性吸附剂吸附DNA的影响不超过13%。
实施例4
1)取4g nFe3O4(粒径为18nm)溶于200mL水后加入16g正硅酸乙酯(TEOS)和120g甘油,用冰硼酸调节混合液pH在4.5。然后将混合液在90℃温度下搅拌加热2小时。反应完成后,将产物冷却后通过磁铁从溶液中分离。将分离的固体分别用水和乙醇洗涤,得到干净的负载SiO2的Fe3O4纳米颗粒(SiO2@nFe3O4)。
2)将2g上述步骤制得的SiO2@nFe3O4分散于200mL乙醇溶液中,加入3g P123,在85℃下连续搅拌回流12h。用磁铁回收固体物质并分别用水和乙醇洗涤。接着将样品分散在100mL含有3g硝酸铵的乙醇中并在85℃下回流24小时。用磁铁回收固体物质并分别用水和乙醇洗涤得到高效吸附胞外DNA的磁珠。
制备的高效吸附胞外DNA的磁珠的平均粒径为18nm,红外光谱图显示该磁珠携带羟基官能团,该磁珠的比表面积为145m2/g;制备的材料对胞外DNA最大吸附量为490mg/g,吸附平衡时间为3min;模拟污染物葡萄糖对该DNA选择性吸附剂吸附DNA的影响不超过11%,模拟污染物蛋白质对该DNA选择性吸附剂吸附DNA的影响不超过12%,模拟污染物腐殖酸对该DNA选择性吸附剂吸附DNA的影响不超过15%。
Claims (6)
1.一种高效吸附环境中胞外DNA的磁珠,其特征在于为纳米球状双层结构,粒径在15-30nm,比表面积在65-210m2/g,其中内层为具有磁性的纳米四氧化三铁nFe3O4,外层为负载介孔的硅烷涂覆层,并含有羟基官能团;该磁珠对胞外DNA的饱和吸附量达365-537mg/g,吸附平衡时间为1-5分钟;该磁珠对胞外DNA呈特异性吸附,有竞争污染物存在时磁珠对DNA的吸附量在没有污染物时的85%以上。
2.一种制备如权利要求1所述的高效吸附环境中胞外DNA的磁珠的方法,其具体步骤如下:
1)将纳米四氧化三铁颗粒nFe3O4溶于水后加入正硅酸乙酯TEOS和甘油,用弱酸调节混合液pH在3-5;然后将混合液在70-95℃温度下搅拌加热1-3小时;反应完成后,将产物冷却后通过磁铁从溶液中分离;将分离的固体洗涤,得到负载SIO2的nFe3O4纳米颗粒SiO2@nFe3O4;其中TEOS和nFe3O4纳米颗粒的质量比为1-5;甘油和nFe3O4纳米颗粒的质量比为10-40;
2)将上述步骤制得的SiO2@n Fe3O4分散于醇溶液中,加入表面活性剂超声溶解,在60-90℃下连续搅拌回流6-12h;用磁铁回收固体物质并洗涤;接着将样品分散在含有硝酸铵的醇溶液中并在60-90℃下回流12-36小时;用磁铁回收固体物质洗涤后得到高效吸附胞外DNA的磁珠;其中表面活性剂与SiO2@Fe3O4的质量比为0.5-2;硝酸铵与SiO2@nFe3O4的质量比为0.5-2。
3.根据权利要求2所述的方法,特征在于步骤1)中nFe3O4的粒径为为15-30nm;所述的弱酸为冰醋酸或硼酸。
4.根据权利要求2所述的方法,特征在于步骤1)中所述的洗涤均为分别用水和乙醇洗涤。
5.根据权利要求2所述的方法,特征在于步骤2)中所述的醇溶液为甲醇或乙醇;表面活性剂优选为十六烷基三甲基溴化铵或聚氧乙烯-聚氧丙烯-聚氧乙烯。
6.根据权利要求2所述的方法,特征在于SiO2@n Fe3O4在醇溶液中的质量浓度为5-10g/L。
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