CN111220610B - 一种基于铁醇盐纳米酶的As5+比色检测法 - Google Patents
一种基于铁醇盐纳米酶的As5+比色检测法 Download PDFInfo
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Abstract
本发明属于分析化学技术领域,涉及As5+的检测方法,尤其涉及一种基于铁醇盐纳米酶的As5+比色检测法,包括:用去离子水配制1 mg·mL‑1的铁醇盐纳米酶溶液,分别取100µL的铁醇盐纳米酶溶液分散在2700µL醋酸‑醋酸盐缓冲溶液中,加入100µL不同浓度的As5+,孵化0.5~5 min;分别将100µL的5 mM的TMB乙醇溶液加入,孵化10~30 min;用紫外‑可见吸收分光光度计测定混合溶液紫外吸收光谱,记录波长为652 nm处的吸光度并绘制标准工作曲线;将待测As5+样品重复上述步骤测定吸光度;并与标准工作曲线比对,即得As5+浓度。本发明利用铁醇盐纳米酶比色检测As5+,检测过程条件温和,实现As5+的方便、快速检测,成本低廉;检测范围宽至3.33~333.33µg·L‑1,满足世界卫生组织对砷离子的最低限制。
Description
技术领域
本发明属于分析化学技术领域,涉及As5+的检测方法,尤其涉及一种基于铁醇盐纳米酶的As5+比色检测法。
背景技术
由于矿产的开发和工业废水的排放,无机砷污染在全球范围内日趋严重。据调查,全球至少有2000万人生活在严重无机砷污染的地区。在自然界中,五价砷酸盐(As5+)和三价亚砷酸盐(As3+)是无机砷的最常见的化学形态。无机砷具有很强的毒性和致癌性,严重威胁人体健康,此世界卫生组织(WHO)严格规定饮用水中的砷含量不得高于10μg·L-1。因此,无机砷的定量检测,对环境保护和公共健康具有重要意义。
目前针对无机砷检测方法主要有仪器分析法、电化学法、生物传感法和比色法等,其中仪器分析法包括原子吸收光谱、原子发射光谱法和原子荧光光谱法等,具备有检测限低、选择性优、精确度好等特点。但适用的线性范围较窄,且操作步骤繁琐等缺点极大地限制了其应用。电化学方法可用于砷的多种形态的分析,由于电化学法在测定无机砷过程中产生的是电信号,因而电化学法适用于无机砷连续不间断检测。但是电化学法检测无机砷容易受电极影响,且电极寿命较短,成本高昂。生物传感分析法检测无机砷具有较高的灵敏度度、可移植性、样本需求量低等优点,可以用于痕量无机砷的定量和定性检测分析。但这种方法需要专门的基因工程和生物技术细胞转化,步骤繁琐,且成本昂贵,应用范围有限。比色法能进行可视化检测,是一种较为直观的无机砷检测方法。
此外,纳米酶作为一种新兴的纳米材料,具有与天然酶类似的催化活性,能够催化一些特定的反应。纳米酶比色法,采用纳米酶来检测目标物具有合成方便、原理简单和成本低廉等优点,因此纳米酶比色法是一种较为理想的无机砷检测方法。
发明内容
针对上述现有技术中存在的不足和限制,本发明的目的在于公开一种基于铁醇盐纳米酶的As5+比色检测法。
技术方案
一种基于铁醇盐纳米酶的As5+比色检测法,步骤如下:
(1)用去离子水配制1mg·mL-1的铁醇盐纳米酶溶液,分别取100μL的铁醇盐纳米酶溶液分散在2700μL醋酸-醋酸盐缓冲溶液中,加入100μL不同浓度的As5+,孵化0.5~5 min,优选2min;
(2)分别将100μL的5mM的TMB乙醇溶液加入上述混合溶液中,孵化10~30min,优选20min;
(3)用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录波长为652nm处的吸光度并绘制As5+浓度-吸光度标准工作曲线;
(4)将待测As5+样品重复步骤(1)~(3),以紫外-可见吸收分光光度计测定652nm处吸光度;
(5)通过计算与As5+浓度-吸光度标准工作曲线比对,即可获得待测As5+样品的As5+浓度。
本发明较优公开例中,步骤(1)所述醋酸-醋酸盐缓冲溶液浓度0.2M,pH为4.0。
本发明较优公开例中,步骤(1)所述As5+溶液浓度为3.33~333.33μg·L-1。
本发明较优公开例中,步骤(4)所述待测As5+样品检测范围为3.33~333.33μg·L-1,检测低限为1.57μg·L-1。
本发明所述铁醇盐纳米酶,制备步骤如下:
A、按固液比为7.5mM:9g:150~450mL,将FeCl3·6H2O或Fe(NO3)3·9H2O和尿素分散在乙二醇里,充分溶解成混合溶液,所述固液比优选7.5mM:9g:300mL;
B、混合溶液在150~250℃溶剂热反应20~40min,优选195℃反应30min;
C、铁醇盐纳米酶离心收集,并用乙醇和去离子水洗净,50~70℃干燥18~30h优选60℃干燥24h后即得。
依据本发明所述方法制得的铁醇盐纳米酶为花状球体,其形貌特征如图1所示。
本发明首先制备铁醇盐(IA)纳米酶,铁醇盐是一种氧化酶模拟物,能够催化溶解氧形成超氧阴离子自由基,氧化3,3',5,5'-四甲基联苯胺(TMB),形成蓝色产物TMBox。当在IA+TMB体系中加入As5+时,As5+能够被吸附在铁醇盐上,抑制其氧化酶活性,引起IA+TMB 体系吸光度变化。然后测试加入不同浓度As5+的IA+TMB体系在652nm的吸光度,绘制 As5+浓度-吸光度标准工作曲线,并检测待测物的As5+含量。
在本说明书中,术语“氧化酶模拟物”是指具有氧化酶催化活性的纳米材料。具体地,本发明中的氧化酶模拟物以氧气作为电子受体,通过氧化TMB底物生成有色物质TMBox,用于比色检测。
在本说明书中,术语“TMB”是化合物“3,3’,5,5’-四甲基联苯胺”的缩写名称,二者可互换使用。
在本说明书中,术语“IA”是指合成的铁醇盐,二者可互换使用。
本发明所用反应物、试剂均为市售。
有益效果
本发明公开了一种基于铁醇盐纳米酶的As5+比色检测方法。利用铁醇盐纳米酶比色检测As5+,检测过程条件温和,不需要其他试剂,实现了As5+的方便、快速检测,且检测成本低廉,操作简单;检测范围宽至3.33~333.33μg·L-1,检测限为1.57μg·L-1,可以完全满足世界卫生组织对砷离子的最低限制(10μg·L-1)。铁醇盐纳米酶比色检测As5+具有较高的选择性,可以避免大量共存离子的干扰。
附图说明
图1.IA的扫描电镜图;
图2.IA+TMB、IA和TMB体系的全谱图;
图3.超氧阴离子的电子自旋共振(ESR)谱图;
图4.As5+浓度-吸光度标准工作曲线图;
图5.As5+传感机理示意图;
图6.IA+TMB体系对As5+检测选择性图。
具体实施方式
下面结合实施例对本发明进行详细说明,以使本领域技术人员更好地理解本发明,但本发明并不局限于以下实施例。
实施例1
1.铁醇盐纳米酶制备
1.2g FeCl3·6H2O和5.4g尿素分散在180mL乙二醇里,机械搅拌20min,混合溶液195℃反应30min;铁醇盐通过离心收集,并用乙醇和去离子水洗三次,在60℃干燥24h。其形貌为花状球体,如图1所示。
取制备好的100μL铁醇盐纳米酶(1mg·mL-1,用去离子水溶解)分散在2700μL的醋酸-醋酸盐缓冲溶液中(0.2M,pH 4.0),将100μL的TMB溶液(5mM,用乙醇溶解)加入上述混合溶液中,并孵化20min。用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录吸光度,全谱图如图2所示。
利用ESR检测体系中产生的超氧阴离子,ESR谱图如图3所示。
2.基于铁醇盐纳米酶的As5+比色检测方法
100μL的IA(1mg mL-1,用去离子水溶解)分散在2700μL的醋酸-醋酸盐缓冲溶液中(0.2M,pH 4.0);然后将100μL的不同浓度的As5+(3.33至333.33μg·L-1)加入混合溶液中,孵化2min;分别将100μL的TMB溶液(5mM,用乙醇溶解)加入上述混合溶液中,孵化20min;用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录波长为652nm 处的吸光度并绘制As5+浓度-吸光度标准工作曲线(图4)。
图5为As5+传感机理示意图。
此外,As5+检测选择性通过检测加入不同离子的IA+TMB体系吸光度来考察,如图6所示,IA+TMB体系对As5+具有较高的检测选择性。
实施例2
1.铁醇盐纳米酶制备
1.2g FeCl3·6H2O和5.4g尿素分散在90mL乙二醇里,机械搅拌10min,混合溶液150℃反应20min;铁醇盐通过离心收集,并用乙醇和去离子水洗三次,60℃干燥24h。
取制备好的100μL铁醇盐纳米酶(1mg·mL-1,用去离子水溶解)分散在2700μL的醋酸-醋酸盐缓冲溶液中(0.2M,pH 4.0)。100μL的TMB溶液(5mM,用乙醇溶解)加入上述混合溶液中,孵化20min。用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录吸光度。
2.基于铁醇盐纳米酶的As5+比色检测方法
100μL的IA(1mg·mL-1,用去离子水溶解)分散在2700μL的醋酸-醋酸盐缓冲溶液中(0.2M,pH 4.0);然后将100μL的不同浓度的As5+(3.33至333.33μg·L-1)加入混合溶液中,孵化0.5min;分别100μL的TMB溶液(5mM,用乙醇溶解)加入上述混合溶液中,孵化10min;用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录波长为652nm 处的吸光度并绘制As5+浓度-吸光度标准工作曲线。
实施例3
1.铁醇盐纳米酶制备
1.2g FeCl3·6H2O和5.4g尿素分散在135mL乙二醇里,机械搅拌15min,混合溶液175℃反应25min;铁醇盐通过离心收集,并用乙醇和去离子水洗三次,60℃干燥24h。
取制备好的100μL铁醇盐纳米酶(1mg·mL-1,用去离子水溶解)分散在2700μL的醋酸-醋酸盐缓冲溶液中(0.2M,pH 4.0);100μL的TMB溶液(5mM,用乙醇溶解)加入上述混合溶液中,孵化20min。用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录吸光度。
2.基于铁醇盐纳米酶的As5+比色检测方法
100μL的IA(1mg·mL-1,用去离子水溶解)分散在2700μL的醋酸-醋酸盐缓冲溶液中(0.2M,pH 4.0);然后将100μL的不同浓度的As5+(3.33至333.33μg·L-1)加入混合溶液中,孵化1min;100μL的TMB溶液(5mM,用乙醇溶解)加入上述混合溶液中,孵化15 min;用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录波长为652nm处的吸光度并绘制As5+浓度-吸光度标准工作曲线。
实施例4
1.铁醇盐纳米酶制备
1.2g FeCl3·6H2O和5.4g尿素分散在225mL乙二醇里,机械搅拌25min,混合溶液225℃反应35min;铁醇盐通过离心收集,并用乙醇和去离子水洗三次,60℃干燥24h。
取制备好的100μL铁醇盐纳米酶(1mg·mL-1,用去离子水溶解)分散在2700μL的醋酸-醋酸盐缓冲溶液中(0.2M,pH 4.0);将100μL的TMB溶液(5mM,用乙醇溶解)加入上述混合溶液中,孵化20min;用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录吸光度。
2.基于铁醇盐纳米酶的As5+比色检测方法
100μL的IA(1mg·mL-1,用去离子水溶解)分散在2700μL的醋酸-醋酸盐缓冲溶液中(0.2M,pH 4.0);然后将100μL的不同浓度的As5+(3.33至333.33μg·L-1)加入混合溶液中,孵化3min;100μL的TMB溶液(5mM,用乙醇溶解)加入上述混合溶液中,孵化25 min;用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录波长为652nm处的吸光度并绘制As5+浓度-吸光度标准工作曲线。
实施例5
1.铁醇盐纳米酶制备
1.2g FeCl3·6H2O和5.4g尿素分散在270mL乙二醇里,机械搅拌30min,混合溶液在250℃反应40min;铁醇盐通过离心收集,并用乙醇和去离子水洗三次,60℃干燥24h。
取制备好的100μL铁醇盐纳米酶(1mg·mL-1,用去离子水溶解)分散在2700μL的醋酸-醋酸盐缓冲溶液中(0.2M,pH 4.0);100μL的TMB溶液(5mM,用乙醇溶解)加入上述混合溶液中,孵化20min;用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录吸光度。
2.基于铁醇盐纳米酶的As5+比色检测方法的建立
100μL的IA(1mg·mL-1,用去离子水溶解)分散在2700μL的醋酸-醋酸盐缓冲溶液中(0.2M,pH 4.0);然后将100μL的不同浓度的As5+(3.33至333.33μg L-1)加入混合溶液中,孵化5min;100μL的TMB溶液(5mM,用乙醇溶解)加入上述混合溶液中,孵化30 min;用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录波长为652nm处的吸光度并绘制As5+浓度-吸光度标准工作曲线。
以上所述,仅为本发明较佳的具体实施方式。当然,本发明还可有其它多种实施例,在不背离本发明精神及其实质的情况下,任何熟悉本技术领域的技术人员,当可根据本发明作出各种相应的等效改变和变形,都应属于本发明所附的权利要求的保护范围。
Claims (9)
1.一种基于铁醇盐纳米酶的As5+比色检测法,其特征在于,检测步骤如下:
(1)用去离子水配制1mg·mL-1的铁醇盐纳米酶溶液,分别取100μL的铁醇盐纳米酶溶液分散在2700μL醋酸-醋酸盐缓冲溶液中,加入100μL不同浓度的As5+,孵化0.5~5min;所述铁醇盐纳米酶,其制备步骤包括:
A、按固液比为7.5mM:9g:150~450mL,将FeCl3·6H2O或Fe(NO3)3·9H2O和尿素分散在乙二醇里,充分溶解成混合溶液;
B、混合溶液在150~250℃溶剂热反应20~40min;
C、铁醇盐纳米酶离心收集,并用乙醇和去离子水洗净,50~70℃干燥18~30h;
(2)分别将100μL的5mM的TMB乙醇溶液加入上述混合溶液中,孵化10~30min;
(3)用紫外-可见吸收分光光度计测定混合溶液紫外吸收光谱,记录波长为652nm处的吸光度并绘制As5+浓度-吸光度标准工作曲线;
(4)取100μL的1mg·mL-1铁醇盐纳米酶溶液分散在2700μL醋酸-醋酸盐缓冲溶液中,加入100μL待测As5+样品,孵化0.5~5min,再加入100μL的5mM的TMB乙醇溶液,孵化10~30min,以紫外-可见吸收分光光度计测定652nm处的吸光度;
(5)通过计算与As5+浓度-吸光度标准工作曲线比对,即可获得待测样品的As5+浓度。
2.根据权利要求1所述基于铁醇盐纳米酶的As5+比色检测法,其特征在于:步骤(1)所述醋酸-醋酸盐缓冲溶液浓度0.2M,pH为4.0。
3.根据权利要求1所述基于铁醇盐纳米酶的As5+比色检测法,其特征在于:步骤(1)所述As5+溶液浓度为3.33~333.33μg·L-1。
4.根据权利要求1所述基于铁醇盐纳米酶的As5+比色检测法,其特征在于:步骤(1)所述用去离子水配制1mg·mL-1的铁醇盐纳米酶溶液,分别取100μL的铁醇盐纳米酶溶液分散在2700μL醋酸-醋酸盐缓冲溶液中,加入100μL不同浓度的As5+,孵化2min。
5.根据权利要求1所述基于铁醇盐纳米酶的As5+比色检测法,其特征在于:步骤(1)A中所述固液比为7.5mM:9g:300mL。
6.根据权利要求1所述基于铁醇盐纳米酶的As5+比色检测法,其特征在于:步骤(1)B中所述混合溶液在195℃反应30min。
7.根据权利要求1所述基于铁醇盐纳米酶的As5+比色检测法,其特征在于:步骤(1)C中60℃干燥24h后即得。
8.根据权利要求1所述基于铁醇盐纳米酶的As5+比色检测法,其特征在于:步骤(2)所述分别将100μL的5mM的TMB乙醇溶液加入上述混合溶液中,孵化20min。
9.根据权利要求1所述基于铁醇盐纳米酶的As5+比色检测法,其特征在于:步骤(4)所述待测As5+样品检测范围为3.33~333.33μg·L-1,检测低限为1.57μg·L-1。
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