CN112557383B - 一种基于MnO2复合酶模拟物的铜离子比色检测方法 - Google Patents
一种基于MnO2复合酶模拟物的铜离子比色检测方法 Download PDFInfo
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Abstract
本发明一种基于MnO2复合酶模拟物的铜离子比色检测方法及应用,包括以下步骤:配置不同浓度的铜离子和L‑cys溶液;合成MB和MB/Au/PANI/MnO2复合材料;配制NaAC‑HAC缓冲液;配制TMB溶液;条件优化;对铜离子比色检测。本发明提供的基于MnO2复合酶模拟物的铜离子比色检测方法及应用,MB/Au/PANI/MnO2复合材料作为复合酶模拟物,具有强催化性,实现对铜离子的高灵敏、定量检测,提高目标物检测的选择性和稳定性,将复合酶模拟物换成掺杂MnO2的其他复合物可应用于新目标物的检测分析,通用性强,检测方法简单、便捷,成本低廉,原料易得、无毒,可应用于实际样品检测,适合工业推广。
Description
技术领域
本发明属于比色检测技术领域,具体涉及一种基于MnO2复合酶模拟物的铜离子比色检测方法及应用。
背景技术
目前,业内常用的现有技术是这样的:
铜是人体必需微量元素,在人体一系列基本生理过程和活动中起着重要的作用。铜参与多种生物酶的形成并保持酶的活性,使电子传递和氧化还原代谢生命活动正常进行。铜还参与红细胞生成,以维持正常的造血功能。此外,儿童抽搐与人体内铜含量密切相关。但是,过量的铜离子(Cu2+)也会导致一系列健康问题,包括缺血性心脏病、贫血、肾病、骨骼疾病、老年痴呆症、朊病毒、帕金森疾病等。更重要的是,在水中Cu2+含量过高会对水生生物有不利影响。世卫组织和美国环境保护署已将饮用水中Cu2+的最大允许限量设定为1.3ppm。中国国家标准表明,自来水中Cu2+的最大浓度限制为1 mg/L。因此,在适当范围内监测环境中Cu2+的浓度至关重要。目前检测Cu2+的传统方法通常使用原子吸收光谱法、原子荧光光谱法和电感耦合等离子体质谱法,但这些方法耗时长、样品制备复杂,需要配备专业操作人员。因此,无论是从生物角度还是从环境角度,都需要设计一种简单定量检测Cu2+的分析方法。比色化学传感器因其操作简单、直观、选择性高、响应时间短等优点,是一种替代性的设备,能够在水介质中通过显示颜色变化检测目标离子,可实现裸眼检测和现场检测。
辣根过氧化物酶(Horseradish Peroxidase,HRP)是一种非常常用的酶,对H2O2的分解具有高效的催化作用,被广泛应用于生物感应的标记,如酶联免疫吸附试验。2019年,研究人员以棉线作为固体吸附剂,提高了棉线上局部Cu2+浓度,利用抗坏血酸将Cu2+还原为Cu+,通过观察HRP催化氧化无色TMB(四甲基联苯胺,3,3’,5,5’-Tetramethylbenzidine)为蓝色氧化态(ox TMB)的颜色变化,得出Cu+具有抑制HRP催化氧化TMB的作用,从而实现定量检测Cu2+,检测限为0.15 nmol/L,与其他分析方法相比,提出了一种灵敏、轻便的在线预浓缩比色检测Cu2+的方法。然而。这个比色化学传感器以HRP作为酶催化反应,HRP具有易变性、成本高、制备复杂、反应孵育时间长等缺点,大大限制了其实际应用。因此,研究具有制备简单、性质稳定、环境耐受性强等优点的酶对于检测生物分子具有重要意义。
模拟酶,又被称为纳米酶,近年来,无机纳米材料作为一种仿生纳米酶的应用在研究领域引起了越来越大的兴趣。仿生纳米酶是一类化学合成的纳米材料,具有与某些天然酶相似的生物催化活性。纳米酶结构比天然酶简单、化学性质稳定,不仅具有酶的功能,还具有批量生产和成本低的优点。此外,纳米酶本质是一种纳米材料,具有较高的比表面积和特殊的理化性质,如光、电、磁等,纳米材料独特的生化特性不仅使纳米酶具有多种功能,而且使其能够进行多种设计和应用。在纳米材料中加入磁性纳米材料,如Fe3O4,利用外加磁场可以有效地将纳米材料从溶液中分离出来,并且很容易回收利用。然而,未修饰的Fe3O4不仅容易团聚和氧化,而且在酸性介质中不稳定,限制了其广泛的应用。聚苯胺(Polyaniline,PANI)是一种高效的Fe3O4纳米粒子保护剂,近年来,PANI因其反应条件稳定性好、无毒、成本低而被开发为催化载体。金Au是一种具有催化活性的金属,具有反应条件温和、操作简单、反应时间短、反应选择性高等特性。MnO2是一种由人工制作的纳米酶,具有较好稳定性,具有大小不均的多孔结构及特定晶体结构,这种构造有助于催化作用。磁珠(MagneticBeads,MB)是一种具有较高导磁率、廉价易用的抗干扰元件,此外还有比表面积大、稳定性好、低毒等特点。因此,将MB、Au、PANI、MnO2制备成复合物对于检测Cu2+具有重要意义。
半胱氨酸(L-Cysteine,L-Cys)可以生成谷胱甘肽,谷胱甘肽是最主要及最强的抗氧化剂。谷胱甘肽是属于含有巯基的、小分子肽类物质,半胱氨酸上的巯基为其活性基团,L-Cys与Cu2+可形成不溶性的硫醇盐(mercaptide)。在L-Cys与Cu2+反应后,溶液在催化材料存在下发生显色。
比色检测是以有色化合物的显色反应为基础,通过比较(目视比色法)或测量(紫外可见光谱法)有色物质溶液颜色深度来确定待测组分含量的方法。比色检测成本低、操作简便、检测时长短、肉眼快速定性检测,适用于快速检测。比色检测是一种广泛使用的检测方法之一,ELISE中复合材料及TMB存在下与待测物的含量呈相关性,酶催化底物生成有色产物,有色产物的量与待测物的量直接相关,可通过颜色深浅进行定性或定量分析。由于酶催化效率高,比色检测灵敏度高,因此,利用比色检测具有重要意义。
综上所述,现有技术存在的问题是:
(1)用于检测Cu2+方法,如吸收光谱法、电化学法、离子色谱法,这些方法成本高、耗时长、需要精密仪器设备及专业操作技术人员。
(2)目前,用于Cu2+检测的简易型分析研究较少,且Cu2+在生物基质中的检测具有一定难度,难以实现低浓度目标物高灵敏度检测。
发明内容
为解决现有技术中存在的技术问题,本发明的目的在于提供一种基于MnO2复合酶模拟物的铜离子比色检测方法及应用,铜离子的检测范围为1.0×102-1.0×107 nM,检测限为0.65 nM。
为实现上述目的,达到上述技术效果,本发明采用的技术方案为:
一种基于MnO2复合酶模拟物的铜离子比色检测方法,包括以下步骤:
S101:配置不同浓度铜离子和L-Cys溶液(配置102-107 nmol/L的Cu2+及L-Cys溶液)
S102:合成磁珠MB和MB/Au/PANI/MnO2复合材料
S103:配制NaAC-HAC缓冲液
S104:配制TMB溶液
S105:条件优化
S106:对铜离子进行比色检测。
进一步的,步骤S101中,称取CuSO4·5H2O,配置成硫酸铜溶液,随后稀释若干次,得100 nmol/L Cu2+,所配溶液置于4℃保存。
进一步的,步骤S101中,称取L-Cys,配成10 mL、400 μmol/L溶液,所配溶液置于4℃保存。
进一步的,步骤S102中,MB的合成步骤包括:
HCl溶液中通氮气20-25 min,称取FeCl2·4H2O和FeCl3·6H2O置于圆底烧瓶中,加入以上通氮气的HCl溶液,充分搅拌至完全溶解,混合液中通氮气除氧20-25 min ,重复通入数次,然后快速加入NaOH溶液,在氮气保护下剧烈搅拌2-2.1 h,用二次水冲洗数次至中性,一部分置于4 ℃保存待用,另一部分自然晾干待用。
进一步的,步骤S102中,MB/Au/PANI/MnO2复合材料的合成步骤包括:
水洗后的磁珠MB加入PVP溶液中,振荡摇匀,加入氯金酸,超声5-6min,备用,记为溶液A;
苯胺与HCL溶液混合搅拌均匀,记为溶液B;
将溶液A与溶液B混合,同时加入过硫酸铵,振匀,静置2-2.1 h,磁性分离、清洗、烘干,再分散于水中,超声均匀,加入KMnO4溶液,振匀,磁性分离、清洗,即得所需MB/Au/PANI/MnO2复合材料。
进一步的,步骤S103中,NaAC-HAC缓冲液的配制步骤包括:
称取乙酸钠,稀释定容至100 mL,用乙酸调pH至4,得NaAC-HAC缓冲液。
进一步的,步骤S104中,TMB溶液要现配现用,TMB溶液的制备步骤包括:
A液:TMB溶于DMSO,搅拌均匀,备用;
B液:柠檬酸加入NaHPO4搅拌均匀,定容至100 mL;
随后将制备好的A液全部加入10 mL B液中,超声均匀,得所需TMB溶液。
进一步的,步骤S105中,对MB/PANI/Au/MnO2复合材料与TMB反应体积的优化、L-Cys与MB/PANI/Au/MnO2复合材料反应体积的优化、Cu2+与L-Cys反应时间的优化、L-Cys与MB/PANI/Au/MnO2复合材料反应时间的优化、MB/PANI/Au/MnO2复合材料与TMB反应时间优化、缓冲液pH的优化。
进一步的,步骤S106中,取NaAC-HAC缓冲液,加入L-Cys溶液与Cu2+反应,随后加入MB/PANI/Au/MnO2复合材料,再加入TMB溶液反应后进行比色检测,测定吸光度。
本发明公开了一种基于MnO2复合酶模拟物的铜离子比色检测方法在检测除铜离子的目标物中的应用,将MB/PAMAM/MnO2复合材料替换成掺杂MnO2的其他复合物。
与现有技术相比,本发明的有益效果为:
MnO2复合酶模拟物,即MB/Au/PANI/MnO2 复合材料,具有强催化性,可催化TMB氧化成蓝色阳离子自由基氧化四甲基联苯胺(oxidized TMB,ox TMB),L-Cys可有效抑制阳离子自由基的生成,使其还原为无色TMB分子,L-Cys与Cu2+可形成不溶性硫醇盐,通过比色分析,实现Cu2+的定量检测,提高了Cu2+检测灵敏度、目标物检测的选择性和稳定性;
相比于HRP,MnO2具有较好化学稳定性、操作简易、成本低、易修饰等特点,可避免使用高成本且不稳定、难操作的生物酶;通过利用MB/Au/PANI/MnO2复合材料,不仅可用于Cu2+定量检测,还可将MB/Au/PANI/MnO2复合材料换成掺杂MnO2的其他复合物并应用于新目标物的检测分析,通用性强,灵敏度高;
纳米酶催化氧化TMB与比色分析方法相结合,构建Cu2+为检测对象的比色分析新方法,提高了分析灵敏度,与传统的Cu2+检测方法相比,其避免了设备昂贵、预处理复杂、操作规程繁琐、毒性大等缺点,检测方法简便、高效、成本低且稳定,原料易得、无毒,可应用于实际样品检测,适合工业化推广使用。
附图说明
图1为本发明的工作原理流程图;
图2为本发明的原理示意图;
图3为本发明在波长652nm下的TMB与MB/Au/PANI/MnO2复合材料反应的不同体积对Cu2+的影响曲线图;
图4为本发明在波长652nm下的TMB与MB/Au/PANI/MnO2复合材料的反应时间对Cu2+的影响曲线图;
图5为本发明Cu2+检测的选择性实验结果图;
图6为本发明的不同Cu2+浓度的标准工作曲线图;
图7为本发明的不同Cu2+浓度的UV-vis响应曲线图。
具体实施方式
下面对本发明的实施例进行详细阐述,以使本发明的优点和特征能更易于被本领域技术人员理解,从而对本发明的保护范围做出更为清楚明确的界定。
实施例1
如图1-7所示,一种基于MnO2复合酶模拟物的铜离子比色检测方法,以铜离子Cu2+检测为例,作用机理为:
MB/PANI/MnO2复合材料作为复合酶模拟物,可以催化TMB氧化成蓝色阳离子自由基,L-Cys可以有效抑制阳离子自由基的生成,使其还原成无色TMB分子,L-Cys与Cu2+可形成不溶性的硫醇盐。在L-Cys与不同浓度的Cu2+反应后,溶液在MB/PANI/MnO2复合材料及TMB的存在条件下变为蓝色,且在652nm波长下的吸光度值随铜离子浓度变化而变化,从而达到检测铜离子的目的,利用这个特点,可以建立一种简便、成本低、快速、高效且稳定的检测新方法。
本发明还公开了一种基于MnO2复合酶模拟物的铜离子比色检测方法在检测除铜离子的目标物中的应用,将MB/PAMAM/MnO2复合材料替换成掺杂MnO2的其他复合物即可进行其他目标物的检测分析。
研究不同浓度的Cu2+对检测的影响及MB和MB/Au/PANI/MnO2复合材料的合成:
(1)配置102-107 nmol/L的Cu2+及L-Cys溶液
称0.0025 g CuSO4·5H2O配成1 mL、107 nmol/L的硫酸铜溶液。取100 μL、107 nmol/L硫酸铜溶液,稀释10倍得106 nmol/L Cu2+;再取100 μL、106 nmol/L稀释10倍得105nmol/L Cu2+;依照此法配制104 nmol/L Cu2+、103 nmol/L Cu2+、100 nmol/L Cu2+。
称0.00048g的L-cys配成10 mL、400 μmol/L溶液,所配溶液均置于4℃保存。
(2)磁珠MB和MB/Au/PANI/MnO2复合材料的合成
MB的合成步骤包括:
取30 mL、1.2 mmol/L HCl通氮气20 min,称取0.29815 g FeCl2·4H2O、0.81087 gFeCl3·6H2O置于容器中,加入以上通氮气的30 mL、1.2 mmol/L HCl溶液,充分搅拌至完全溶解,混合液通氮气20 min,重复通入三次,然后快速加入30 mL、1.25 mol/L NaOH,在氮气保护下剧烈搅拌2 h,用二次水冲洗三次至中性,一部分置于4 ℃保存待用,另一部分自然晾干待用。
MB/Au/PANI/MnO2复合材料的合成步骤包括:
水洗后的MB取1ml加入PVP溶液中(0.1 g PVP+10 ml水),置于恒温振荡器震荡1h,滴加2 mL质量分数1%的HAuCl4,超声5 min,备用,记为溶液A;
将0.3 mL苯胺与10 mL 1 mol/L HCl搅拌混合均匀,记为溶液B;
溶液A与溶液B混合,加入0.18 g过硫酸铵振荡1 h,静止2 h后进行磁性分离,用乙醇清洗沉淀物1~2次,在50~60℃下烘干;
取烘干产物5 mg分散于5 mL水中,超声,加入5 mg KMnO4,振荡1 h后进行磁性分离,水洗沉淀物1~2次,装入2 mL的离心管中,即得MB/Au/PANI/MnO2复合材料,随后将合成的MB/Au/PANI/MnO2复合材料稀释四倍,得实验用MB/Au/PANI/MnO2复合材料,每次取实验用MB/Au/PANI/MnO2复合材料时均需混匀25 s。
(3)缓冲溶液制备
称取乙酸钠(NaAC)8.3 g,稀释定容至100 mL,用1.75 mol/L乙酸(HAC)调pH至4,得0.6 mol/L、pH=4的NaAC-HAC缓冲液。
实施例2
TMB溶液的制备步骤包括:
TMB溶液要现配现用。
A液:0.0143 g TMB溶于100 μL DMSO,搅拌均匀,备用;
B液:24.3 mL、0.1 mol/L 柠檬酸加入5.7 mL、0.2 mol/L NaH2PO4搅拌均匀,定容至100 mL;
随后将制备好的A液全部加入10 mL B液中,超声均匀,得实验用6 mmol/L TMB溶液,4℃避光保存。
实施例3
Cu2+的比色检测:
S101:称0.0025 g CuSO4·5H2O配成1 mL、107 nmol/L的硫酸铜溶液。取100 μL、107 nmol/L的硫酸铜溶液,稀释10倍得106 nmol/L Cu2+;取100 μL、106 nmol/L稀释10倍得105 nmol/L Cu2+;依照此法配制104 nmol/L Cu2+、103 nmol/L Cu2+、102 nmol/L Cu2+。
称0.00048g的L-cys配成10 mL、400 μmol/L溶液,所配溶液均置于4℃保存。
S102:MB的合成步骤包括:
取30 mL、1.2 mmol/L HCl通氮气20 min,称取0.29815 g FeCl2·4H2O、0.81087 gFeCl3·6H2O置于容器中,加入以上通氮气的30 mL、1.2 mmol/L HCl溶液,充分搅拌至完全溶解,混合液通氮气20 min,重复通入三次,然后快速加入30 mL、1.25 mol/L NaOH,在氮气保护下剧烈搅拌2 h,用二次水冲洗三次至中性,一部分置于4 ℃保存待用,另一部分自然晾干待用。
MB/Au/PANI/MnO2复合材料的合成步骤包括:
水洗后的MB取1ml加入PVP溶液中(0.1 g PVP+10 ml水),振荡摇床1 h,加入2 ml1 %氯金酸,超声5min,备用,记为溶液A;
0.3 ml 苯胺+10 ml、1 M HCL,混合搅拌均匀,记为溶液B;
溶液A与溶液B混合,同时加入过硫酸铵0.18 g,振荡1 h,静置2 h,磁性分离,用乙醇清洗1~2次,在50~60 ℃的温度下烘干、称量,取5 mg上述制品分散于5 ml水中,超声均匀,加入5 mg KMnO4,振荡1 h,磁性分离,水洗1~2次,即得所需MB/Au/PANI/MnO2复合材料。
S103:NaAC-HAC缓冲液的配制步骤包括:
称取乙酸钠(NaAC)8.3 g,稀释定容至100 mL,用1.75 mol/L乙酸(HAC)调pH至4,得0.6 mol/L、pH=4的NaAC-HAC缓冲液。
S104:TMB溶液的制备步骤包括:
TMB溶液要现配现用。
A液:0.0143 g TMB溶于100 μL DMSO,搅拌均匀,备用;
B液:24.3 mL、0.1 mol/L 柠檬酸加入5.7 mL、0.2 mol/L NaH2PO4搅拌均匀,定容至100 mL;
随后将制备好的A液全部加入10 mL B液中,超声均匀,得实验用6 mmol/L TMB溶液,4℃避光保存。
S105:条件优化:
MB/PANI/Au/MnO2复合材料与TMB反应体积的优化、L-Cys与MB/PANI/Au/MnO2反应体积的优化、Cu2+与L-Cys反应时间的优化、L-Cys与MB/PANI/Au/MnO2复合材料反应时间的优化、MB/PANI/Au/MnO2复合材料与TMB反应时间优化、缓冲液pH的优化。
图3为本发明在波长652nm下的TMB与MB/Au/PANI/MnO2复合材料反应的不同体积对Cu2+的影响曲线图,结果显示,吸光度信号大小与TMB与MB/Au/PANI/MnO2复合材料反应的体积线性相关,相关系数为0.9817,体积越大,信号值越大。
图4为本发明在波长652nm下的TMB与MB/Au/PANI/MnO2复合材料的反应时间对Cu2+的影响曲线图,结果显示,吸光度信号基本不受反应时间影响。
S106:对Cu2+进行选择性检测:
最佳优化条件下,取线性范围内一浓度(1mM)的铜离子和其10倍量的其他离子(镁离子、铁离子、锌离子、钠离子、钙离子、镉离子、钾离子、锰离子)用本体系反应(L-Cys、MB/PANI/MnO2材料与TMB),检测其吸光度值,紫外扫描波长范围为200~800 nm。
图5所示的结果显示,在镁离子、铁离子、锌离子、钠离子、钙离子、镉离子、钾离子、锰离子的干扰下,本发明的检测方法对铜离子具有良好的选择性。
图6为不同Cu2+浓度的标准工作曲线图,图7为不同Cu2+浓度的UV-vis响应曲线图,吸光度信号大小随铜离子浓度增大而增大。
本发明未具体描述的部分采用现有技术即可,在此不做赘述。
以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
Claims (1)
1.一种基于MnO2复合酶模拟物的铜离子比色检测方法,其特征在于,包括以下步骤:
S101:配置102-107 nmol/L的Cu2+及L-Cys溶液;
S102:合成磁珠和MB/Au/PANI/MnO2复合材料;
S103:配制NaAC-HAC缓冲液;
S104:配制TMB溶液;
S105:条件优化;
S106:对铜离子进行比色检测;
步骤S101中,称取CuSO4·5H2O,配置成硫酸铜溶液,随后稀释若干次,得100 nmol/LCu2+,所配溶液置于4℃保存;
步骤S101中,称取L-Cys,配成10 mL、400 μmol/L溶液,所配溶液置于4℃保存;
步骤S102中,磁珠的合成步骤包括:
HCl溶液中通氮气20-25 min,称取FeCl2·4H2O和FeCl3·6H2O置于圆底烧瓶中,加入以上通氮气的HCl溶液,充分搅拌至完全溶解,混合液中通氮气除氧20-25 min ,重复通入数次,然后快速加入NaOH溶液,在氮气保护下剧烈搅拌2-2.1 h,用二次水冲洗数次至中性,一部分置于4 ℃保存待用,另一部分自然晾干待用;
步骤S102中,MB/Au/PANI/MnO2复合材料的合成步骤包括:
水洗后的MB加入PVP溶液中,振荡摇匀,加入氯金酸,超声5-6min,备用,记为溶液A;
苯胺与HCL溶液混合搅拌均匀,记为溶液B;
将溶液A与溶液B混合,同时加入过硫酸铵,振匀,静置2-2.1 h,磁性分离、清洗、烘干,再分散于水中,超声均匀,加入KMnO4溶液,振匀,磁性分离、清洗,即得所需MB/Au/PANI/MnO2复合材料;
步骤S103中,NaAC-HAC缓冲液的配制步骤包括:
称取乙酸钠,稀释定容至100 mL,用乙酸调pH至4,得NaAC-HAC缓冲液;
步骤S104中,TMB溶液要现配现用,TMB溶液的制备步骤包括:
A液:TMB溶于DMSO,搅拌均匀,备用;
B液:柠檬酸加入NaHPO4搅拌均匀,定容至100 mL;
随后将制备好的A液全部加入10 mL B液中,超声均匀,得所需TMB溶液;
步骤S105中,对MB/PANI/Au/MnO2复合材料与TMB反应体积的优化、L-Cys与MB/PANI/Au/MnO2复合材料反应体积的优化、Cu2+与L-Cys反应时间的优化、L-Cys与MB/PANI/Au/MnO2复合材料反应时间的优化、MB/PANI/Au/MnO2复合材料与TMB反应时间优化、缓冲液pH的优化;
步骤S106中,取NaAC-HAC缓冲液,加入L-Cys溶液与Cu2+反应,随后加入MB/PANI/Au/MnO2复合材料,再加入TMB溶液反应后进行比色检测,测定吸光度;
MB/PANI/Au/MnO2复合材料复合材料具有强催化性,催化TMB氧化成蓝色阳离子自由基氧化四甲基联苯胺,L-Cys有效抑制阳离子自由基的生成,使其还原为无色TMB分子,L-Cys与Cu2+形成不溶性硫醇盐,通过比色分析,实现Cu2+的定量检测,铜离子的检测范围为1.0×102-1.0×107 nM,检测限为0.65 nM。
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