CN107764763B - 碘离子信号增强的双氧水比色检测方法 - Google Patents
碘离子信号增强的双氧水比色检测方法 Download PDFInfo
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Abstract
碘离子信号增强的双氧水比色检测方法,属于紫外比色可见检测技术领域。合成Pt/COF‑LZUl、Cu(Ⅱ)‑HKUST‑1、Pt/Ru/C纳米材料中的至少一种,将其中一种加入蒸馏水,超声分散得分散液,将分散液加入到TMB中,加入待测浓度的双氧水,加入Tris缓冲溶液,加入KI水溶液,在650nm处用紫外分光光度计测其吸光度值。由于不依赖于过氧化物酶等蛋白酶的催化活性,稳定性高,不会因高浓度的盐离子发生团聚而产生假阳(阴)性信号。在温和条件下,纳米框架材料能增强碘离子的催化活性,能催化溶解氧氧化显色底物TMB,这表明碘离子能够信号放大,可提高体系的灵敏度,方便成本低稳定性好。
Description
技术领域
本发明属于紫外比色可见检测技术领域,具体为一种可视化检测生物分子的方法。
背景技术
生物体内存在各种各样的生物分子,如蛋白质、氨基酸以及小分子代谢物等,它们有着特殊的生理功能,并在生命活动中发挥着至关重要的作用。因此,准确实时监控体内这些生物分子的浓度变化和分布情况,对于研究生物体的各种生理和病理变化、以及进行疾病的早期诊断等都很重要。比色法(colorimetry)是通过比较或测量有色物质溶液的颜色深度来确定待测组分含量的方法。比色法显色稳定、重现性好、可视化、操作简便、价格低廉。目前,常见的比色法大致可分为:金属纳米粒子介导的可视化策略、刻蚀金银纳米粒子的可视化策略和基于过氧化物酶催化活动的可视化策略。其中基于过氧化物酶催化活动的可视化策略包括酶联免疫法(ELISA)、脱氧核酶(DNAzyme)法、纳米模拟酶法等。虽然上述方法应用广泛,但存在以下难题:
(1)金属纳米比色检测法采用金属纳米颗粒为信号探针,识别分子修饰的纳米金颗粒的聚集而导致的表面等离子体共振特性的颜色变化,然而在高浓度盐离子情况下,纳米金颗粒易团聚而产生假阳或阴性信号。
(2)在ELISA中,其底物一般为无色化合物,经蛋白酶作用后成为有色的产物,通常是过氧化物酶(HRP)或碱性磷酸酶来催化底物转化成有色的产物,从而进行定量分析。在一些苛刻的条件,例如当高温或重金属离子(如汞)存在时,蛋白质酶的活性会发生不可逆的变性,从而限制了ELISA在实际中的广泛应用。
(3)灵敏度不高,且需要复杂的设计及优化过程。
(4)金银纳米粒子的形貌容易受环境、pH值以及谷胱甘肽等的影响,从而导致刻蚀金银纳米粒子的可视化策略容易产生假阳性信号。
有机框架包括共价有机框架(COFs)和金属有机框架(MOFs)。由于有机框架具有孔道结构高度有序、孔径可调、比表面积较大、合成方法多样和易于功能化修饰等优点,吸引了越来越多的科研工作者的兴趣,并已成功的应用于氢气存储、气体吸附与分离、传感器、药物缓释、催化反应等领域。当纳米框架材料存在时,碘催化氧化还原性底物四甲基联苯胺(TMB)的催化能力能够显著地增强。TMB是非常好的酶显色试剂,能溶解于多种有机溶剂和蒸馏水中,为稳定的无色溶液,与适量双氧水与缓冲液混匀后,能与过氧化物酶作用产生清晰的蓝色产物,极易观察。由于其灵敏度高,稳定性好,配制后的工作液稳定,显色终止后读数稳定,背景低(底物溶液在650nm检测时检测LOD值小于0.04),加上无致癌性,被广泛用于紫外可见的比色检测反应。在H2O2存在的情况时,碘的催化反应是一种经典和常规的比色法,具有高灵敏度,稳定性好,成本低,方便快捷,目标分析响应快,反应前显无色,反应后显蓝色,因此碘催化反应似乎是一种有效的构建比色传感器的平台。近年来,报道了基于使用碘介导的金纳米棒蚀刻的高灵敏比色检测方法[Zhang,Z.;Chen,Z.;Wang,S.;Cheng,F.;Chen,L.Iodine-Mediated Etching of Gold Nanorods for Plasmonic ELISA Based onColorimetric Detection of Alkaline Phosphatase.ACS Appl.Mater.Interfaces2015,7,27639-27645.],也报道了碘化物反应性Cu-Au纳米颗粒型的无标记比色检测平台,用于靶细胞的超敏检测[Ye,X.;Shi,H.;He,X.;Wang,K.;He,D.;Yan,L.;Xu,F.;Lei,Y.;Tang,J.;Yu,Y.Iodide-Responsive Cu–Au Nanoparticle-Based Colorimetric Platformfor Ultrasensitive Detection of Target Cancer Cells.Anal.Chem.2015,87,7141-7147.]。然而这些方法每次检测都需要约55℃的高温和高的离心速度,并且金纳米棒的形貌也容受环境、pH值以及谷胱甘肽等的影响。因此,利用碘化物催化的反应来发展无标记,简单和高灵敏度的比色生物传感器仍然是非常有价值的。换言之,基于碘的催化反应,利用纳米框架材料的信号增强构建新型的灵敏度高、特异性好、简便的检测体内生物分子的比色探针对于疾病的早期诊断具有重要的实际意义,也是十分有必要的。
发明内容
本发明的目的在于克服现有技术的不足,提供一种碘离子信号增强的双氧水比色检测方法。
本发明包括以下步骤:
1、合成Pt/COF-LZUl、Cu(Ⅱ)-HKUST-1、Pt/Ru/C纳米材料中的至少一种;
2、将上述已合成纳米材料中的一种加入蒸馏水,所述纳米材料与蒸馏水的质量体积比0.5~1.5毫克:1毫升,超声分散得分散液;
3、将所述分散液加入到TMB中,分散液与TMB的体积比为1~2:10;
4、向步骤3所得液体中加入待测浓度的双氧水,所述液体与双氧水的体积比为4.5~5:1;
5、向步骤4所得液体中加入Tris缓冲溶液,所述液体与缓冲溶液的体积比为1~1.5:10;
6、向步骤5所得液体中加入KI水溶液,所述液体与KI水溶液的体积比为15.5~16:1,KI水溶液中的KI与蒸馏水的摩尔体积比90~110毫摩尔:1升,
7、最后将上述液体移至比色皿中,并且在650nm处用紫外分光光度计测其吸光度值。
合成Pt/COF-LZUl、Cu(Ⅱ)-HKUST-1、Pt/Ru/C纳米材料中的方法为已有技术,实施例给出了其文献出处的举例。
本发明的优点:由于不依赖于过氧化物酶等蛋白酶的催化活性,稳定性高,不会因高浓度的盐离子发生团聚而产生假阳(阴)性信号。在温和条件下,纳米框架材料能增强碘离子的催化活性,能催化溶解氧氧化显色底物TMB,这表明碘离子能够信号放大,可以提高体系的灵敏度,是一种新型的比色检测方法。灵敏度高,同时也显示了过氧化物酶活性介导法的优点,如方便、成本低、稳定性好等。依据氧化后溶液在652nm处的吸光度与碘离子浓度的关系,建立了一种简易的可视化检测生物分子的方法。
附图说明
图1为实施例不同纳米材料的透射电镜图(TEM图),其中的(a)为MOFs材料,40000X;(b)为Pt/Ru/C材料,80000X;(c,d)为COFs-Pd材料,15000X,400000X。
图2为实施例不同条件下的紫外吸收峰值,其中的a、b、c、d、e、f分别为a:KI+H2O2;b:TMB+H2O2;c:TMB+H2O2+KI;d:COFs-Pd+TMB+H2O2+KI;e:MOFs+TMB+H2O2+KI;f:Pt/Ru/C+TMB+KI+H2O2。
图3为实施例的传感体系存在与不存在KI时的紫外吸收光谱图,其中a为不存在KI时空白样品的紫外吸收光谱图;b为不存在KI且H2O2的浓度为50μM的紫外吸收光谱图;c为存在KI时空白样品的紫外吸收光谱谱图;d为存在KI且H2O2的浓度为50μM的紫外吸收光谱图。
图4显示实施例不同的碘离子的浓度对传感性能的影响。
图5显示实施例不同的温度条件对传感性能的影响,其中连接实心点的曲线表示没加KI的情况下温度对传感性能的影响,其中连接空心点的曲线表示加KI的情况下温度对传感性能的影响,连接实心点的曲线是表示选择的是37C°,为传感系统提供了最大的信号与背景比值。
图6为实施例有KI时,加入不同浓度的H2O2之后传感器的紫外吸收光谱图,其中自下到上的12条曲线H2O2的浓度分别为0nM;5nM;25nM;50nM;250nM;500nM;2500nM;5000nM;25000nM;50000nM;250000nM;500000nM。
图7为实施例有KI时,紫外吸收增强值与H2O2的浓度之间的关系图,其中插入了传感器对低浓度目标H2O2的校准曲线。
图8为实施例无KI时,加入不同浓度的H2O2之后传感器的紫外吸收光谱图,其中自下到上的6条曲线分别为5μM;25μM;50μM;250nM;500nM;2500nM。
图9为实施例无KI时,紫外吸收增强值与H2O2的浓度之间的关系图。
图10显示实施检测方法的特异性,control为背景实验,H2O2为双氧水,Gly为甘氨酸,Glu为谷氨酸,Ala为丙氨酸。
具体实施方式
实施例:见图1~10。
1.仪器和试剂
Pd(OAc)2购于昆明铂锐金属材料有限公司(中国,昆明);1,3,5-均三苯甲醛(1,3,5-Triformylbenzene)购于百灵威科技有限公司(中国,北京);1,4-对苯二胺(1,4-diaminobenzene)、1,4-二氧六环(1,4-dioxane)购于阿拉丁试剂有限公司(中国,上海);冰醋酸(CH3COOH)购于成都化学试剂,醋酸钠(CH3COONa)、硼氢化钠(NaBH4)购于麦克林试剂有限公司(中国,上海);甲醇(CH3OH)、无水乙醇(CH3CH2OH)购于成都格雷西亚化学技术有限公司(中国,成都);二氯甲烷(DCM)、四氢呋喃(THF)、N,N-二甲基甲酰胺(DMF)均购于西陇化工有限公司(中国,广州);一水合乙酸铜(Cu(CH3COO)2 .H2O)购于上海MACKLIN生化有限公司(中国,上海);均苯三酸(C9H6O6)购于萨恩化学技术有限公司(中国,上海);XC-72R活性碳购于上海Cabot Corp(中国,上海);氢氧化钾购于北京化工厂(中国,北京);30%过氧化氢(30%H2O2)、柠檬酸三钠和乙二醇购于天津市风船化学试剂科技有限公司(中国,天津);三氯化钌(Ⅲ)水合物(RuCl3 .xH2O)购于上海思域化工科技有限公司(中国,上海);30%过氧化氢(30%H2O2)购于天津市大茂化学试剂厂(中国,天津);氯化钠购于广东光华化学厂有限公司(中国,广州);TMB显色液购于上海碧云天生物技术有限公司(中国,上海)。
TGL16离心机为长沙湘智离心机仪器有限公司产品;PHS-29A型pH计为上海精科雷磁产品;真空干燥箱为上海博迅实业有限公司产品;扫描电子显微镜(SEM)S-3000N为日本Hitachiscience systemsltd公司产品;TEM2100透射电镜为日本电子株式会社产品;ST2200HP超声波清洗器为上海科导超声仪器有限公司产品;TU-1901双光束紫外可见分光光度计为北京普析通用仪器有限公司产品。
2.材料的合成
2.1Pt/COF-LZUl的合成
共价有机框架材料COF-LZUl的合成按现有技术[例如文献:Wang,W.;Ding,S.Y.;Gao,J.;Wang,Q.;Zhang,Y.;Song,W.G.;Su,C.Y.;Wang,S.J.Am.Chem.Soc.2011,133,19816-19822.]:准确称取1,4-苯二胺(0.16g,15mmol),1,3,5-均三三苯甲醛(0.16g,10mmol)于反应管中,加入1,4-二氧六环(10mL)将其溶解混匀,然后缓慢滴加醋酸(2mL,3M),随着醋酸的滴入立即有黄色固体产生,将反应管接入真空线,在液氮冷冻条件下抽真空,赶尽气泡,封管,自然升至室温,然后将其转移至120℃恒温烘箱内反应三天,停止加热,待体系冷却至室温时,打开反应管,加入THF,离心,依次用DMF,THF洗涤,索氏提取(THF作溶剂),60℃真空干燥12小时,得淡黄色固体。
Pt/COF-LZUl的合成:常温常压条件下,称取50mg的COF-LZUl材料分散于25mL甲醇溶液中,超声2h,分散均匀,加入0.04mmol/L H2PtCl6甲醇溶液,搅拌混合均匀,再慢慢加入0.5mmol/L NaBH4溶液,反应2h,离心,采用乙醇多次洗涤,干燥。
2.2Cu(Ⅱ)-HKUST-1的合成
共价有机框架材料MOFs的合成按现有技术[例如文献:Chui,S.S.Y.;Lo,S.M.F.;Charmant,J.P.H.;Orpen,A.G.;Williams,I.D.Science 1999,283,1148-1150.]称取0.420g均苯三甲酸于100mL烧杯中,溶于40mL无水乙醇得到溶液A;再称取0.600g醋酸铜倒入另一烧杯中,同时加入40mL蒸馏水和4mL冰醋酸使其溶解得到溶液B。把B溶液转移至锥形瓶中,在常温搅拌的条件下,通过分液漏斗向B溶液中逐滴加入A溶液,溶液滴加完毕,继续搅拌1h后,离心,水和乙醇各洗3次,于80℃真空干燥12h,即制得Cu(Ⅱ)-HKUST-1纳米颗粒。
2.3Pt/Ru/C纳米复合材料的合成
本实验中所用到的Pt/Ru/C纳米材料催化剂的合成前体为H2PtCl6.6H2O和RuCl3.xH2O。在100mL烧杯中依次加入25.0mL乙二醇、1.0mL的0.05mol/LH2PtCl6.6H2O、1.0mL0.05mol/LRuCl3.xH2O水溶液、0.5mL 0.40mol/LKOH溶液以及0.040g的XC-72R活性炭,超声分散两小时,使其充分混匀。之后将烧杯置于微波炉中间利用微波加热90s,离心,分别用丙酮、去离子水洗涤数次后,分散保存于去离子水中保存备用。
检测方法
具体操作如下:
Ⅰ.在离心管中注入含有纳米材料的溶液。
Ⅱ.加入40μL TMB显色液,轻微振荡使之混合均匀。
Ⅲ.滴加相应浓度和体积的H2O2,轻轻摇晃使其充分反应,遮光反应10min。
Ⅴ.加入Tris缓冲溶液稀释至500μL,轻微振荡,使之混合均匀。
Ⅵ.加入30μL的100mM的碘化钾溶液,轻微振荡,使之充分混合均匀。
Ⅶ.最后将上述混合溶液移至比色皿中,并且在650nm处用紫外分光光度计测其吸光度值。
4.结果与分析
4.1.材料表征
本实施例选取COFs-Pd和MOFs材料作为纳米框架材料的研究对象。利用高倍透射电镜(TEM)确定本实施例中的纳米材料是否合成成功,并观察纳米颗粒的微观形貌特征。由图1(a)可知中MOFs颗粒的平均粒径为100-200nm,呈中空状,分散性好。由图1(b)可知Pt/Ru/C的结构呈球形状,粒径为50nm(2b)。COFs的结构如图1(c)所示,COFs的平均粒径为200nm,与Pt/Ru/C的结构类似呈球形状。当Pd颗粒负载至COFs上后,从图1(d)中可观察到许多小黑点在COFs材料上,说明Pd已负载成功。以上结果证明纳米材料是合成成功的。
4.2.控制实验
为了证明新传感平台的可行性,在相同的条件下,本实施例进行了多个控制实验。a为KI+H2O2;b为TMB+H2O2;c为TMB+H2O2+KI;d为COFs-Pd+TMB+H2O2+KI;e为MOFs+TMB+H2O2+KI;f为Pt/Ru/C+TMB+KI+H2O2。从图2中可知,KI、H2O2、TMB本身不能引起紫外吸收峰的变化(a,b)。当体系加入50μM H2O2,TMB+H2O2+KI同时存在时,紫外吸收值为0.19,混匀后产生清晰的蓝色产物,碘催化氧化还原性底物TMB在H2O2存在时。当体系中存在纳米框架材料(MOFs、COFs-Pd或Pt/Ru/C)时,碘催化显著地增强,紫外吸收值从0.3增加至1.06。COFs-Pd存在时,紫外吸收值为0.25;MOFs存在时,紫外吸收值为0.57;Pt/Ru/C存在时,紫外吸收值为1.06。因此,纳米框架材料确实能提高碘离子的催化反应。
4.3.信号放大
为了考察纳米框架材料能够增强碘离子的催化活性用于信号增强的检测,体系考察了KI存在和不存在时的紫外吸收值,以MOFs材料为例。当体系加入目标双氧水和KI时,该混合物在室温下下孵育10分钟。实验结果如图3所示,当体系没有KI时,传感器的信号为(174±12)%;相反,在相同条件下,采用KI的信号放大策略,传感器的信号为(306±21)%。这些结果证实,此测定法可用于信号放大的检测。
4.4.碘离子的浓度对传感性能的影响
为了达到最佳的传感性能,体系对碘离子的浓度进行了优化。实验结果表明随着碘离子浓度的增大,体系的信号值增大。但是当浓度增加时,背景信号也逐渐增大,6mM的碘离子浓度,为传感系统提供了最大的信号与背景的差值(见图4)。因此,使用6mM的碘离子的浓度进行以后的实验。
4.5不同温度条件对传感性能的影响
为了达到最佳的传感性能,本实例进行不同的温度条件下的优化。实验结果表明随着温度的增大,体系的信号值增大。但是当温度超过37℃时,背景信号也逐渐增大,37℃条件为传感系统提供了最大的信号与背景比值(见图5)。因此,使用37℃的温度进行以后的实验。
4.6.分析性能
为了考察该传感器可以用于生物分子的定量分析,在最佳的反应条件下,传感体系检测了一系列不同浓度的双氧水。如见图6所示,随着双氧水的浓度的增加,从5nM到500μM时时,体系的紫外吸收峰强度逐渐地增大;见图7描述了紫外吸收响应与双氧水浓度的关系。所提出的传感器灵敏高,根据3δ/斜率计算(δ,空白样品的标准偏差),检测下限为1nM。这个检测下限比传统的检测双氧水的方法好了一个数量级。
除此之外,我们进行了无KI的对照实验。随着双氧水的浓度的增加,从5μM到2.5mM时,体系的紫外吸收峰强度逐渐地增大检测下限为1μM(见图8和图9)。这个结果比KI参与的放大的灵敏度差了3个数量级。这些结果表明引入KI显著提高了生物传感器的灵敏度。
特异性是一个成功的实验体系的另一个关键因素。在相同的实验条件下,我们研究了由各种非目标物质引起的非特异性吸附的紫外吸收的强度变化。如见图10所示,当加入高浓度的非目标物质时,只有双氧水才能引起紫外吸收的增强。上述结果表明,上述检测方法具有很好的特异性,所提出的检测平台能够满足生物医学应用的选择性要求。
本实施例为一种新型纳米框架材料的碘离子信号增强的新型比色检测方法,在温和条件下,纳米框架材料能增强碘离子的催化活性,能催化溶解氧氧化显色底物TMB,碘离子能够信号放大。基于碘增强的比色法测定不仅拥有响应速度快,灵敏度高的特性,同时也显示了过氧化物酶的活性介导法的优点,如方便、成本低、稳定性好等。依据氧化后溶液在652nm处的吸光度与碘离子浓度的关系,建立了一种简易的可视化检测生物分子的方法。
Claims (1)
1.一种碘离子信号增强的双氧水比色检测方法,其特征在于包括以下步骤:
(1)、合成Pt/COF-LZUl、Cu(Ⅱ)-HKUST-1、Pt/Ru/C纳米材料中的至少一种;
(2)、将上述已合成纳米材料中的一种加入蒸馏水,所述纳米材料与蒸馏水的质量体积比0.5~1.5毫克:1毫升,超声分散得分散液;
(3)、将所述分散液加入到TMB中,分散液与TMB的体积比为1~2:10;
(4)、向步骤(3)所得液体中加入待测浓度的双氧水,所述液体与双氧水的体积比为4.5~5:1;
(5)、向步骤(4)所得液体中加入Tris缓冲溶液,所述液体与缓冲溶液的体积比为1~1.5:10;
(6)、向步骤(5)所得液体中加入KI水溶液,所述液体与KI水溶液的体积比为15.5~16:1,KI水溶液中的KI与蒸馏水的摩尔体积比90~110毫摩尔:1升,
(7)、最后将上述液体移至比色皿中,并且在650nm处用紫外分光光度计测其吸光度值。
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