CN111744472A - 一种多孔硅铂金纳米酶材料的制备方法及应用 - Google Patents
一种多孔硅铂金纳米酶材料的制备方法及应用 Download PDFInfo
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Abstract
本发明涉及一种多孔硅铂金纳米酶材料的制备方法及应用。以介孔SiO2纳米球为载体负载Au‑Pt纳米粒子，制备了多孔硅铂金纳米酶，通过加入DNA互补链，获得多孔硅铂金纳米酶信号标签。该材料可用于生物催化和免疫测定中，具有成本低、稳定性好、可重复使用、高催化效率等优点，且具有可控的组成和结构，还能进行修饰和改性。
Description
技术领域
本发明涉及化学测试分析技术领域，尤其涉及一种多孔硅铂金纳米酶材料的制备与应用。
背景技术
天然酶作为一种高效的绿色催化剂，在食品、环境、生物、化工等领域具有重要应用。但天然酶在实际应用中受到pH、温度、金属离子和时间等众多环境因素的限制，使其易失活、稳定性差，同时还具有提纯成本高、难回收、价格昂贵等缺陷。因此，纳米酶材料的发展有望解决天然酶目前存在的问题。
纳米酶材料因其具备独特的物理化学性质，同时与天然酶的尺寸、形状、催化活性等具有相似性而被广泛研究。纳米酶材料不仅具有成本低、稳定性好、可重复使用、高催化效率等优点，而且具有可控的组成和结构，还能进行修饰和改性，引起了研究者们的极大兴趣，已被广泛研究并应用于生物催化和免疫测定中。例如，Pt NP，尤其是掺杂有其他金属，如Au或Ni，在电化学催化，催化加氢和生物传感方面引起了广泛关注。Pt NP具有出色的氧化酶样，过氧化物酶样和过氧化氢酶样活性，有望作为纳米酶有前途的候选物。此外，适体作为一种寡核苷酸或肽分子，可以通过非共价相互作用(例如静电相互作用，疏水相互作用和互补形状)与靶分子特异性结合。适体在生物测定中被广泛使用，因为它们可以提供与抗体相当的分子识别特性。此外，适体提供了优于抗体的优势，因为它们易于工程改造，易于通过化学合成制备，具有所需的存储特性，并且在治疗应用中几乎没有免疫原性。因此，将纳米酶与适体整合可以解决天然酶的不足，提供更高的灵敏度和选择性。
发明内容
本发明的目的在于在现有技术的基础上开发一种新型的多孔硅铂金纳米酶材料，该材料可用于生物催化和免疫测定中，具有成本低、稳定性好、可重复使用、高催化效率等优点，且具有可控的组成和结构，还能进行修饰和改性。
本发明解决上述技术问题所采用的方案是：
一种多孔硅铂金纳米酶材料的制备方法，包括如下步骤：
(1)金纳米颗粒的制备
取四氯金酸和柠檬酸三钠用去离子水定容后置于冰水浴中，加入新配置的硼氢化钠溶液搅拌至其从浅黄色变为酒红色，然后将溶液静置一段时间进行还原，得到金纳米颗粒溶液；
(2)氨基化二氧化硅纳米微球(m-SiO2)的制备
将三乙胺加入水中搅拌，再加入十六烷基三甲基溴化铵、水杨酸钠、正硅酸乙酯分别搅拌反应，所得混合液用乙醇稀释后离心收集沉淀物，将所得沉淀物用HCl与甲醇的混合溶液洗涤，然后分散在乙醇中，得到m-SiO2乙醇分散液；向所得m-SiO2乙醇分散液中加入3-氨丙基三乙氧基硅烷，搅拌，离心取沉淀物清洗后分散在乙醇溶液中，得到氨基化二氧化硅纳米微球溶液；
(3)金二氧化硅纳米微球(m-SiO2/Au)的制备
向步骤(2)所得氨基化二氧化硅纳米微球溶液中加入步骤(1)所得金纳米颗粒溶液，进行超声搅拌反应，得到红色的m-SiO2/Au溶液；
(4)多孔硅铂金纳米酶(m-SAP NPs)的制备
将上述步骤(3)中制备的m-SiO2/Au溶液在室温条件下搅拌，然后加入氯铂酸和柠檬酸钠，搅拌，再加入新制备的硼氢化钠冰水溶液，搅拌反应后静置至溶液从棕色变为深色，将所得溶液离心洗涤，再分散在水中，得到m-SAP NPs溶液。
优选地，步骤(2)中十六烷基三甲基溴化铵与水杨酸钠的质量比为2:1。
优选地，步骤(2)所述HCl与甲醇的混合溶液中HCl：甲醇体积比为1：9～11，洗涤时间为5～6h，洗涤温度60～80℃。
优选地，步骤(1)所得金纳米颗粒溶液中金纳米粒子的浓度为4～4.9mg/L；步骤(2)所得氨基化二氧化硅纳米微球溶液中氨基化二氧化硅纳米微球的浓度为86～100mg/L；步骤(3)所述氨基化二氧化硅纳米微球与金纳米粒子溶液的体积比为1：5～8。
本发明还提供一种多孔硅铂金纳米酶材料，采用上述的方法制备得到。
本发明的另一目的是提供上述多孔硅铂金纳米酶材料的应用：将戊二醛原液加入到步骤(4)所得m-SAP NPs溶液中孵育，将所得溶液离心洗涤，分散到水中，得到戊二醛修饰过的铂金二氧化硅纳米微球溶液，再加入氨基化后的DNA互补链并搅拌，获得铂金二氧化硅纳米微球信号标签m-SAP/cDNA，将所述信号标签应用于生物催化和免疫测定。
优选地，DNA互补链在最终所得溶液中的浓度为0.46～0.60nmol/mL，最终得到的信号标签的浓度为0.19～0.30mg/L。
本发明先合成多孔二氧化硅，然后通过静电吸附再原位还原的方法负载铂金纳米颗粒，相比于刻蚀，用量更少；铂金二氧化硅纳米微球的引入在很大程度上解决了天然酶在实际应用中易失活、稳定性差，提纯成本高、耐酸碱性差、难回收、价格昂贵等缺陷。且本发明中引入适配体，其对于小分子的结合力强，空间位阻小，很适合对小分子进行检测。
附图说明
图1是本申请多孔硅铂金纳米酶制备示意图；
图2是MNP-适体制备示意图；
图3是本申请实施例1所得多孔硅铂金纳米酶的TEM和SEM图像，其中(A)m-SiO2 NP的TEM图像、(B)m-SiO2 NP放大版TEM图像、(C)m-SiO2NPs的SEM图像、(D)m-SiO2/Au杂化物TEM图像、(E)m-SAP NPTEM图像、(F)MNP/m-SAP复合体的TEM图像；
图4为适体及其结合过程的表征，其中图示分别为(A)m-SAP NP的HAADF-STEM图像；(B)Si，(C)Au，(D)Pt和(E)Si，Au，Pt重叠图像的元素映射图像；(F)m-SAP NP的EDS光谱；
图5是本申请实施例1所得多孔硅铂金纳米酶流体力学尺寸以及Zeta电位，其中图示分别为(A)MNP的TEM图像；(B)与(A)相对应的MNPTEM图像的放大版本；(C)流体力学尺寸(a)m-SiO2，(b)MNP适体，(c)m-SAP/c-DNA，(d)m-SAP/MNP；(D)Au NP，m-SiO2 NP，m-SiO2-NH2，m-SAP，MNP-NH2和m-SAP/MNP的Zeta电位；
图6是在APTS，OPD和TMB三种显色剂下颜色变化的照片，其中，a、b、c分别代表APTS、OPD、TMB，图示(A)m-SAP+H2O2，(B)无H2O2的m-SAP和(C)仅H2O2；
图7是m-SAP对TMB的催化活性表征，其中，(a)TMB，(b)TMB+m-SAP，(c)以H2SO4终止的TMB+m-SAP的溶液的UV-vis光谱，插图为相应溶液的照片。
具体实施方式
为更好的理解本发明，下面的实施例是对本发明的进一步说明，但本发明的内容不仅仅局限于下面的实施例。
实施例1
制备所述多孔硅铂金纳米酶材料具体包括以下步骤：
(1)取1.04mL浓度为24mmol/L的四氯金酸，加入7.3535mg柠檬酸三钠后定容至100mL，在不断搅拌的条件下加入3mL冰浴过浓度为0.1mol/L的硼氢化钠溶液，搅拌10min后获得金纳米粒子溶液，颜色从浅黄色到酒红色。将最终溶液储存在4℃下使用。
(2)取68mg三乙胺加入25mL水，80℃下磁力搅拌30min后，加入368mg十六烷基三甲基溴化铵和184mg水杨酸钠80℃下反应1h后，加入4mL正硅酸乙酯80℃下反应2h后，取出冷却后离心清洗取沉淀，后加入25mL盐酸/甲醇溶液，盐酸甲醇溶液体积比为1：10，在60℃下反应6h，取出冷却后离心清洗取沉淀分散于25mL乙醇中；取其中5mL加入600μL 3-氨丙基三乙氧基硅烷，在室温下搅拌6h，后离心清洗取沉淀分散于30mL乙醇中得到m-SiO2微球溶液。取m-SiO2微球溶液2mL，加入15mL步骤(1)所得金纳米粒子溶液后，超声搅拌反应15min，得到红色的m-SiO2/Au溶液。
(3)将红色的m-SiO2/Au离心洗涤后分散在8mL水中，在搅拌条件下加入200μL浓度为19.3mmol/L的氯铂酸和1mL 1％的柠檬酸钠，搅拌15min后迅速加入1mL现配冰浴过浓度为0.1mol/L的硼氢化钠，反应15min，静置1h后，溶液从棕色变为深色，反应所得的溶液离心洗涤获得铂金二氧化硅纳米微球，然后再分散在水中至40mL，得到m-SAP NPs溶液。通过TEM图像表征所制备的m-SAP NPs的形态和结构，如图3所示，m-SiO2 NP分布均匀，尺寸均匀，平均直径约为190nm。图4通过HAADF-STEM分析了m-SiO2 NP，m-SiO2/Au和m-SAP NP更详细的特征，进一步证明了双金属Au/Pt纳米颗粒成功负载在m-SiO2 NP上。
实施例2
铂金二氧化硅纳米微球信号标签的制备及其与MNP(磁性纳米粒子Magnetic nanoparticles)-适体的共轭
取1mL戊二醛原液加入到5mL铂金二氧化硅纳米微球溶液中并在室温下孵育1h。将反应所得的戊二醛修饰过的铂金二氧化硅纳米微球溶液离心洗涤，分散到5mL水中；然后加入0.5mL浓度为5.1nmol/mL氨基化修饰的DNA互补链并在室温下搅拌反应1h，互补链序列为：5'－ACACGTGCCCAACAAAAAA－3'。将反应所得的溶液离心洗涤获得铂金二氧化硅纳米微球信号标签m-SAP/cDNA，分散到5mL水中备用。通过传统的EDC/NHS生物缀合方法，在MNP表面修饰能特异性识别AFB1的适体。简要地说，将1mL MNP移到PE管中，然后加入50μL EDC(10mg/mL)和25μL磺基-NHS(10mg/mL)，在室温下超声混合20分钟。磁分离后，将MNP重悬于1mL PBS溶液(pH＝7.4，0.01M)中。然后将100μL适体(1.0mg/mL)引入MNP溶液中，室温下振摇1h。将所得溶液用水洗涤3次，并用PBS溶液分散，在4℃下保存以备使用。然后将100μLMNP-适体与上述m-SAP溶液混合，并在室温下搅拌1h。用水洗涤后，将100μL MNP-适体标记的m-SAP最终分散在PBS溶液中，并保存在4℃进行检测。
图4对适体及其结合过程进行表征。为了证明m-SiO2 NP和MNP-适体的共轭过程，进行了水动力直径和Zeta电位的测定，如图5所示，m-SiO2 NP，MNP-适体和m-SAP/cDNA的流体力学直径分别为201.09、117.95、220.82nm。当MNP-适体和m-SAP/cDNA温育并反应时，m-SAP/MNP复合物产生并显示出大的尺寸增加至457.43nm，这意味着适体和cDNA成功杂交。如图5D，对于Au NP，m-SiO2 NP和MNP-NH2，它们的Zeta电位分别为–32.9，–34.6和24.2mV，在用氨基修饰m-SiO2 NP，加载Au-Pt纳米粒子并与MNP-适体一起孵育后，它们的Zeta电位首先从–34.6mV升高到26.8mV，然后下降到–26.7mV和–18.7mV，这表明m-SiO2-NH2，m-SAP和m-SAP/MNP的成功结合。
实施例3
m-SAP的过氧化氢酶样活性
本发明制备的多孔硅铂金纳米酶的氧化酶和过氧化氢酶样活性，通过以下方法进行证明：我们在ELISA中采用传统的显色剂APTS，OPD和TMB作为底物，分别在不存在和存在H2O2的情况下与m-SAP反应。如图6。结果表明，m-SAP对着色剂具有氧化酶和过氧化氢酶样活性，因此，将其称为纳米酶。
由于TMB是ELISA中最常用的显色剂，我们进一步评估了m-SAP对TMB的催化活性。如图7。m-SAP可以催化TMB的氧化，产生蓝色，其吸收峰在650nm左右(b)。此外，可以通过添加H2SO4终止蓝色反应，以产生黄色，其吸收峰在450nm处(c)。相反，在没有m-SAP的情况下，TMB溶液(a)没有观察到明显的颜色变化。
为了讨论m-SAP与TMB的关系，我们计算了H2O2存在和不存在下TMB氧化的动力学参数以及比较了m-SAP与其他纳米酶和HRP的动力学参数。如表1。根据Michaelise-Menten方程v＝Vmax×[S]/(Km+[S])计算动力学参数。此处，Km表示与底物的亲和力：较低的Km表示较高的亲和力。
显然，在存在(0.016mM)和不存在(0.058mM)H2O2的情况下，m-SAP的Km均低于HRP(0.125mM)，这表明m-SAP对TMB具有良好的亲和力。与其他报告的材料相比，m-SAP的Km值最低。可归为以下原因：(1)m-SiO2 NP可以负载大量Au/Pt纳米粒子杂化物，(2)具有大比表面积的Au/Pt纳米颗粒显示出优异的催化活性，(3)二氧化硅纳米球的亲水性表面为催化反应提供了更多的可及位置。
表1：使用TMB作为底物比较m-SAP与其他纳米酶和HRP的动力学参数(未给出：-)
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对比例1
本实施例与实施例1的区别仅在于：步骤(2)中取68mg三乙胺加入25mL水，80℃下磁力搅拌30min后，加入368mg十六烷基三甲基溴化铵和92mg水杨酸，其余操作步骤如实施例1。得到的多孔二氧化硅孔径大小如下表2所示，多孔二氧化硅用于负载纳米金及纳米铂，孔径越大意味着可以搭载更多的铂金纳米颗粒。
表2实施例1和对比例1所得多孔二氧化硅的孔径对比
| 样品 | 粒径 | 孔径大小 |
| 实施例1 | 100nm | 小 |
| 对比例1 | 200nm | 大 |
采用上述方法合成的多孔硅铂金纳米酶，通过修饰互补DNA链，获得了多孔硅铂金纳米酶信号标签，可以与其适体DNA构建形成新的识别模型，用来检测AFB1等。其中，多孔硅铂金纳米酶取代了HRP作为酶标记，适体取代了用于AFB1识别的抗体，解决了酶标抗体难以制备和纯化的问题，具有低成本，高稳定性、可重复使用、高催化效率的优点。
以上所述是本发明的优选实施方式而已，当然不能以此来限定本发明之权利范围，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和变动，这些改进和变动也视为本发明的保护范围。
Claims (7)
1.一种多孔硅铂金纳米酶材料的制备方法，其特征在于，包括如下步骤：
(1)金纳米颗粒的制备
取四氯金酸和柠檬酸三钠用去离子水定容后置于冰水浴中，加入新配置的硼氢化钠溶液搅拌至其从浅黄色变为酒红色，然后将溶液静置一段时间进行还原，得到金纳米颗粒溶液；
(2)氨基化二氧化硅纳米微球(m-SiO2)的制备
将三乙胺加入水中搅拌，再加入十六烷基三甲基溴化铵、水杨酸钠、正硅酸乙酯分别搅拌反应，所得混合液用乙醇稀释后离心收集沉淀物，将所得沉淀物用HCl与甲醇的混合溶液洗涤，然后分散在乙醇中，得到m-SiO2乙醇分散液；向所得m-SiO2乙醇分散液中加入3-氨丙基三乙氧基硅烷，搅拌，离心取沉淀物清洗后分散在乙醇溶液中，得到氨基化二氧化硅纳米微球溶液；
(3)金二氧化硅纳米微球(m-SiO2/Au)的制备
向步骤(2)所得氨基化二氧化硅纳米微球溶液中加入步骤(1)所得金纳米颗粒溶液，进行超声搅拌反应，得到红色的m-SiO2/Au溶液；
(4)多孔硅铂金纳米酶(m-SAP NPs)的制备
将上述步骤(3)中制备的m-SiO2/Au溶液在室温条件下搅拌，然后加入氯铂酸和柠檬酸钠，搅拌，再加入新制备的硼氢化钠冰水溶液，搅拌反应后静置至溶液从棕色变为深色，将所得溶液离心洗涤，再分散在水中，得到m-SAP NPs溶液。
2.根据权利要求1所述的制备方法，其特征在于：步骤(2)中十六烷基三甲基溴化铵与水杨酸钠的质量比为2:1。
3.根据权利要求1所述的制备方法，其特征在于：步骤(2)所述HCl与甲醇的混合溶液中HCl：甲醇体积比为1：9～11，洗涤时间为5～6h，洗涤温度60～80℃。
4.根据权利要求1所述的制备方法，其特征在于：步骤(1)所得金纳米颗粒溶液中金纳米粒子的浓度为4～4.9mg/L；步骤(2)所得氨基化二氧化硅纳米微球溶液中氨基化二氧化硅纳米微球的浓度为86～100mg/L；步骤(3)所述氨基化二氧化硅纳米微球与金纳米粒子溶液的体积比为1：5～8。
5.一种多孔硅铂金纳米酶材料，其特征在于，采用权利要求1～4所述的方法制备得到。
6.如权利要求5所述的多孔硅铂金纳米酶材料的应用，其特征在于，将戊二醛原液加入到步骤(4)所得m-SAP NPs溶液中孵育，将所得溶液离心洗涤，分散到水中，得到戊二醛修饰过的铂金二氧化硅纳米微球溶液，再加入氨基化后的DNA互补链并搅拌，获得铂金二氧化硅纳米微球信号标签m-SAP/cDNA，将所述信号标签应用于生物催化和免疫测定。
7.根据权利要求6所述的应用，其特征在于：DNA互补链在最终所得溶液中的浓度为0.46～0.60nmol/mL，最终得到的信号标签的浓度为0.19～0.30mg/L。
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| CN113325179B (zh) * | 2021-04-14 | 2024-02-27 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | 一种基于Au@Pt酶的免疫层析试纸条及其制备方法 |
| CN114570362A (zh) * | 2022-03-03 | 2022-06-03 | 武汉轻工大学 | 一种纳米酶的制备方法及其应用 |
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