CN112362626A - 正电性Ag@Au核壳结构纳米粒子在核酸酶活性检测中的应用 - Google Patents
正电性Ag@Au核壳结构纳米粒子在核酸酶活性检测中的应用 Download PDFInfo
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Abstract
本发明提供了一种基于正电性Ag@Au核壳结构纳米粒子((+)Ag@Au CSNPs)的核酸酶活性检测新方法。利用正电性Ag@Au核壳结构纳米粒子作为荧光淬灭和信号放大平台,荧光染料标记的DNA探针可通过静电作用吸附在(+)Ag@Au CSNPs表面,荧光淬灭。核酸酶加入导致DNA探针水解,荧光恢复,利用荧光信号强度与核酸酶浓度之间的线性关系实现定量检测。本发明制备的(+)Ag@Au CSNPs在含盐、蛋白质或金属离子的复杂体系中具有很高的稳定性,在均相体系可以实现“混合+检测”的一步检测法,该方法具有良好的选择性,在生物分析和医疗诊断等领域具有潜在的应用价值。
Description
技术领域
本发明涉及荧光探针领域,具体涉及一种正电性Ag@Au核壳结构纳米粒子在核酸酶活性检测中的应用。
背景技术
S1核酸酶是一种单链特异性核酸内切酶,能水解单链DNA或单链RNA中的磷酸二酯键,在DNA复制、重组、修复和分子克隆等生物过程中发挥着重要作用,其检测对医疗诊断、药物研发和生物传感领域具有重要作用。因此,迫切需要建立一种简单、快速、有效的核酸酶活性检测方法。现有的核酸酶活性检测方法主要有电化学、比色法、荧光法等。其中,荧光法由于快速、灵敏、选择性好、成本低和操作简便等内在优点受到了研究者广泛的关注,成为最有潜力的方法之一。
金纳米粒子(AuNPs)和银纳米粒子(AgNPs)作为常用的金属纳米粒子,由于稳定性好、独特的光电和催化性能,常用作荧光淬灭基团构建荧光传感器。其中,银纳米粒子消光系数高于同尺寸的金纳米粒子,可以与荧光染料发生更有效的能量转移,而金纳米粒子比银纳米粒子稳定性和生物相容性好,易于修饰DNA、RNA和蛋白质等生物分子。因此,研究人员提出了Ag@Au核壳复合金属纳米粒子,该类复合纳米粒子兼具银纳米粒子高的荧光淬灭能力和金纳米粒子好的生物相容性的优点,比单一的金属纳米粒子具有更强的光电催化性能和更大的比表面积,作为固定基质既能提高生物分子的固载量,又可以有效提高方法的灵敏度。然而,据我们所知,利用多功能复合金属纳米材料作为有效的荧光猝灭平台检测S1核酸酶活性的研究尚未见报道。
发明内容
本发明提出了一种正电性Ag@Au核壳结构纳米粒子((+)Ag@Au CSNPs)在检测核酸酶活性中的应用,(+)Ag@Au CSNPs在含有盐、蛋白质或金属离子的复杂体系中具有很高的稳定性,在均相体系可以实现“混合+检测”的一步检测模式。
实现本发明的技术方案是:
正电性Ag@Au核壳结构纳米粒子在核酸酶活性检测中的应用,其中正电性Ag@Au核壳结构纳米粒子简称为(+)Ag@Au CSNPs(带正电)。
利用(+)Ag@Au CSNPs的荧光淬灭和信号放大特性检测S1核酸酶活性。
将荧光染料(FAM)标记的DNA作为探针,通过静电作用吸附在Ag@Au核壳结构纳米粒子表面,(+)Ag@Au CSNPs能淬灭标记在单链DNA上荧光染料的荧光。
加入S1核酸酶后,DNA探针被水解成DNA片段并从正电性Ag@Au核壳结构纳米粒子表面脱附,荧光恢复。
荧光信号强度与核酸酶浓度在2.5×10-4-3.0×10-2 U/mL范围内呈良好的线性关系,检出限为6.0×10-5 U/mL。
(+)Ag@Au核壳结构纳米粒子的制备如下:
(1)将新制备的5.0 mL、100 mM的NaBH4加入到30 mL、5.0 mM AgNO3溶液中,剧烈搅拌直至溶液颜色变为黄绿色,制得Ag纳米粒子溶液;
(2)将3.0 mL、1.0 mM的HAuCl4、3.0 mL、1.0 mM的NH2OH•HCl和2.0 mL的CTAB逐滴加入到15 mL步骤(1)制备的Ag纳米粒子溶液,在室温下搅拌45 min制得正电性Ag@Au核壳结构纳米粒子。
一步法定量检测S1核酸酶:
将不同浓度的S1核酸酶分别加入到10 μL、12 μM的DNA探针、190 μL PBS缓冲溶液(10mM、pH 7.0)和200 μL (+)Ag@Au CSNPs混合液中,室温条件下孵育40 min。随后,用荧光分光光度计在500 ~ 700 nm范围内测定上述溶液的荧光强度,激发和发射波长分别为480和520 nm,狭缝宽度为10 nm。
本发明的有益效果是:
(1)本发明制备的(+)Ag@Au CSNPs由于修饰了十六烷基三甲基溴化铵(CTAB)表面活性剂,稳定性优于负电性的Ag@Au CSNPs,在含盐、蛋白质或金属离子的复杂体系中具有很高的稳定性,在均相体系中可以实现“混合+检测”的模式;
(2)(+)Ag@Au CSNPs核壳结构复合纳米粒子的生物相容性和荧光淬灭能力优于单一的金属纳米粒子,从而可以增强方法的灵敏度;
(3)(+)Ag@Au CSNPs与带负电的DNA通过静电作用结合,方法更加简便;
(4)该方法具有良好的选择性,在生物分析和医疗诊断等领域具有潜在的应用价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1A和图1B分别为(+)AuNPs和(+)Ag@Au CSNPs的透射电镜图;
图2为(+)AuNPs(a)和(+)Ag@Au CSNPs(b)的zeta电位分析;
图3为AgNPs(a)、(+)AuNPs(b)和(+)Ag@Au CSNPs(c)的紫外-可见吸收光谱图,其中插图:(+)Ag@Au CSNPs图片;
图4为(+)Ag@Au CSNPs在不同条件下的稳定性研究;(a)(+)Ag@Au CSNPs,(b)a+10 mMNaCl,(c)a+10 mM牛血清白蛋白,(d)a+10 mM金属离子(Mg2+:Ca2+=1:1);
图5为不同浓度(+)AuNPs(A)和(+)Ag@Au CSNPs(B)对300 nM DNA探针的荧光淬灭效果,其中(+)AuNPs和(+)Ag@Au CSNPs的添加量为(a:0.0 nM、b:3.0 nM、c:5.0 nM、d:10nM);(C)不同条件下体系的荧光光谱图,(a)300 nM DNA探针;(b)a+ (+)Ag@Au CSNPs;(c)b+ 2.0×10-2 U/mL S1核酸酶;
图6是(A)(+)Ag@Au CSNPs与DNA探针反应时间以及(B)DNA探针浓度(a:DNA探针、b:a+(+)Ag@Au CSNPs)对荧光强度的影响;
图7是一步法测定核酸酶活性的(A)荧光光谱图和(B)工作曲线;
图8是核酸酶活性检测选择性考察;(A)加入不同底物后传感体系的荧光强度(a:S1核酸酶,b:DNase I,c:Exo III,d:λexo,e:胃蛋白酶、f:BSA,g:空白);(B)S1核酸酶活性检测中干扰物的影响(a:S1核酸酶,b:a+DNase I,c:a+Exo III,d:a+λexo,e:a+胃蛋白酶、f:a+BSA);实验条件为2.0×10-2 U/mL S1核酸酶、2.0 U DNase I、2.0 U Exo III、2.0 U λexo、2.0 mg/mL胃蛋白酶和2.0 mg/mL BSA。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例
采用种子生长法合成(+)Ag@Au核壳结构纳米粒子,步骤如下:
(1)将5.0 mL、100 mM的NaBH4 加入到30 mL、5.0 mM AgNO3溶液中,剧烈搅拌直至溶液颜色变为黄绿色,制得Ag纳米粒子溶液;
(2)将3.0 mL、1.0 mM的HAuCl4,3.0 mL、1.0 mM NH2OH•HCl和2.0 mL的CTAB逐滴加入到15 mL制备的Ag纳米粒子溶液,在室温下搅拌45 min制得(+)Ag@Au CSNPs。
对比例
(+)AuNPs制备:
1.8 mL NaBH4(100 mM)溶液逐滴加入到15 mL HAuCl4(1.0 mM)和2.0 mL的CTAB(10mM)的混合溶液中,不断搅拌直到溶液颜色从橘黄色变为橙红色。
一步法定量检测S1核酸酶:
将不同浓度的S1核酸酶分别加入到10 μL的DNA探针(12 μM)、190 μL PBS缓冲溶液(10mM、pH 7.0)和200 μL (+)Ag@Au CSNPs的混合液中,室温下孵育40 min的。然后用荧光分光光度计在500 ~ 700 nm范围内测定上述溶液的荧光强度,激发和发射波长分别为480 nm和520 nm,狭缝宽度为10 nm。
结果
1. (+)Ag@Au CSNPs表征
如图1所示:与(+)AuNPs的透射电镜图(图1A)相比,(+)Ag@Au CSNPs(图1B)出现了中间亮边缘暗的核-壳结构,粒径分布均匀,平均粒径为36 nm。
如图2所示,利用动态光散射仪进行电位分析:(+)Ag@Au CSNPs的电位为+43.4 mV(b),比(+)AuNPs的电位(+30.7 mV,a)高,更有利于通过静电作用与带负电的DNA探针结合。
如图3所示,与AgNPs(a)和(+)AuNPs(b)的紫外-可见吸收光谱图比较,(+)Ag@AuCSNPs(c)在391nm和521nm处分别有一个银纳米粒子和金纳米粒子的特征吸收峰,可以证明成功的合成了Ag@Au复合纳米粒子。
如图4所示,在(+)Ag@Au CSNPs溶液(a)中分别加入NaCl(b)、牛血清白蛋白(c)或金属离子(Mg2+:Ca2+=1:1,d)后,吸收峰位置和强度几乎无变化,说明(+)Ag@Au CSNPs在盐、蛋白或金属离子的复杂体系中具有很高的稳定性。
核酸酶检测的可行性
如图5所示,探究不同浓度的(+)AuNPs(图5A)和(+)Ag@Au CSNPs(图5B)对300 nM DNA探针的荧光淬灭效果。结果表明:随着纳米粒子的浓度增大,荧光淬灭能力增强,且(+)Ag@Au CSNPs对DNA的探针荧光淬灭能力优于单一的(+)AuNPs。
为了验证该方法的可行性,研究不同条件下体系的荧光光谱图。图5C中显示,当DNA探针单独存在时,具有明显的荧光吸收峰(a)。加入(+)Ag@Au CSNPs后,体系荧光很弱(b),说明(+)Ag@Au CSNPs能吸附DNA探针并淬灭标记在探针上的荧光分子的荧光。进一步加入2.0×10-2 U/mL的S1核酸酶后,DNA探针被S1核酸酶水解成DNA片段,从(+)Ag@Au CSNPs表面脱附,荧光信号部分恢复(c)。
实验优化
如图6A所示,当(+)Ag@Au CSNPs和DNA探针的孵育时间在0-40 min范围内变化时,荧光强度随孵育时间的增加而减小,随后达到一个平台。因此,最终选择40 min作为最佳反应时间。
如图6B所示,研究了(+)Ag@Au CSNPs加入前后,荧光强度随DNA探针浓度的改变。荧光强度随DNA探针浓度在0-300 nM范围内的增加而增加。当DNA探针浓度>300 nM时,△F有所下降(△F=F0-F,F0和F分别为(+)Ag@Au CSNP加入前后体系的荧光强度)。因此,选择300nM的DNA探针作为最佳浓度值。
工作曲线和方法选择性
在最佳实验条件下,考察了该传感器荧光强度与S1核酸酶浓度之间的关系。如图7所示,体系荧光强度与S1核酸酶浓度在2.5×10-4-3.0×10-2 U/mL范围内呈良好的线性关系,检出限为6.0×10-5 U/mL(按CDL=3δ/S计算,其中δ为20份空白溶液的标准偏差,S是回归方程的斜率)。与其他荧光法相比(表1),该荧光法检测S1核酸酶具有线性范围、高灵敏性和更加简便的优点,展现出良好的应用前景。
表1 荧光法测定核酸酶活性的对比
5. 方法选择性
为了验证该传感器对S1核酸酶检测的特异性,实验选取了脱氧核糖核酸酶I(DNaseI)、核酸外切酶III(Exo III)、λ噬菌体核酸外切酶(λexo)、胃蛋白酶和牛血清白蛋白(BSA)作为对照实验。如图8A所示,将S1核酸酶或干扰组分别加入反应体系,只有添加了S1核酸酶的体系具有明显的荧光信号,干扰组仍然处于荧光淬灭状态,这主要是由于S1核酸酶对富含胸腺嘧啶(T)的DNA探针的特异性识别和切割作用。当干扰物与S1核酸酶同时加入反应体系时,体系荧光强度与S1核酸酶单独存在时无明显差异(图8B)。该结果表明在其它干扰物存在的情况下,此传感体系对S1核酸酶的检测依然灵敏,证实了此传感其具有良好的选择性。
回收率实验结果
将三种不同浓度的S1核酸酶添加到15%的胎牛血清中,以确认传感器在实际样品应用中的可行性。如表2所示,通过加标回收的方法测得S1核酸酶的回收率为98%~106%,相对标准偏差为2.8%~4.1%,说明了本方法在实际血清样品核酸酶活性检测中具有潜在应用价值。
表2 荧光法测定S1核酸酶活性的回收率实验结果
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.正电性Ag@Au核壳结构纳米粒子在核酸酶活性检测中的应用。
2.根据权利要求1所述的应用,其特征在于:利用正电性Ag@Au核壳结构纳米粒子的增强荧光淬灭特性检测核酸酶活性。
3.根据权利要求1所述的应用,其特征在于:将荧光染料标记的DNA探针通过静电作用吸附在正电性Ag@Au核壳结构纳米粒子表面,正电性Ag@Au核壳结构纳米粒子能淬灭标记在单链DNA上荧光染料的荧光。
4.根据权利要求3所述的应用,其特征在于:加入S1核酸酶后,DNA探针被水解成DNA片段并从正电性Ag@Au核壳结构纳米粒子上脱附,荧光恢复。
5.根据权利要求1-4任一项所述的应用,其特征在于:荧光信号强度与核酸酶浓度在2.5×10-4-3.0×10-2 U/mL范围内呈良好的线性关系,检出限为6.0×10-5 U/mL。
6.根据权利要求5所述的应用,其特征在于,正电性Ag@Au核壳结构纳米粒子的制备如下:
(1)将NaBH4加入到AgNO3溶液中,剧烈搅拌直至溶液颜色变为黄绿色,制得Ag纳米粒子溶胶;
(2)将HAuCl4、NH2OH•HCl和CTAB逐滴加入到步骤(1)制备的Ag纳米粒子溶液中,在室温下搅拌45 min制得正电性Ag@Au核壳结构纳米粒子。
7.根据权利要求6所述的应用,其特征在于:所述步骤(1)将5.0 mL、100 mM的NaBH4加入到30 mL、5.0 mM AgNO3溶液中。
8.根据权利要求6所述的应用,其特征在于:所述步骤(2)中将3.0 mL、1.0 mM的HAuCl4、3.0 mL、1.0 mM的NH2OH•HCl和2.0 mL的CTAB逐滴加入到15 mL步骤(1)制备的Ag纳米粒子溶胶中。
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