CN113324933B - 一种检测血管紧张素转换酶的方法及其应用 - Google Patents
一种检测血管紧张素转换酶的方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种检测血管紧张素转换酶的方法及其应用,将ACE在去离子水中稀释成最终浓度从0.5mU/L到60mU/L,每个96孔板中含有ACE 25μL、5μM的Cys‑Ang(I)‑Cys肽40μL,在37℃下孵育,然后在96孔板中加入50μL的AuNPs,通过检测Cys‑Ang(I)‑Cys肽的残留量来放大ACE活性信号,最后在5min内记录AuNPs溶液在400‑800nm范围内的紫外可见吸收光谱。本发明设计了一种基于血管紧张素I的Cys‑Ang(I)‑Cys肽,Cys‑Ang(I)‑Cys肽每一端都连接一个半胱氨酸(Cys),Cys‑Ang(I)‑Cys肽可以通过Au‑S键聚集在两端AuNPs的表面,并刺激AuNPs的聚集呈现紫色或蓝色,ACE引入后Cys‑Ang(I)‑Cys肽被水解,巯基从肽的一端解离,导致AuNPs分散,呈现红色,通过实现ACE催化肽的水解以及肽介导的AuNPs组装/分解,以一种快速、直接的方式进行ACE检测,以及运行该方法进行ACE抑制剂或抑制肽的筛选。
Description
技术领域
本发明涉及血管紧张素转换酶活性检测技术领域,更具体地说,本发明涉及一种检测血管紧张素转换酶的方法及其应用。
背景技术
血管紧张素转换酶(ACE)在肾素-血管紧张素***中起着调节血压的主导作用,其产物直接影响心血管疾病(CVD)的发病机制。ACE本质上是一类依赖于锌的肽酶,能将血管紧张素(Ang)I水解成血管紧张素II,血管紧张素II被ACE2进一步裂解形成更短的Ang-(1-7)肽。
ACE除了能水解血管紧张素I外,还能水解许多其他多肽,如P物质、脑啡肽和缓激肽,影响生殖、肾脏代谢功能、造血和免疫反应等多种生理过程。ACE活性升高导致大量血管紧张素II的产生,这是一种使高血压成为可能的强效血管收缩剂,因此,ACE是抗高血压药物的潜在靶点。ACE是由两个不同生物功能的独立催化结构域组成,针对ACE的特定结构域的抑制剂被开发成有效的降压药物已被证明具有强效降血压作用。ACE抑制剂,如食品衍生肽正在被开发,更安全,更有益于人类健康。因此,高效、快速检测人血清中ACE水平对于预防高血压和发现新型抑制剂是至关重要的。
测量ACE活性的传统方法主要依赖于荧光测定法,其中ACE催化特定底物水解产生荧光底物,以及分光光度测定法以确定生成的水解物数量。基于高效液相色谱(HPLC)和质谱等其他方法也已经发展出来。然而,大多数检测ACE的方法可靠性和敏感性有限。更重要的是,大量的试剂要求、复杂的操作和先进的仪器限制了它们在一些资源贫乏的环境下的应用。因此,迫切需要开发一种简单有效的血管紧张素转换酶(ACE)检测技术,在生物医学诊断中具有重要价值。迅速发展的纳米技术为开发生物传感技术以应对这些挑战提供了一个有前景的平台。纳米结构在纳米尺度上的独特现象和非凡性能对生物传感器的性能有重要的帮助。一个显著的材料是等离子体金纳米颗粒(AuNPs),由于其独特的表面化学、电子和可调光学特性,已被广泛应用于生物医学分析。基于AuNPs的等离子体传感器可以有效、简单、方便地用肉眼读取,无需借助先进仪器,这为生物医学诊断中的多种生物分析方法带来了巨大的前景。
发明内容
为了克服现有技术的上述缺陷,本发明的实施例提供一种检测血管紧张素转换酶的方法及其应用,本发明所要解决的技术问题是:更加简单、方便、有效的检测血管紧张素转换酶的活性。
为实现上述目的,本发明提供如下技术方案:一种检测血管紧张素转换酶的方法,检测ACE活性时,将ACE在去离子水中稀释成最终浓度从0.5mU/L到60mU/L,每个96孔板中含有ACE 25μL、Cys-Ang(I)-Cys肽40μL,在37℃下孵育,然后在96孔板中加入50μL的AuNPs,通过检测Cys-Ang(I)-Cys肽的残留量来放大ACE活性信号,最后在5min内记录AuNPs溶液在400-800nm范围内的紫外可见吸收光谱;
检测ACE活性前需要对AuNPs进行合成,AuNPs的合成方法为:
称取100毫升1mM的氯金酸加入到圆底烧瓶中,通过连续搅拌加热到120℃,然后迅速加入10毫升38.8mM的柠檬酸钠溶液并持续加热,以确保氯金酸完全还原,当溶液由无色变为深红色时意味着AuNPs的形成,移去热源,在室温下冷却得到AuNPs备用。
在一种优选的实施方式中,检测ACE活性前需要利用Cys-Ang(I)-Cys肽诱导AuNPs的聚集,在96孔板中将不同浓度的Cys-Ang(I)-Cys肽加入150μL的去离子水中,然后加入50μL的AuNPs,平衡5分钟后,由于Au-S相互作用,AuNPs溶液发生了不同程度的聚集,随着Cys-Ang(I)-Cys肽浓度的增加,AuNPs溶液的颜色由红色变为蓝色或紫色,然后利用紫外-可见光谱、动态光散射和透射电子显微镜对溶液进行进一步的分析和表征,确定每个96板孔中加入Cys-Ang(I)-Cys肽的浓度为5μM。
在一种优选的实施方式中,检测ACE活性时在37℃下孵育时间为30分钟,确保Cys-Ang(I)-Cys肽的充分水解。
在一种优选的实施方式中,所述ACE合成过程中移去热源冷却后采用聚醚砜膜去除AuNPs中的较大杂质,得到实验所需的AuNPs。
在一种优选的实施方式中,利用Cys-Ang(I)-Cys肽诱导AuNPs的聚集时Cys-Ang(I)-Cys肽的添加量为50μL,确保96板孔中最终体积为250μL,所述96板孔中不同浓度的Cys-Ang(I)-Cys肽的浓度范围为0-1000nM。
本发明还提供一种检测血管紧张素转换酶方法的应用,
(一):检测ACE抑制剂,将40μL的ACE与40μL不同浓度的依那普利、雷米普利混合,在37℃下孵育,然后加入Cys-Ang(I)-Cys肽40μL和去离子水30μL,按照检测ACE活性的方法测试剩余的ACE活性,随后加入50μL的AuNPs作为信号放大工具,采用400-800nm范围内的AuNPs紫外可见吸收光谱进行ACE抑制活性检测;
(二):筛选ACE抑制肽,采用9种不同的活性肽与30mU/mL的ACE在去离子水中孵育,孵育15分钟后,加入5μM的Cys-Ang(I)-Cys与未失活的ACE在37℃下反应15分钟,然后将50μL的AuNPs加入到150μL的混合物中,以颜色的变化来筛选出具有ACE抑制活性的多肽。
在一种优选的实施方式中,所述检测ACE抑制剂时在37℃下孵育时间为20分钟,确保ACE活性被充分抑制。
在一种优选的实施方式中,所述筛选ACE抑制肽时具有ACE抑制功能的肽能诱导AuNPs聚集,呈紫色,而无ACE抑制功能的肽不能诱导AuNPs聚集,呈红色;9种不同的活性肽分别为KAFAF、CAAAA、VPP、IPP、RPP、IPA、LEP、LQP和GSH,且9种活性肽的浓度均为15μM。
与现有技术相比,本发明的技术效果和优点:
本发明设计了一种基于血管紧张素I的Cys-Ang(I)-Cys肽,Cys-Ang(I)-Cys肽每一端都连接一个半胱氨酸(Cys),Cys-Ang(I)-Cys肽可以通过Au-S键聚集在两端AuNPs的表面,并刺激AuNPs的聚集呈现紫色或蓝色,ACE引入后Cys-Ang(I)-Cys肽被水解,巯基从肽的一端解离,导致AuNPs分散,呈现红色,通过实现ACE催化肽的水解以及肽介导的AuNPs组装/分解,以一种快速、直接的方式进行ACE检测,以及运行该方法进行ACE抑制剂或抑制肽的筛选。
附图说明
图1为本发明检测血管紧张素转换酶活性以及抑制剂或抑制肽的示意图;
图2为本发明对ACE检测的线性范围示意图;
图3为本发明雷米普利抑制率测定结果示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中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做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。
如图1所示,一种检测血管紧张素转换酶的方法,检测ACE活性时,将ACE在去离子水中稀释成最终浓度从0.5mU/L到60mU/L,每个96孔板中含有ACE 25μL、Cys-Ang(I)-Cys肽40μL,在37℃下孵育,然后在96孔板中加入50μL的AuNPs,通过检测Cys-Ang(I)-Cys肽的残留量来放大ACE活性信号,最后在5min内记录AuNPs溶液在400-800nm范围内的紫外可见吸收光谱;
检测ACE活性前需要对AuNPs进行合成,AuNPs的合成方法为:
称取100毫升1mM的氯金酸加入到圆底烧瓶中,通过连续搅拌加热到120℃,然后迅速加入10毫升38.8mM的柠檬酸钠溶液并持续加热,以确保氯金酸完全还原,当溶液由无色变为深红色时意味着AuNPs的形成,移去热源,在室温下冷却得到AuNPs备用。
在一种优选的实施方式中,检测ACE活性前需要利用Cys-Ang(I)-Cys肽诱导AuNPs的聚集,在96孔板中将不同浓度的Cys-Ang(I)-Cys肽加入150μL的去离子水中,然后加入50μL的AuNPs,平衡5分钟后,由于Au-S相互作用,AuNPs溶液发生了不同程度的聚集,随着Cys-Ang(I)-Cys肽浓度的增加,AuNPs溶液的颜色由红色变为蓝色或紫色,然后利用紫外-可见光谱、动态光散射和透射电子显微镜对溶液进行进一步的分析和表征,确定每个96板孔中加入Cys-Ang(I)-Cys肽的浓度为5μM。
在一种优选的实施方式中,检测ACE活性时在37℃下孵育时间为30分钟,确保Cys-Ang(I)-Cys肽的充分水解。
在一种优选的实施方式中,所述ACE合成过程中移去热源冷却后采用聚醚砜膜去除AuNPs中的较大杂质,得到实验所需的AuNPs。
在一种优选的实施方式中,利用Cys-Ang(I)-Cys肽诱导AuNPs的聚集时Cys-Ang(I)-Cys肽的添加量为50μL,确保96板孔中最终体积为250μL,所述96板孔中不同浓度的Cys-Ang(I)-Cys肽的浓度范围为0-1000nM。
本发明还提供一种检测血管紧张素转换酶方法的应用,
(一):检测ACE抑制剂,将40μL的ACE与40μL不同浓度的依那普利、雷米普利混合,在37℃下孵育,然后加入Cys-Ang(I)-Cys肽40μL和去离子水30μL,按照检测ACE活性的方法测试剩余的ACE活性,随后加入50μL的AuNPs作为信号放大工具,采用400-800nm范围内的AuNPs紫外可见吸收光谱进行ACE抑制活性检测;
(二):筛选ACE抑制肽,采用9种不同的活性肽与30mU/mL的ACE在去离子水中孵育,孵育15分钟后,加入5μM的Cys-Ang(I)-Cys与未失活的ACE在37℃下反应15分钟,然后将50μL的AuNPs加入到150μL的混合物中,以颜色的变化来筛选出具有ACE抑制活性的多肽。
在一种优选的实施方式中,所述检测ACE抑制剂时在37℃下孵育时间为20分钟,确保ACE活性被充分抑制。
在一种优选的实施方式中,所述筛选ACE抑制肽时具有ACE抑制功能的肽能诱导AuNPs聚集,呈紫色,而无ACE抑制功能的肽不能诱导AuNPs聚集,呈红色;9种不同的活性肽分别为KAFAF、CAAAA、VPP、IPP、RPP、IPA、LEP、LQP和GSH,且9种活性肽的浓度均为15μM。
通过含有多肽介导的AuNPs组装传感器来检测ACE活性,设计了一种基于血管紧张素I的Cys-Ang(I)-Cys肽,每一端都连接一个半胱氨酸,半胱氨酸包含巯基,可以通过Au-S键连接到AuNPs上,因此Cys-Ang(I)-Cys肽可以诱导AuNPs的组装,由于AuNPs的表面等离子体共振(SPR)特性,分散的AuNPs和聚集的AuNPs在溶液中表现出不同的表面等离子体(SPR)吸收,并伴随着颜色的变化,这种变化可以用肉眼直观地观察到。Cys-Ang(I)-Cys肽可以通过Au-S键聚集在两端AuNPs的表面,并刺激AuNPs的聚集呈现紫色或蓝色,ACE引入后,Cys-Ang(I)-Cys肽被水解,巯基从肽的一端解离,导致AuNPs分散,呈现红色。通过实现ACE催化肽的水解以及肽介导的AuNPs组装/分解,以一种快速、直接的方式进行ACE检测,考虑到ACE抑制剂对ACE催化活性的抑制作用,该等离子体生物传感器也可用于筛选高择性的ACE抑制剂,用于抗高血压药物的开发。
如图2所示:本发明紫外-可见光谱显示,随着ACE浓度从5mU/mL增加到60mU/mL,SPR吸收峰红移,溶液逐渐变红,A580/A520值用于评价用于ACE检测的AuNPs聚集状态,根据A580/A520值,ACE检测的线性范围为5-40mU/mL,R2=0.9743,ACE的检出限为0.40mU/mL(S/N=3)。
进一步使用该等离子体纳米传感器检测ACE抑制剂雷米普利,因为它们可以抑制ACE的酶活性,从而导致肽诱导AuNPs的聚集,加入ACE抑制剂后,AuNPs的颜色由红色变为蓝色,说明ACE催化的水解被抑制,剩余的Cys-Ang(I)-Cys导致AuNPs聚集,A580/A520值随雷米普利浓度的增加而增加,且当雷米普利的浓度达到31.62μM时,ACE活性被完全抑制,雷米普利的半数抑制浓度(IC50值)为6.65μM,如图3所示。
最后应说明的几点是:首先,在本申请的描述中,需要说明的是,除非另有规定和限定,术语“安装”、“相连”、“连接”应做广义理解,可以是机械连接或电连接,也可以是两个元件内部的连通,可以是直接相连,“上”、“下”、“左”、“右”等仅用于表示相对位置关系,当被描述对象的绝对位置改变,则相对位置关系可能发生改变;
其次:本发明公开实施例附图中,只涉及到与本公开实施例涉及到的结构,其他结构可参考通常设计,在不冲突情况下,本发明同一实施例及不同实施例可以相互组合;
最后:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,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)
1.一种检测血管紧张素转换酶的方法,其特征在于:
检测ACE活性时,将ACE在去离子水中稀释成最终浓度从0.5mU/L到60mU/L,每个96孔板中含有ACE 25μL、Cys-Ang(I)-Cys肽40μL,在37℃下孵育,使得Cys-Ang(I)-Cys肽充分水解,然后在96孔板中加入50μL的AuNPs,通过检测Cys-Ang(I)-Cys肽的残留量来放大ACE活性信号,最后在5min内记录AuNPs溶液在400-800nm范围内的紫外可见吸收光谱;
检测ACE活性前需要对AuNPs进行合成,AuNPs的合成方法为:
称取100毫升1mM的氯金酸加入到圆底烧瓶中,通过连续搅拌加热到120℃,然后迅速加入10毫升38.8mM的柠檬酸钠溶液并持续加热,以确保氯金酸完全还原,当溶液由无色变为深红色时意味着AuNPs的形成,移去热源,在室温下冷却得到AuNPs备用。
2.根据权利要求1所述的一种检测血管紧张素转换酶的方法,其特征在于:检测ACE活性前需要利用Cys-Ang(I)-Cys肽诱导AuNPs的聚集,在96孔板中将不同浓度的Cys-Ang(I)-Cys肽加入150μL的去离子水中,然后加入50μL的AuNPs,平衡5分钟后,由于Au-S相互作用,AuNPs溶液发生了不同程度的聚集,随着Cys-Ang(I)-Cys肽浓度的增加,AuNPs溶液的颜色由红色变为蓝色或紫色,然后利用紫外-可见光谱、动态光散射和透射电子显微镜对溶液进行进一步的分析和表征,确定每个96板孔中加入Cys-Ang(I)-Cys肽的浓度为5μM。
3.根据权利要求1所述的一种检测血管紧张素转换酶的方法,其特征在于:检测ACE活性时在37℃下孵育时间为30分钟。
4.根据权利要求1所述的一种检测血管紧张素转换酶的方法,其特征在于:所述ACE合成过程中移去热源冷却后采用聚醚砜膜去除AuNPs中的较大杂质,得到实验所需的AuNPs。
5.根据权利要求2所述的一种检测血管紧张素转换酶的方法,其特征在于:利用Cys-Ang(I)-Cys肽诱导AuNPs的聚集时Cys-Ang(I)-Cys肽的添加量为50μL,确保96板孔中最终体积为250μL,所述96板孔中不同浓度的Cys-Ang(I)-Cys肽的浓度范围为0-1000nM。
6.根据权利要求1-5任意一项所述的一种检测血管紧张素转换酶方法的应用,其特征在于:
(一):检测ACE抑制剂,将40μL的ACE与40μL不同浓度的依那普利、雷米普利混合,在37℃下孵育,然后加入Cys-Ang(I)-Cys肽40μL和去离子水30μL,按照检测ACE活性的方法测试剩余的ACE活性,随后加入50μL的AuNPs作为信号放大工具,采用400-800nm范围内的AuNPs紫外可见吸收光谱进行ACE抑制活性检测;
(二):筛选ACE抑制肽,采用9种不同的活性肽与30mU/mL的ACE在去离子水中孵育,孵育15分钟后,加入5μM的Cys-Ang(I)-Cys与未失活的ACE在37℃下反应15分钟,然后将50μL的AuNPs加入到150μL的混合物中,以颜色的变化来筛选出具有ACE抑制活性的多肽。
7.根据权利要求6所述的一种检测血管紧张素转换酶方法的应用,其特征在于:所述检测ACE抑制剂时在37℃下孵育时间为20分钟,确保ACE活性被充分抑制。
8.根据权利要求6所述的一种检测血管紧张素转换酶方法的应用,其特征在于:所述筛选ACE抑制肽时具有ACE抑制功能的肽能诱导AuNPs聚集,呈紫色,而无ACE抑制功能的肽不能诱导AuNPs聚集,呈红色;9种不同的活性肽分别为KAFAF、CAAAA、VPP、IPP、RPP、IPA、LEP、LQP和GSH,且9种活性肽的浓度均为15μM。
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