CN114456801B - 一种Ag+@CA-CdSe荧光探针及其在GSH检测中的应用 - Google Patents
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Abstract
本发明公开了一种Ag+@CA‑CdSe荧光探针及其在GSH检测中的应用，其中Ag+@CA‑CdSe荧光探针的结构式如下所示：
本发明Ag+@CA‑CdSe荧光探针在545nm处随着GSH浓度逐渐增大的荧光强度逐渐增强，检测范围为GSH为1‑7×10‑5M，检出限为0.741μM。本发明Ag+@CA‑CdSe荧光探针具较高的灵敏性和专一的选择性，并成功应用在细胞中GSH的检测。
Description
技术领域
本发明属于小分子氨基酸检测技术领域，具体涉及一种Ag+@CA-CdSe荧光探针及其在GSH检测中的应用。
背景技术
谷胱甘肽(GSH)是一种具有巯基与谷氨酸、半胱氨酸和甘氨酸结合的三肽，具有抗氧化和综合解毒作用。因此，谷胱甘肽可以参与生物转化，从而将体内有害的毒物转化为无害的物质，排出体外。谷胱甘肽还有助于维持免疫***的正常功能，在生理过程中具有重要作用。其中，谷胱甘肽类药物广泛应用于临床。除了利用其巯基螯合重金属外，还用于肝炎、溶血性疾病、角膜炎、白内障、视网膜疾病等作为治疗或辅助治疗药物。最新研究表明，GSH可以纠正乙酰胆碱和胆碱酯酶的失衡，起到抗过敏作用，还可以防止皮肤老化和色素沉着，减少黑色素的形成，提高皮肤的抗氧化能力，使皮肤有光泽。GSH浓度的变化与帕金森病、免疫功能障碍、肝病和多种癌症有关。GSH的快速、灵敏、选择性检测在医学上具有重要意义。
现有检测GSH的方法主要有：酶循环法、分光光度法、流式细胞术法、高效液相色谱(HPLC)法、高效毛细管电泳(HPCE)法、电化学法、同位素法、质谱法、荧光光谱法等，这些检测方法各有千秋。有的方法灵敏度高、速度快、可靠，但有的方法复杂，实验难度大，甚至成分无法分离。
量子点具有良好的发光性能、稳定性，生物相容性及波长可调节性等特点，并且有灵敏度高、选择性好、响应时间短、操作简单、成本低等特点。因此，用无机金属离子和量子点结合使表面功能化形成新的探针，再去检测生物小分子，在环境监测、生物成像及纳米材料等诸多领域具有及其广泛的应用前景。
发明内容
本发明旨在提供一种Ag+@CA-CdSe荧光探针及其在GSH检测中的应用，所要解决的技术问题是通过分子设计结合能够检测识别GSH的荧光探针。
本发明Ag+@CA-CdSe荧光探针的结构式如下所示：
本发明Ag+@CA-CdSe荧光探针的制备方法，首先在CdSe量子点的表面接枝配体半胱胺，然后再和Ag+离子反应使量子点表面功能化而获得Ag+@CA-CdSe荧光探针结构；具体包括如下步骤：
步骤1：将4.03mmolNaBH4和0.38mmol硒粉(Se)放入10ml离心管中，用5ml移液枪注入5ml去离子水至黑色粉末消失，得到无色透明的NaHSe溶液；
步骤2：将0.75mmol CdCl2·2.5H2O和2.03mmol半胱胺(CA)加入去离子水，完全溶解后迅速加入步骤1获得的溶液，得到CA-CdSe QDs；
步骤3：在1×10-5M的CA-CdSe QDs溶液中加入Ag+(1×10-5M)，从而得到新型的Ag+@CA-CdSe荧光探针。
本发明合成路线示意如下：
本发明Ag+@CA-CdSe荧光探针的应用，是在GSH的检测过程中作为检测试剂使用。
进一步地，本发明Ag+@CA-CdSe荧光探针能特异性识别GSH，可以通过测定荧光强度变化的方式可检测GSH，随着GSH浓度逐渐增大，Ag+@CA-CdSe荧光探针在545nm处的荧光强度逐渐增大。可以应用在水介质进行荧光光谱检测，通过荧光强度的变化实现对水溶液中GSH的定性或定量检测。所述Ag+@CA-CdSe荧光探针的检测限为0.741μM。
本发明Ag+@CA-CdSe荧光探针具较高的灵敏性和专一的选择性，并成功应用在细胞中GSH的检测。
附图说明
图1为CA-CdSe量子点透射电镜图。
图2为本发明Ag+@CA-CdSe荧光探针中前驱体CdSe量子点加入不同金属离子的荧光光谱(λex＝350nm)。
图3为本发明Ag+@CA-CdSe荧光探针中前驱体CdSe量子点加入不同浓度Ag+荧光变化图，以及荧光强度增强值(F-F0)与Ag+浓度之间关系图。
图4为本发明Ag+@CA-CdSe荧光探针中加入不同氨基酸后的通过荧光变化对其选择图。
图5为本发明Ag+@CA-CdSe荧光探针中加入不同浓度GSH的荧光变化图，以及荧光强度增强值(F-F0)与GSH浓度之间关系图。
图6为本发明Ag+@CA-CdSe荧光探针在365nm紫外灯照射下的图片。其中1.CA-CdSe，2.Ag+@CA-CdSe，3.Ag+@CA-CdSe+GSH，4.CA-CdSe+GSH。
图7为本发明Ag+@CA-CdSe荧光探针检测细胞内GSH的荧光成像图。
具体实施方式
本发明可以通过以下的实施例进一步说明，但不仅仅局限于实施例。
实施例1：Ag+@CA-CdSe荧光探针的制备与表征
NaHSe前驱体制备：将4.03mmolNaBH4和0.38mmol硒粉放入10ml离心管中，用5ml移液枪注入5ml去离子水至黑色粉末消失，得到无色透明的溶液。
Cd前驱体制备：取80ml超纯水于三颈烧瓶中通入N240分钟，然后加入0.75mmolCdCl2·2.5H2O和2.03mmol半胱胺(CA)，搅拌使其完全溶解，同时在通入N2氛围中搅拌30分钟后，将NaHSe溶液迅速倒入Cd的前驱体中搅拌，用1M NaOH溶液调节pH＝5,在40℃回流4h得到半胱胺为配体的CdSe量子点，即CA-CdSe QDs。
在1×10-5M的CA-CdSe QDs溶液中加入Ag+(1×10-5M)，得到Ag+@CA-CdSe荧光探针。
本实验在浓度为10-6M-10-4M的CA-CdSe QDs溶液中加入浓度10-6M到10-4MAg+进行了多组对比实验，发现只有在10-5M CA-CdSe QDs溶液中加入1×10-5MAg+去检测GSH效果最佳。其他组结果相同，检测效果不佳。
图1为CdSe量子点透射图。粒径大小约为3.4nm。
实施例2：Ag+@CA-CdSe荧光探针中前驱体CdSe量子点对Ag+的特异性选择(λex＝350nm)。
图2是CA-CdSe量子点中加入不同金属离子(Ag+,Fe3+,Hg2+,Cr3+,Cu2+,Mn2+,Ni2+,Pb2 +,Al3+,Mg2+,Zn2+,Ca2+,Li+,Co2+,Fe2+,K+)后荧光光谱图，从图中可看出量子点加入Ag+离子溶液后，量子点荧光发生了很大程度的增强，而加入其他金属离子没有增强量子点的荧光强度，甚至有的金属离子还略微减弱了量子点的荧光。以上可以说明CA-CdSe量子点对Ag+具有专一性识别。图3是Ag+浓度范围为2-18μM，并根据检测限方程L＝3σ/K，计算出检测限为5.41×10-8M。并且荧光强度发生了一定增强。
实施例3：Ag+@CA-CdSe荧光探针中加入不同氨基酸后的通过荧光变化对GSH特异选择。
发现只有在Ag+@CA-CdSe荧光探针中加入GSH后荧光强度发生了很大的增强，而其他氨基酸都没有这个效果，这个结果说明Ag+@CA-CdSe荧光探针对GSH具有很强的特异性识别(如图4所示)。
实施例4：Ag+@CA-CdSe荧光探针中加入不同浓度GSH的荧光变化，以及荧光强度增强值(F-F0)与GSH浓度之间相关性。
对其进行荧光检测，从图5中可以看出，随着GSH浓度增大，Ag+@CA-CdSe荧光探针的荧光在不断的增强。GSH浓度在1-7×10-5M浓度范围内有线性关系，可根据这个线性回归方程计算要检测GSH的浓度，简单方便。根据检测限方程L＝3σ/K，计算得出对于GSH的检测限为7.414×10-7M。
实施例5：Ag+@CA-CdSe荧光探针在365nm紫外灯照射下荧光强度变化。
如图6所示，从图中可以明显的发现Ag+@CA-CdSe荧光探针对GSH有识别性能。其中1.CA-CdSe，2.Ag+@CA-CdSe，3.Ag+@CA-CdSe+GSH，4.CA-CdSe+GSH。
实施例6：Ag+@CA-CdSe荧光探针的细胞成像图。
将CA-CdSe量子点放在细胞中，孵育两小时，使CA-CdSe量子点充分分布到细胞质区域。在绿色通道中细胞没有显示绿色荧光，加入银离子后，绿色通道显示微弱的荧光，继而加入谷胱甘肽后，绿色荧光有明显的增强。观察绿色通道与明场的合并图中，可以观察到先后加入Ag+与GSH出现绿色通道荧光连续增强的变化，说明Ag+@CA-CdSe荧光探针可以在Hela细胞中专一检测GSH。
Claims (7)
1.一种Ag+@CA-CdSe荧光探针，其特征在于其结构式如下所示：
2.一种权利要求1所述的Ag+@CA-CdSe荧光探针的制备方法，其特征在于：
首先在CdSe量子点的表面接枝配体半胱胺，然后再和Ag+离子反应使量子点表面功能化而获得Ag+@CA-CdSe荧光探针结构。
3.根据权利要求2所述的制备方法，其特征在于包括如下步骤：
步骤1：将NaBH4和硒粉加入去离子水中，黑色粉末消失，得到无色透明的NaHSe溶液；
步骤2：将CdCl2·2.5H2O和半胱胺加入去离子水中，完全溶解后迅速加入步骤1获得的NaHSe溶液中，得到半胱胺为配体的CdSe量子点CA-CdSeQDs；
步骤3：向CA-CdSeQDs溶液中加入Ag+，从而得到Ag+@CA-CdSe荧光探针。
4.根据权利要求3所述的制备方法，其特征在于：
步骤3中，CA-CdSeQDs溶液的浓度为1×10-5M，Ag+的浓度为1×10-5M。
5.一种权利要求1所述的Ag+@CA-CdSe荧光探针的应用，其特征在于：
所述Ag+@CA-CdSe荧光探针用于制备检测GSH的检测试剂。
6.根据权利要求5所述的应用，其特征在于：
所述Ag+@CA-CdSe荧光探针在pH＝7.4、常温下作为检测试剂使用，在水介质中进行荧光光谱检测，通过荧光强度的变化实现对水溶液中GSH的定性或定量检测。
7.根据权利要求6所述的应用，其特征在于：
所述Ag+@CA-CdSe荧光探针的检测限为0.741μM。
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| CN111795958A (zh) * | 2020-07-14 | 2020-10-20 | 南宁师范大学 | 特异性检测Ag+的CdSe量子点的制备及其检测方法和应用 |
| CN111826155A (zh) * | 2020-07-23 | 2020-10-27 | 安徽大学 | 一种CdS量子点-荧光素FRET荧光探针及其制备方法和用途 |
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| CN111795958A (zh) * | 2020-07-14 | 2020-10-20 | 南宁师范大学 | 特异性检测Ag+的CdSe量子点的制备及其检测方法和应用 |
| CN111826155A (zh) * | 2020-07-23 | 2020-10-27 | 安徽大学 | 一种CdS量子点-荧光素FRET荧光探针及其制备方法和用途 |
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