CN111004186A - 一种小分子荧光探针及其制备方法与应用 - Google Patents
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Abstract
本发明公开了一种小分子荧光探针及其制备方法与应用，用于检测样品中次氯酸离子以及生物成像应用，该探针母体为6‑羟基‑2‑萘甲醛和2‑硫代巴比妥酸，该分子探针可实现紫外和荧光精确检测次氯酸离子，可以用于检测溶液、活细胞以及斑马鱼中外源性的次氯酸离子，本发明合成方法简单，操作方便，不需要苛刻的条件，而且合成产率和纯度都很高，因此在铜离子和锌离子检测方面具有良好的应用前景。
Description
技术领域
本发明涉及荧光成像分子探针领域，尤其是涉及一种利用荧光成像技术检 测次氯酸离子的探针，具体涉及一种小分子荧光探针及其制备方法与应用。
背景技术
次氯酸是一种不稳定的弱酸，常用于消毒剂和漂白剂的主要成分。而且， 在生物体内具有重要的生理功能，如免疫系统中可以充当抗菌抗病毒等重要功 能。此外，次氯酸在维持生物体内环境中起着重要角色，如平衡体内氧化还原 过程。但是，次氯酸在生物体内的量一旦超过机体所需的正常范围，便会对机 体内环境造成一定损害，例如，过量的次氯酸也可能对一下磷脂，蛋白以及核 酸物质产生严重损伤。因此，开发高灵敏度和高选择性的小分子探针来检测次 氯酸仍然是一个艰巨的挑战。
目前，用于检测次氯酸的方法很多，例如比色法，电位分析法，化学发光 发以及库仑滴定发等。但是，基于荧光探针的次氯酸检测技术正在成为一种重 要研究方法。近几年报道了很多小分子探针对次氯酸选择性识别，尽管如此多 的次氯酸识别探针被报道，但是目前对于双功能识别的探针报道很少，而且该 类探针对次氯酸识别能力和选择性都不是很理想，鉴于此，本研究发明一种高 选择性和高灵敏度的双功能次氯酸识别荧光探针。
发明内容
针对现有技术存在的不足，本发明的目的在于提供一种制备工艺简单、操 作方便的针对次氯酸离子的荧光探针。
为实现上述目的，本发明提供一种小分子荧光探针，所述小分子荧光探针 分子式为C13H10N2SO3，其结构式为：
本发明还提供上述小分子荧光探针的制备方法，具体包括如下步骤：
步骤一、将6-羟基-2-萘甲醛和2-硫代巴比妥酸溶解在乙醇溶液中；
步骤二、将上述反应物混合均匀，并在85℃下反应2-24小时，反应结束后 过滤，干燥得到红色沉淀；
步骤三、上述红色沉淀溶解在乙醇中，反复洗涤，滤液缓慢挥发后得到红 色结晶性固体。
作为本发明的进一步改进，所述6-羟基-2-萘甲醛和2-硫代巴比妥酸的摩尔 比为1:1。
作为本发明的进一步改进，所述乙醇与6-羟基-2-萘甲醛和2-硫代巴比妥酸 混合物的质量比为10:1。
本发明还提供上述小分子荧光探针检测、识别环境中或生物样品中次氯酸 离子的应用。
作为本发明的一种应用范围,所述小分子荧光探针利用荧光成像检测正常细 胞和癌细胞中外源性的次氯酸离子的应用。
作为本发明的一种应用范围,所述小分子荧光探针利用荧光成像检测斑马鱼 体内外源性的次氯酸离子的应用。
作为本发明的一种应用方式,通过在440nm和515nm紫外光源下测定吸 光度值检测、识别环境中或生物样品中次氯酸离子应用，该小分子荧光探针在 次氯酸离子的存在下，在440nm处的吸收峰下降；并在515nm处产生新的吸 收。
作为本发明的一种应用方式，以及以440nm为激发波长，在535nm的波 长处测定检测、识别环境中或生物样品中次氯酸离子溶液的吸光度应用。
本发明具有如下优点：本发明的小分子探针以萘甲醛和硫代巴比妥酸反应 为原料，萘甲醛和硫代巴比妥酸结构通过碳碳键连接的母体结构，具有很强共 轭π电子，萘甲醛和硫代巴比妥酸结构通过碳碳键连接的母体结构中碳碳键容 易断裂，结构中碳碳键断裂后后能发射很强的荧光，小分子探针在次氯酸的存 在下结构中碳碳键发生断流，使得其荧光从无到有，紫外吸收峰红移，实现荧 光技术精确检测次氯酸离子；且均可以检测活细胞和斑马鱼体内外源性的次氯 酸离子。因此在次氯酸离子离子检测方面具有良好的应用前景。同时，本发明 的合成方法简单、操作方便，不需要苛刻的条件。
附图说明
图1为实施例1中合成小分子探针的路线；
图2为实施例1中合成小分子探针的核磁氢谱；
图3为实施例1中合成小分子探针的核磁碳谱；
图4为实施例1中合成小分子探针的质谱；
图5为实施例1中小分子探针对次氯酸识别的机理图；
图6为实施例2中小分子探针对次氯酸识别的紫外和荧光光谱；
图7为实施例4中小分子探针对次氯酸的选择性；
图8为实施例3中验证小分子探针对次氯酸识别的密度泛函理论计算；
图9为实施例5中小分子探针在人胚肺细胞(MRC-5)中检测内源性次氯 酸的生物成像；
图10为实施例6中小分子探针在斑马鱼体内检测内源性次氯酸的生物成 像。
具体实施方式
下面将结合实施例和效果例对本发明做进一步的详述，而非限制本发明的 范围。
实施例1中小分子荧光探针的制备
将6-羟基-2-萘甲醛(0.300g,1.47mmol)和2-硫代巴比妥酸(0.376g,261 mmol)溶解在20mL乙醇溶液中，并在85℃条件下搅拌4小时，产生淡红色 沉淀；将上述红色沉淀过滤，用乙醇反复洗涤，干燥得到黄色固体粉末0.441g， 产率85％，通过核磁和质谱可以确定该产物即为目标小分子探针。合成小分子 探针的路线为图1，核磁表征为图2和图3，质谱表征为图4以及小分子荧光探 针对次氯酸识别机制模型图为图5。
1H NMR(500MHz,DMSO)δ12.42(s,1H),12.32(s,1H),10.43(br s,1H),8.74(s,1H),8.40(s,1H),8.32(dd,J＝8.9,1.7Hz,1H),7.90(d,J＝8.8Hz,1H),7.72(d,J＝ 8.8Hz,1H),7.18–7.14(m,2H)；
13C NMR(126MHz,DMSO)δ178.56,162.29,159.99,159.17,156.70,138.31,137.53,132.16,129.86,127.47,126.88,125.84,119.76,116.98,109.23；
HRMS(ESI)m/z[M+1]+:Calcd for C15H11N2O3S,299.0485,found,487.0489。
实施例2中小分子探针对次氯酸离子识别的紫外和荧光光谱
制备1mL小分子探针(10μM和5μM)的水溶液。同浓度的ClO-溶液滴 加到探针溶液中，
如图6(a)所示，在探针溶液中加入ClO-后，在440nm处的吸收带逐渐减少， 515nm处有一个新的吸收峰。在荧光滴定实验中，制备1mL小分子探针(5μM) 的水溶液。同浓度的ClO-溶液滴加到探针溶液中，以440nm为激发波长测量探 针从400nm到700nm的荧光值，实验结果见图6(b)。可观察到探针的荧光强 度随ClO-浓度增加而增强，最大发射光谱在535nm。
实施例3小分子探针对ClO-识别的密度泛函理论研究
为了进一步了解探针的分子结构，荧光和吸收光谱的传感机理，进行了有 无ClO-情况下探究了小分子探针的理论计算，以密度泛函理论深入了解它们的 电子跃迁(DFT).如图6所示，提出了该小分子荧光探针和ClO-反应前后的分 子轨道图，并且优化的结构显示探针可以与ClO-反应发生碳碳键断裂。这与上 述光谱实验的结果一致。此外，荧光探针基态的电子云主要位于分子的香豆素 结构中，当转移并分散到整个共轭体系和周围原子激发时。此外，反应前后结 构的能级从HOMO(-0.2243/-0.2197eV)到LUMO(-0.1067/-0.0635eV)的 水平为0.1176eV/0.1562eV。
实施例4中验证小分子荧光探针对次氯酸离子和其他离子的荧光光谱图、 荧光变化图和紫外照射下小分子荧光探针对不同阴离子溶液中选择性实验
如图7所示，小分子荧光探针和其他离子混合时，荧光基本上不发生变化， 小分子荧光探针和次氯酸离子反应时，荧光强度变强。
制备10mL分子探针(2μM)的EtOH-H2O溶液(v/v＝1:10)。通过将相应 的盐溶于去离子水制备各种阴离子溶液(ClO-，SO3 2-，NO,1O2,·OH,PBO·,TBHP,DBTP,Cys, GSH,H2O2，1.0eq)。随后，将1.0eq阴离子分别加入到探针溶液中。通过荧光光 谱进行检测，实验结果见图7a。取荧光最大波长进行对比，如图7b所示，离子 包括ClO-，SO3 2-，NO,1O2,·OH,PBO·,TBHP,DBTP,Cys,GSH,H2O2。除ClO-外，这些阴离子 对探针的荧光都没有产生明显变化。如图7C所示，在ClO-的加入后，荧光强度 明显，结果表明探针对ClO-具有高选择性。
实施例5小分子探针的在细胞中成像效果
为了探索小分子探针对于活细胞中亚细胞标记和保护ClO-的潜在生物学可 用性，在MRC-5细胞系中进行细胞生物成像。为了证明探针检测内源性ClO- 细胞的能力，我们进行了阴性和阳性对照的成像分析作为有力的证据。当细胞 与游离小分子探针一起温育时，观察到可见荧光信号，图9所示。之后，将这 些预处理细胞与探针一起温育20分钟，加入ClO-(6.0μM)立即观察到红色通 道中的显着荧光强度。这些结果清楚地表明SPTPA探针满足监测活细胞中内源 ClO-的要求。
实施例6小分子探针的在斑马鱼中成像效果
在正常的代谢过程中，生物体的器官可以自发地产生各种ROS。到目前为 止，大多数研究报告证明荧光成像的可能性是通过预先喂入/注射或将ROS诱导 的化学物质添加到生物体中而引入额外的ROS。然而，荧光探针在正常生物中 对自发ROS的生物成像仍然面临探针的灵敏度和稳定性及其通过各种生物屏障 的能力的问题。在活体动物的数量上产生了许多生物过程，导致ROS(ClO-) 在那里自我产生。因此，为了研究探针对生物体内源性和外源性ClO-的潜在生 物学应用，5日龄斑马鱼是一种流行的脊椎动物模型，它们被选为我们的研究模 型系统。图10所示，斑马鱼在胚胎培养基中培养，并与游离探针(2.0μM)孵 育20分钟，以确保探针渗透到斑马鱼的整个组织中，在腹部显示出弱而可见的 红色荧光。此外，斑马鱼用探针预处理，用纯水洗涤三次，分别与ClO-(6.0μM) 一起孵育，在斑马鱼的头部和腹部显示出显着的红色荧光。因此，这些结果表 明探针具有高组织穿透能力，并且可以实现斑马鱼中ClO-的可视化。
本发明具有如下优点：通过本发明所述制备方法合成小分子探针，还可以 实现紫外和荧光光谱法精确传感ClO-，并且可以快速、准确的检测细胞和斑马 鱼体内中内源性的ClO-。因此在ClO-检测方面具有良好的应用前景。同时，本 发明的合成方法简单、操作方便，不需要苛刻的条件。
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实 施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。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前提下的若干改进 和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.一种小分子荧光探针，其特征在于：所述小分子荧光探针分子式为C13H10N2SO3，其结构式为：
2.权利要求1所述的小分子荧光探针的制备方法，其特征在于，具体包括如下步骤：
步骤一、将6-羟基-2-萘甲醛和2-硫代巴比妥酸溶解在乙醇溶液中；
步骤二、将上述反应物混合均匀，并在85℃下反应2-24小时，反应结束后过滤，干燥得到红色沉淀；
步骤三、上述红色沉淀溶解在乙醇中，反复洗涤，滤液缓慢挥发后得到红色结晶性固体。
3.权利要求2所述的小分子荧光探针的制备方法，其特征在于，所述6-羟基-2-萘甲醛和2-硫代巴比妥酸的摩尔比为1:1。
4.权利要求3所述的小分子荧光探针的制备方法，其特征在于，所述乙醇与6-羟基-2-萘甲醛和2-硫代巴比妥酸混合物的质量比为10:1。
5.根据权利要求1-4任一项所述的小分子荧光探针在检测、识别环境中或生物样品中次氯酸离子的应用。
6.根据权利要求5所述的应用，其特征在于,所述小分子荧光探针利用荧光成像检测正常细胞和癌细胞中外源性的次氯酸离子的应用。
7.根据权利要求6所述的应用，其特征在于,所述小分子荧光探针利用荧光成像检测斑马鱼体内外源性的次氯酸离子的应用。
8.根据权利要求1-4任一项所述的小分子荧光探针的检测方法，其特征在于,通过在440nm和515nm紫外光源下测定吸光度值检测、识别环境中或生物样品中次氯酸离子应用，该小分子荧光探针在次氯酸离子的存在下，在440nm处的吸收峰下降；并在515nm处产生新的吸收。
9.根据权利要求1-4任一项所述的小分子荧光探针的检测方法，其特征在于,以及以440nm为激发波长，在535nm的波长处测定检测、识别环境中或生物样品中次氯酸离子溶液的吸光度应用。
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