CN114656495B - 一种用于监测斑马鱼胚胎发育阶段中锌离子浓度变化的荧光探针 - Google Patents
一种用于监测斑马鱼胚胎发育阶段中锌离子浓度变化的荧光探针 Download PDFInfo
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Abstract
本发明公开了一种用于监测斑马鱼胚胎发育阶段中锌离子浓度变化的荧光探针。所述的探针化合物结构如式I所示。该荧光探针由荧光团8‑氨基‑BODIPY和识别基团二吡啶胺两部分组成,探针本身荧光极弱,与Zn2+特异性结合后荧光显着增强。该荧光探针不仅制备方法简单,具有高灵敏度和高选择性,而且还可以成功追踪斑马鱼胚胎发育过程中Zn2+的分布和变化,在生物领域具有广阔的应用前景。
Description
技术领域
本发明涉及小分子荧光探针原位检测锌离子在细胞中的位置和表达水平,具体地说是一种基于8-甲硫基-BODIPY为荧光团的荧光探针BDA可以对内源锌离子含量进行精确检测,属于荧光探针技术领域。
背景技术
Zn2+是一种的重要的微量元素,其浓度在哺乳动物细胞中维持在100-500μM之间,参与人体正常生理功能的稳态。在细胞中,大部分Zn2+能够与功能性蛋白质或酶结合,是金属蛋白酶参与内稳态、转录和翻译的关键辅助因子。剩余的游离Zn2+含量较低,主要分布于脑、肠、胰腺和视网膜等人体组织中,与神经信号传导和细胞凋亡密切相关。重要的是,细胞内Zn2+含量的变化已被认为是哺乳动物卵母细胞周期中的关键事件。许多研究表明,高水平的Zn2+对胎儿和儿童的生长发育至关重要,持续缺乏会导致胚胎发育迟缓甚至发育迟缓。因此,准确检测体内Zn2+浓度和分布,特别是动态监测胚胎发育过程中Zn2+的变化,这对于全面了解Zn2+相关生理病理过程具有重要意义。
分子成像技术可以无创地观察体内生理病理指标和过程。其中,荧光探针因其高灵敏度、高选择性和高时空分辨率而被广泛应用于生物分子成像。近年来,人们更加重视开发对Zn2+具有高灵敏度和特异性的荧光探针,有望用于实时监测体内Zn2+的分布。目前已报道了几种基于各种荧光基团的荧光探针检测Zn2+,但大部分工作仍存在合成繁琐、对生物环境易感性和一定的生物毒性等问题,尤其是大部分工作仅限于体外成像Zn2+和在活细胞中。
为克服难以活体内检测Zn2+的问题,实现对活体斑马鱼胚胎和胚胎发育中Zn2+分布和水平的动态监测,迫切需要开发适用于斑马鱼模型的Zn2+高特异性和高灵敏度探针并且允许以高空间和时间精度进行荧光检测,以确定活体内痕量Zn2+含量变化。
发明内容
本发明要解决的技术问题:一是提供一种检测Zn2+的荧光探针及其制备方法和生物应用,所述探针化合物具有对癌细胞和斑马鱼内源性Zn2+进行荧光原位成像的优点。二是提供一种灵敏度高、选择性好的荧光探针,通过荧光成像阐明Zn2+在斑马鱼胚胎发育至幼虫阶段的生物学作用的能力。斑马鱼胚胎发育过程中Zn2+的分布和变化。
为了解决上述技术问题,采取的技术方案如下:
本发明提供一种检测Zn2+的荧光探针,具有如下分子结构式:
化合物BDA
本发明还提供一种检测Zn2+的荧光探针的制备方法,步骤包括:
在N2气氛下将8-甲硫基-BODIPY(1eq)、N,N-二(2-吡啶甲基)乙二胺(3eq)和三乙胺(3eq)在无水乙腈溶液中混合进行亲和取代反应,室温条件下充分反应2~3h。将混合液减压旋蒸后得到粗产物,粗产物经硅胶柱层析纯化(甲醇/二氯甲烷=1/9)得到黄色固体,即锌离子荧光探针BDA。
其中,锌离子近红外荧光探针反应式如下:
本发明的另一目的在于提供一种锌离子荧光探针在细胞中锌离子的检测应用。更重要的目的是实时监测斑马鱼胚胎发育过程中Zn2+的分布和变化。
相较于现有技术,本发明的有益效果在于:
本发明提供的锌离子荧光探针能够特异性与锌离子发生反应,其荧光强度显着增加16倍,具有良好的稳定性和生物相容性。
本发明的荧光探针分子,对Zn2+的响应速度非常快,在10分钟内即可完全响应,可以应用于快速检测复杂样品中的Zn2+含量。
本发明的荧光探针分子,具有良好的灵敏度和选择性,荧光信号仅在Zn2+存在的条件下发生,其它常见的金属离子均无法使探针溶液产生荧光光谱的变化。
本发明的荧光探针分子,提供了活细胞中Zn2+的可逆可视化监测。更重要的是,该探针可以实现对斑马鱼胚胎发育阶段中Zn2+分布的动态监测。
因此,本发明为非侵入性监控活体内Zn2+含量的变化提供了一种可靠的手段。在生物分析检测领域具有广阔的应用前景。
附图说明
图1为实施例1中制得的锌离子荧光探针BDA在(PBS,pH=7.4)溶液中对Zn2+(30μM)的紫外吸收光谱图。
图2为实施例1中制得的锌离子荧光探针在(PBS,pH=7.4)溶液中对Zn2+(30μM)的荧光光谱图。
图3为实施例1中制得的锌离子荧光探针BDA(10μM)对Zn2+(30μM)的时间依赖性荧光响应。
图4为实施例1中制得的锌离子荧光探针BDA(10μM)与不同浓度(0-30μM)的Zn2+反应10min后的荧光光谱响应图
图5为实施例1中制得的锌离子荧光探针BDA(10μM)与浓度为0-18μM的Zn2+反应10min后在450nm处荧光强度的线性拟合曲线。
图6为实施例1中制得的荧光探针BDA对不同金属离子选择干扰性检测的荧光响应图。
图7为实施例1中制得的不同浓度的荧光探针BDA在24小时内对HeLa细胞的毒性检测。
图8为实施例1中制得的荧光探针BDA在HeLa细胞中的荧光成像图。
图9为实施例1中制得的荧光探针BDA在斑马鱼胚胎发育阶段中的荧光成像图。
图10为实施例1中制得的荧光探针BDA在斑马鱼胚胎发育阶段中的荧光放大成像图。
图11为实施例1中制得的荧光探针BDA的核磁共振1H-NMR谱图。
图12为实施例1中制得的荧光探针BDA的核磁共振13C-NMR谱图。
具体实施方式
下面结合具体实施例对本发明做进一步的详细说明。
实施例1
一种检测Zn2+的荧光探针BDA的制备方法,步骤包括:
探针BDA的合成:
将8-甲硫基-BODIPY(15mg,0.063mmol)、N,N-二(2-吡啶甲基)乙二胺(45mg,0.18mmol)和三乙胺(18μL,0.13mmol)在在无水乙腈溶液中(1mL)混合进行亲和取代反应。室温条件下充分反应2h。将混合溶液减压旋蒸后得到粗产物,粗产物经硅胶柱层析纯化(DCM:MeOH=9:1)纯化粗产物,得到黄色固体BDA(24.3mg,89%)。1H NMR(400MHz,DMSO-d6)δ10.11(s,1H),8.48(d,J=4.1Hz,2H),7.69(td,J=7.7,1.7Hz,2H),7.59(d,J=4.7Hz,2H),7.46(d,J=7.8Hz,2H),7.41(s,1H),7.28–7.21(m,3H),6.48(ddd,J=7.6,3.9,2.2Hz,2H),3.95(s,4H),3.90(t,J=6.0Hz,2H),2.95(t,J=6.0Hz,2H).13C NMR(101MHz,DMSO-d6)δ158.68,148.79,147.94,136.72,133.42,130.41,125.13,123.18,122.93,122.33,122.10,115.83,114.14,113.08,58.80,50.04,44.48.ESI-MS m/z(C58H65N3O10S2)calculated(M-H)-:431.1967,found(M-H)-:431.1972.
实施例1中制得的荧光探针的氢谱(1H NMR)和碳谱(13C NMR),分别如图11和图12所示,说明本发明的荧光探针BDA成功合成。
实施例2
探针BDA与Zn2+螯合前后的紫外光谱研究
在相同实验条件下,向PBS(pH=7.4)的缓冲溶液中加入10μL探针分子储备液(1mM),随后滴加3μL Zn2+储备液(10mM),并测试其反应前后的紫外吸收光谱。参见图1,图1为探针BDA(10μM)与浓度为30μM锌离子反应10min的紫外吸收的变化图,由图可知,探针BDA的最大吸收峰出现在320nm和390nm附近。随着Zn2+的加入,最大吸收有轻微的红移,结果证明,BDA能够与Zn2+发生反应。
实施例3
探针分子与Zn2+螯合前后的荧光光谱研究
在相同实验条件下,向PBS(pH=7.4)的缓冲溶液中加入10μL探针分子储备液(1mM),随后滴加3μL Zn2+储备液(10mM),并测试其反应前后的荧光光谱。参见图2,图2为探针BDA(10μM)与浓度为30μM锌离子反应10min的荧光吸收的变化图,由图可知,BDA本身的荧光信号可以忽略不计,随着Zn2+的加入,BDA在450nm处的荧光强度显着增加了16倍;同时,在365nm的手提紫外灯照射下,探针BDA与Zn2+共孵育后的产物Zn:BDA和BDA的荧光强度存在显着差异(图2的插图)。这种Zn2+诱导的荧光增强可归因于识别基团N,N-二(2-吡啶甲基)乙二胺螯合Zn2+触发的PeT效应的阻断。结果证明,荧光探针BDA能够与Zn2+结合,并使其荧光显著增加。该探针BDA是荧光增强型探针。
实施例4
探针BDA与Zn2+螯合前后的动力学研究
在相同实验条件下,向PBS(pH=7.4)的缓冲溶液中加入10μL探针分子储备液(1mM),随后滴加3μL Zn2+储备液(10mM),并测试其荧光响应动力学。参见图3,图3为探针BDA对Zn2+(30μM)的时间依赖性荧光响应。额外添加Zn2+后,BDA在450nm处的最大荧光强度增加,并在10分钟时达到最大值且探针的荧光强度逐渐趋于稳定,结果证明,该探针具有良好的稳定性,且对Zn2+响应速度快。
实施例5
探针BDA对不同浓度Zn2+的荧光强度变化的研究
相同实验条件下,向PBS(pH=7.4)的缓冲溶液中加入10μL探针分子储备液(1mM),进行Zn2+荧光滴定实验,并测试其荧光光谱。并参见图4,图4为本发明制得的荧光探针(10μM)与不同浓度的Zn2+(0、2、4、6、8、10、12、14、16、18、20、30μM)反应10min后的荧光光谱响应图,由图4可知,随着加入的Zn2+浓度的升高,荧光探针BDA在450nm处的荧光强度逐渐增强;当Zn2+的浓度达到30μM时,荧光信号达到最大值,此外,从图5中可以看出,在0-18μM范围内,荧光强度与Zn2+的浓度具有很好的线性关系。结果表明,荧光探针BDA能灵敏地响应低浓度的Zn2+,表明探针对Zn2+具有很高的灵敏度。
实施例6
荧光探针BDA对Zn2+的选择性识别
在相同实验条件下,向PBS(pH=7.4)的缓冲溶液中加入10μL探针分子储备液(1mM),随后滴加和3μL的各种金属离子(Na+、K+、Ca2+、Mg2+、Fe2+、Fe3+、Ni2+、Co2+、Cd2+、Mn2+、Sn2+、Sn4+、Li+)储备液(10mM),随后测试其在450nm处的荧光强度;紧接着向上述溶液中加入3μL的Zn2+储备液(10mM),再次测试其在450nm处的荧光强度。参见图6,图6为本发明制得的荧光探针BDA对不同金属离子的荧光响应,由图可知,在BDA与潜在干扰离子孵育后,荧光几乎没有变化。只有与Zn2+结合的BDA显示出强烈的荧光增强。另一方面,除了Co2+、Ni2+和Cu2+对本发明荧光探针BDA识别Zn2+有轻微干扰外,其他金属离子预处理的BDA进一步与Zn2+孵育时,本发明探针BDA表现为良好的荧光开启。然而,这些金属离子(Co2+、Ni2+和Cu2+)在生物体内几乎全部以结合形式存在,对后续的体内应用几乎没有影响,这说明本发明制备的探针具有良好的选择性。
综上所述,本发明制得的荧光探针BDA在体外具有灵敏的Zn2+检测能力,可以达到细胞和活体成像的要求。
实施例7
细胞毒
实施例7依据实施例1制得的荧光探针BDA对HeLa细胞进行CCK-8毒性测试。将HeLa细胞接种于96孔板上,密度为每孔6×105个细胞,37℃孵育24小时,将细胞用培养基洗涤一遍,然后在37℃下与各种浓度的探针BDA(1、2、4、8、12、16、20μM)孵育24小时,随后弃去培养基,在每个孔中加入含有10μL CCK-8的100μL培养基的混合溶液,在37℃培养箱中孵育60分钟。使用酶标仪测量450nm处的吸光度并记录。用以下公式计算细胞存活率:
细胞存活率计算:细胞存活率(%)=[A(实验组)-A(空白组)]/[A(对照组)-A(空白组)]×100%。
参见图7,图7为细胞毒结果。不同浓度的BDA与HeLa细胞共同孵育后,发现即使BDA浓度高达28μM,BDA对细胞的毒性仍然较小,测得的细胞活力可以达到95%以上,这说明合成的BDA具有良好的生物相容性,可以在细胞水平上验证探针BDA的成像能力。
实施例8
HeLa细胞中Zn2+的荧光成像研究
将HeLa细胞铺板至激光共聚焦皿中并培养过夜以达到80%的密度。细胞成像分为三组。在第一组中,BDA(10μM)与HeLa细胞孵育30分钟。在第二组中,BDA+Zn2+/吡啶硫酮(Pyr)组中,细胞先与BDA(10μM)一起孵育30分钟后,PBS清洗两遍,除去残留探针,然后细胞与Zn2+:吡啶硫酮(Pyr)(50μM:100μM)溶液进一步孵育30分钟,最后成像前PBS清洗两遍除去多余离子。在第三组中,首先将细胞与BDA一起孵育30分钟,PBS清洗两遍,除去残留探针,然后与Zn2+:Pyr(50μM:100μM)溶液一起孵育30分钟,然后用PBS缓冲液洗涤三遍后与100μM N,N,N',N'-四(2-吡啶基甲基)-乙二胺(TPEN)孵育10分钟,细胞成像前用PBS缓冲液洗涤细胞三遍。使用带有DAPI通道(λex=404nm和λem=425-475nm)的Nikon Ti-e显微镜进行细胞荧光成像。
参见图8,HeLa细胞与BDA共孵育后,由于HeLa细胞内源性Zn2+水平低,出现微弱的荧光。而利用离子载体吡啶硫酮(Pyr)将外源性Zn2+输送到细胞内后发现外源性Zn2+处理组显示出更高的蓝色荧光。TPEN处理细胞后荧光信号大幅下降70%以上,这些结果明确表明BDA可有效用于检测细胞中的Zn2+。
实施例9
荧光探针BDA追踪斑马鱼胚胎发育过程中Zn2+的分布和变化
为了跟踪斑马鱼发育阶段Zn2+的分布,将斑马鱼胚胎在28.5℃的纯水中培养。孵化过程持续了4天。在特定的时间(0、18、24、48、72、96h)在培养液中加入荧光探针BDA(6μM)溶液共孵育1小时后,进行斑马鱼胚胎发育阶段中特定时间的荧光成像。使用多功能连续变焦显微镜(AZ100,Nikon,Japan)成像进行观察,采集参数为激发330-380nm,发射420nm。
参见图9,通过在不同时间点与BDA孵育来跟踪斑马鱼胚胎发育阶段Zn2+的分布。在最初的发育阶段,在胚膜边缘观察到微弱的荧光。孵化18小时后,蓝色条带变亮并延伸至胚胎中心,表明BDA可以穿透卵子的屏障。18小时内卵黄囊具有明显的蓝色荧光,说明卵黄囊中产生了高含量的Zn2+。此外,心脏和眼睛显示出微弱的荧光。孵育24小时后,卵黄囊处的荧光略有增加。进一步孵育后,心脏和眼睛出现蓝色点状荧光。另外,眼球中央瞳孔处明亮的蓝色荧光表面高浓度Zn2+的富集,这表明Zn2+可能在视觉信号转导中起重要作用。在48小时内,卵黄囊中的Zn2+水平达到最大值。72小时后,斑马鱼幼虫消化系统肠道内出现蓝色荧光。96小时后,在斑马鱼幼虫的卵黄囊、心脏、眼睛和肠道中可以清楚地观察到蓝色荧光。图10的斑马鱼放大图中可以更清晰地看出Zn2+在不同时间点中不同部位的分布以及浓度变化情况。这些结果表明,BDA具有生物相容性和优异的稳定性,可以作为有效的荧光探针检测斑马鱼胚胎发育阶段中Zn2+的变化和分布。
上述仅为本发明的优选具体实施方式,并非是对本发明作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但本发明的设计构思并不局限于此,凡利用此构思对本发明进行非实质性的改动,均应属于侵犯本发明保护范围的行为。
Claims (4)
1.一种用于监测斑马鱼胚胎发育阶段中锌离子浓度变化的荧光探针,其特征在于,该探针结构如下所示:
。
2.制备如权利要求 1 所述的荧光探针的方法,其特征在于,包括如下步骤: 将 8-甲硫基-BODIPY、N,N-二(2-吡啶甲基)乙二胺和三乙胺TEA在无水乙腈MeCN中混合,所述 8-甲硫基-BODIPY、N,N-二(2-吡啶甲基)乙二胺和三乙胺TEA的摩尔比为 1:3:3;在氮气保护下进行亲和取代反应,反应 2h;将混合液减压旋蒸后得到粗产物,粗产物经硅胶柱层析纯化得到黄色固体,即荧光探针;其反应路线如下所示:
3.如权利要求 2 所述的方法,其特征在于:柱纯化中甲醇和二氯甲烷体积比为 1:9。
4. 根据权利要求 1 所述荧光探针在锌离子的荧光检测中的应用,其特征在于,用于锌离子含量的荧光检测,所述检测不涉及疾病诊断或治疗。
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