CN111925316B - 一种基于4-氟苯乙炔基的双光子荧光极性探针及其制备方法和用途 - Google Patents
一种基于4-氟苯乙炔基的双光子荧光极性探针及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种基于4‑氟苯乙炔基的双光子荧光极性探针及其制备方法和用途,其中基于4‑氟苯乙炔基的双光子荧光极性探针的结构如下:本发明基于4‑氟苯乙炔基的双光子荧光极性探针分子在与其他干扰因素共存的体系中,对极性表现出专一性响应。细胞毒性测试表明该探针对于细胞具有较低的生物毒性,双光子共聚焦荧光显微成像实验表明该探针在细胞内光稳定性性好,适用于细胞内极性的双光子荧光成像和检测。
Description
技术领域
本发明涉及一种基于4-氟苯乙炔基的双光子荧光极性探针及其制备方法和用途,以实现双光子荧光成像检测细胞内的极性,具有选择性专一、灵敏度高、生物毒性低的优点。
背景技术
极性是生物微环境中重要组成部分,细胞的极性是细胞操作和调节机制的反馈。正是这些机制诱导和维持亚细胞器的功能或功能,导致各种生理和病理活动。不同的生理和病理活动对应于不同的细胞状态。当细胞状态改变时,极性也在一定程度上变化。因此,细胞极性的检测对于监测不同的细胞状态是必要的。并且,细胞凋亡过程中极性会有很大的改变,因此可以通过极性来检测凋亡,这对于研究细胞凋亡提供了一个很好的思路。
细胞凋亡,是程序性死亡的其中一个过程,在生物过程中扮演重要的角色,检测细胞凋亡对于生物学研究具有重要的意义。目前检测凋亡的方法是基于传统方法,例如检测半胱天冬酶蛋白的活性等。但这些方法的成本很高,周期很长和敏感性很差,并且它们成本很高不能满足评估细胞凋亡的要求。在过去十年中,荧光探针方法已经被广泛应用于监测生物过程,这主要基于荧光探针技术价格便宜,使用简单,灵敏度高,响应速度快。目前,已经开发非常多的荧光探针,但是检测极性的探针还在发展,因此开发性能优异的极性探针是非常有必要的。并且,细胞凋亡过程中极性会有很大的改变,因此可以通过极性来评估凋亡,这对于研究细胞凋亡提供了一个很好的思路。
发明内容
本发明旨在提供一种基于4-氟苯乙炔基的双光子荧光极性探针及其制备方法和用途,所要解决的技术问题是通过分子设计得到一种可以专一检测极性的双光子荧光探针结构,以实现双光子荧光成像检测细胞凋亡过程中的极性变化,具有选择性专一、灵敏度高的优点,细胞毒性测试表明本发明荧光探针对细胞几乎没有毒性作用。
本发明基于4-氟苯乙炔基的双光子荧光极性探针,简记为POLAR-QF,其结构式如下:
本发明基于4-氟苯乙炔基的双光子荧光极性探针的制备方法,包括如下步骤:
步骤1:化合物9-乙基-6-碘-9H-咔唑-3-甲醛(6g,17.2mmol)投入到干燥的三口瓶中,加入DMF(60mL)溶清后,滴3-5滴的三乙胺(2-3mL)当催化剂,加入丙二腈(1.36g,20.6mmol),保持常温搅拌1小时,观察瓶内温度颜色变化,反应2小时点板观察反应状况。反应2小时后,加入1L的H2O搅拌10-20min,用EA(乙酸乙酯)反复萃取直到水层点板观察不到产品点。旋干萃取得到粗产物,粗产物用柱层析(正己烷:乙酸乙酯:二氯乙烷=10:1:3)过得到化合物1(暗红色固体,5.9g,产率86.4%)。
步骤2:将化合物1(1.5g,3.778mmol)、Pd2(PPh3)2Cl2(12.40mg 0.018mmol),CuI(6.98mg,0.036mol)当催化剂置入斯莱克瓶,用泵反复置换反应瓶里面的气体环境,充入氩气,使反应在干燥的氛围下进行反应,用注射器将溶解对氟苯乙炔(0.544g,4.53mmol)的THF(8mL)、Et3N(2mL)溶液注射入反应瓶中,升温到45℃反应5小时,瓶内颜色加深点板观察原料反应完,等待反应液降温到室温后加水800-1000mL搅拌20-30min,用二氯乙烷反复萃取3-4次直到水层点板无产品点,加热浓缩得粗产物2.3g,经柱层析(正己烷:乙酸乙酯=7:1)过滤获取暗红色固体目标化合物POLAR-QF(1.2g,产率59.7%)。
本发明基于4-氟苯乙炔基的双光子荧光探针POLAR-QF的合成过程如下。
本发明基于4-氟苯乙炔基的双光子荧光极性探针的用途,是以非治疗或诊断为目的,在检测活细胞中的极性变化作为检测试剂使用。检测方法如下:
将本发明探针POLAR-QF溶于DMSO中制得2mM的母液,各取15μL母液于3mL不同极性的溶剂中,获得探针POLAR-QF在不同溶剂中的紫外谱图。其在I370max/I370min处的荧光强度随着测试体系极性的增大逐渐减弱。并且荧光强度(I370max/I370min)和Δf之间存在线性关系,这表明POLAR-QF可用于检测常见溶液的极性。为了进一步验证探针POLAR-QF对极性的响应特性,在具有不同比例的水和四氢呋喃极性范围内,测量POLAR-QF的吸收和荧光光谱。当溶剂的极性从含10%水(Δf≈0.2556)依次增大到含80%水(Δf≈0.3103)时,在紫外吸收光谱看到微小的变化与测不同溶剂极性的结果一致。当溶液的极性(Δf)从0.3103(80%水)降低到0.2556(10%水)时,POLAR-QF在I370max/I370min处的荧光强度增大了5.4倍。上述结果还显示荧光强度I370max/I370min与Δf具有良好的线性相关性,这表明POLAR-QF对溶剂极性高度敏感。在四氢呋喃为90%时,探针的有效双光子吸收截面在780nm出现最大,为88GM。我们还探究了探针POLAR-QF在HeLa细胞中光学的稳定性,这是非常重要的实验,因为在细胞凋亡过程中,细胞内环境被破坏,这会影响探针的光稳定性能,从而关系到能否实时监测细胞凋亡过程。此外,利用探针POLAR-QF测试了依托泊苷(etoposide)诱导HeLa细胞凋亡过程细胞内的极性变化。
本发明探针分子POLAR-QF在与其他干扰因素共存的体系中,表现出对极性的专一性响应。细胞毒性测试表明该探针对于细胞几乎没有什么毒副作用,双光子共聚焦荧光显微成像实验表明该探针在HeLa细胞中光稳定性好,适用于细胞内极性的双光子荧光成像和原位检测,可以原位检测依托泊苷(etoposide)诱导细胞凋亡过程中的细胞内极性变化趋势。
附图说明
图1是10μM探针在不同极性有机溶剂中的(a)紫外吸收光谱图;(b)荧光发射光谱图;(c)荧光强度(I370max/I370min)和Δf之间的线性关系图。
图2是10μM探针在不同体积比水/四氢呋喃混合溶剂中的(a)紫外吸收光谱图;(b)荧光发射光谱图;(c)荧光强度(I370max/I370min)和Δf之间的线性关系图。
图3是10μM探针在水/四氢呋喃不同比例的pH稳定性图。
图4是0.1mM探针在不同体积比水/四氢呋喃混合溶剂中的(a)有效双光子吸收截面图;(b)相对双光子荧光强度(Iout)与输入功率(Iin)的对数关系图。
图5是在不同浓度(0μM、10μM、20μM、30μM)的探针分子的作用下的HeLa细胞存活率图。
图6是10μM探针在HeLa细胞中培养时间的共聚焦荧光成像图,探究探针Mito-PF在细胞中的光稳定性。
图7是10μM探针在50μM依托泊苷(etoposide)诱导的HeLa细胞凋亡共聚焦荧光成像图。
具体实施方式
下面通过具体的实施例对本发明技术方案做进一步说明。
实施例1:荧光探针分子POLAR-QF的合成
将化合物2-((9-乙基-6-碘-9H-咔唑-3-基)亚甲基)丙二腈(1.5g,3.778mmol)、Pd2(PPh3)2Cl2(12.40mg,0.018mmol),CuI(6.98mg,0.036mol)当催化剂置入斯莱克瓶,用泵反复置换反应瓶里面的气体环境,充入氩气,使反应在干燥的氛围下进行反应,用注射器将溶解对氟苯乙炔(0.544g,4.53mmol)的THF(8ml)、Et3N(2ml)溶液注射入反应瓶中,升温到45摄氏度反应5小时,瓶内颜色加深点板观察原料反应完,等待反应液降温到室温后加水搅,用二氯乙烷反复萃取直到水层点板无产品点,加热浓缩得粗产物2.3g,经柱层析(正己烷:乙酸乙酯=7:1)过滤获取暗红色固体目标化合物POLAR-QF(1.2g,产率59.7%)。1HNMR(400MHz,DMSO-d6)δ8.74(d,J=1.8Hz,1H),8.48(s,1H),8.32(m,1H),8.14(m,1H),7.86(d,J=8.8Hz,1H),7.76(m,1H),7.69(m,1H),7.62(m,2H),7.25(t,J=8.9Hz,2H),4.50(q,J=7.1Hz,2H),1.32(t,J=7.1Hz,3H).ESI-MS m/z:{[M+H]+}calcd,389.1328;found,389.1247.
实施例2:荧光探针分子的光谱测试
将本发明探针POLAR-QF溶于DMSO中制得2mM的母液,各取15μL母液于3mL不同极性的溶剂中,获得探针POLAR-QF在不同溶剂中的紫外谱图(图1a)。其在I370max/I370min处的荧光强度随着测试体系极性的增大逐渐减弱(图1b)。并且荧光强度(I370max/I370min)和Δf之间存在线性关系(图1c),这表明POLAR-QF可用于比率检测常见溶液的极性。为了进一步验证探针POLAR-QF对极性的响应特性,在具有不同比例的水和四氢呋喃极性范围内,测量POLAR-QF的吸收和荧光光谱(图2)。当溶剂的极性从含10%水(Δf≈0.2556)依次增大到含80%水(Δf≈0.3103)时,在紫外吸收光谱看到微小的变化与测不同溶剂极性的结果一致(图2a)。当溶液的极性(Δf)从0.3103(80%水)降低到0.2556(10%水)时,POLAR-QF在I370max/I370min处的荧光强度增大了5.4倍(图2b)。上述结果还显示荧光强度I370max/I370min与Δf具有良好的线性相关性(图2c),这表明POLAR-QF对溶剂极性高度敏感。为了排除pH的影响,测试其pH稳定性,在水/四氢呋喃体系中,在相同的含水量(80%的含水量)不同的pH值下(pH=6-9)下,探针POLAR-QF在370nm的荧光强度数值几乎都没有多大的变化,却在相同的pH值(pH=7.0),不同的含水量下(50%、80%),发射峰在370nm处的荧光强度数值变化很大。(图3)此实验结果表明pH值对探针POLAR-QF的影响较小。也从侧面说明在不同的pH值环境中,探针POLAR-QF对极性特异性响应。
实施例3:荧光探针分子的双光子性能测试
POLAR-QF在不同水和四氢呋喃混合溶剂中(四氢呋喃的含量分别为90%,50%和20%),有效双光子吸收截面在780nm出现最大并随着四氢呋喃含量的降低,逐渐从88GM降到32GM(图4a)。观察到了POLAR-QF在不同溶剂中的双光子激发荧光强度与输入功率(300-800mw)成平方的关系(图4b)。证明POLAR-QF有能力用于细胞内极性的双光子共聚焦荧光成像。
实施例4:细胞毒性测试
我们用MTT(5-二甲基噻唑-2-基-2,5-二苯基四唑溴化物)方法进行了细胞毒性实验。POLAR-QF在活HeLa细胞中加入各种浓度(0μM,10.0μM,20.0μM,30.0μM),24小时后测试,结果如图5所示,以上显示POLAR-QF的生物毒性很小,可以进行生物应用。
实施例5:细胞光稳定性测试
研究POLAR-QF在细胞中的光稳定性能,这对于探究细胞凋亡过程中极性变化是非常有必要的,因为凋亡需要一定的时间,这会影响探针的光稳定性能,从而关系到能否实时长时间监测细胞凋亡过程。我们结果表明POLAR-QF的蓝色通道(λem=420-460nm)的荧光图像在加入后10min就有明显的荧光产生,到40min以后荧光强度稳定没有很大的改变(图6)。这些结果表明,POLAR-QF可以很好地在活细胞中长时间成像。
实施例6:细胞凋亡共聚焦荧光成像
依托泊苷(etoposide)能够引起细胞凋亡,是一种公认的细胞凋亡试剂。从已有的文献报道来看,细胞凋亡会引起细胞内微环境的变化,例如:极性。由于细胞凋亡过程中极性会发生变化,因此可以检测极性波动来评估凋亡。因此,我们做了以下实验(图7)。将POLAR-QF(10μM,0.5小时)入细胞内孵育。之后将etoposide(50μM)加入细胞内进行成像。通过成像可以发现在蓝色通道中的荧光强度增强。这与体外测试的荧光数据保持一致。通过以上数据的分析,我们能够清晰的发现,随着加入依托泊苷(etoposide),即凋亡深入,这时细胞内的极性减小(蓝色通道荧光增强)。这说明依托泊苷诱导细胞凋亡会引起细胞内极性减小。这些数据证明我们通过细胞内极性变化来评估细胞凋亡是可行的。这为以后监测细胞凋亡提供了一个好的方法,也为以后荧光探针在生物方面的应用提供了一个好的思路。
Claims (1)
1.一种基于4-氟苯乙炔基的双光子荧光极性探针的应用,其特征在于:
是以非治疗或诊断为目的,在检测活细胞中的极性变化时作为检测试剂使用;
所述双光子荧光极性探针的结构式为:
所述双光子荧光极性探针在I370 max/I370 min处的荧光强度随着体系极性的增大逐渐减弱,并且荧光强度I370 max/I370 min和极性Δƒ之间存在线性关系。
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