CN111892923B - 一种基于二腈乙烯基的双光子荧光粘度探针及其制备方法和用途 - Google Patents
一种基于二腈乙烯基的双光子荧光粘度探针及其制备方法和用途 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一种基于二腈乙烯基的双光子荧光粘度探针及其制备方法和用途,以实现双光子荧光成像定性检测细胞内的粘度,具有选择性专一、灵敏度高、生物毒性低的优点。
背景技术
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,因此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,敏感性很差,基本不能满足评估细胞凋亡的要求。
在细胞内环境成像科学的极速进步的时代下,小分子荧光探针以其低毒性、价格低廉、合成简单以及高的灵敏度等优势被作为生物细胞可视化研究的重要工具,被广泛应用在生命健康、细胞内环境监测和生命医学等各大领域。同时伴随着各种技术的革新,对双光子荧光探针的发展也进入了更深层次的研究,与单光子荧光探针相比,双光子荧光探针有着更深的穿透度以及更强的光漂白抗性,同时又可以避免细胞以及生物组织的自荧光作用。但迄今为止,用于评估细胞凋亡的荧光探针分析方法仍然很少见,因此,需要尽快开发性能优异的荧光探针来监测细胞凋亡。
在细胞的微环境中,粘度、极性、温度和pH等都是极为重要的因素。其中粘度是影响和控制质量与信号传递过程以及生物大分子之间传输过程的一个至关重要的因素,稳定的粘度环境是细胞内物质传输、能量输送以及信号分子传递的重要基础。另外随着科技发展进步以及各种仪器的发明创造,学者们对粘度有着更深入的研究,现已经被证实其在微环境中会影响细胞膜的蛋白质之间的相互作用,进而可能引起一系列的疾病,其中包括糖尿病、肿瘤等疾病。且在细胞凋亡过程中粘度会有很大的改变,因此可以通过粘度来监测凋亡,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二腈乙烯基的双光子荧光粘度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双光子荧光粘度探针的结构,以实现双光子荧光成像检测细胞凋亡过程的粘度变化,具有选择性专一、灵敏度高、实时检测的优点,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。
本发明基于二腈乙烯基的双光子荧光粘度探针,简记为JCN,以咔唑为母体,其结构式如下:
本发明基于二腈乙烯基的双光子荧光粘度探针的制备方法,包括如下步骤:
步骤1:将化合物9-乙基-6-碘-9H-咔唑-3-甲醛(2.0g,5.73mmol)、丙二腈(1.5g,22.72mmol)、DMF10 ml和三乙胺2ml加入反应器中,常温下搅拌,点板监控至反应过程结束;反应后处理:将200ml乙酸乙酯加入反应烧瓶中充分溶解后倒入装有100ml水的烧杯中,搅拌均匀,用分液漏斗萃取,收集有机相,再用100ml乙酸乙酯对水层再萃取一次,两次有机相合并后再用100ml H2O洗一次后旋蒸制样,并通过柱色谱法(石油醚:乙酸乙酯=5:1作为洗脱剂)纯化粗产物,得到中间体1,1.8g,产率为78%。
步骤2:将化合物对醛基苯乙炔(1.2g,9.22mmol)、双三苯基膦二氯化钯(0.1g,0.14mmol)、碘化亚铜(0.5g,2.63mmol)和中间体1(1.5g,3.78mmol)加入到100ml施莱克瓶中,氩气保护,再分别加入三乙胺5ml和四氢呋喃20ml,80℃下搅拌反应24h,将混合物冷却至室温后,通过旋转蒸发仪除去溶剂,将得到的粗产物萃取制样,然后通过柱色谱法(石油醚:乙酸乙酯=4:1作为洗脱剂)纯化粗产物,得到目的产物JCN,0.8g,产率为53.3%。
本发明基于二腈乙烯基的双光子荧光粘度探针JCN的合成过程如下:
本发明基于二腈乙烯基的双光子荧光粘度探针的用途,是以非治疗或诊断为目的,在定性检测活细胞中的粘度变化时作为检测试剂使用。检测方法如下:
将本发明双光子荧光探针溶于DMSO中制得2mM的母液,各取15μL母液于3mL不同的溶剂中。首先研究了探针分子JCN在不同溶剂中的光学性能,选择了1,4-二氧六环,乙酸乙酯(EA)、四氢呋喃(THF)、苯甲腈、正己醇、二甲基亚砜(DMSO)、乙二醇、乙腈、甲醇、水、甘油等不同溶剂作为考察对象。观察到JCN的主要紫外吸收峰在330nm和407nm左右,选择330nm作为激发波长,其在375nm和390nm左右出现了两个主要荧光发射肩峰,虽然JCN的紫外峰和荧光峰因溶剂的极性不同存在一定的位移,但其在各溶剂中的荧光强度均不高,只有在甘油中才显示出明显的荧光强度,说明探针对粘度响应。进一步测试了JCN在不同水/甘油体系(从1.03cp到1410.00cp)中的荧光响应详细情况,结果显示其在390nm处的荧光强度随着测试体系粘度的增大逐渐增强,增大了近20倍,利用福特斯-霍夫曼方程对荧光强度(LogI390nm)和粘度(Logη)进行了线性拟合,R2=0.98,具有良好的线性关系,这表明JCN可用于检测溶液的粘度大小。接下来进一步测定了探针JCN在不同粘度体系下的荧光寿命。结果显示在不同水和甘油混合溶剂体系中,其荧光寿命随着粘度的增加而不断增加,同时对荧光寿命数据与粘度数值进行了线性拟合,R2=0.99,呈现出很好的线性关系。在不同水和甘油混合溶剂体系中,探针JCN的有效双光子吸收截面在820nm处,甘油含量为99%数值最大,为87GM,表明该探针具有较好的双光子吸收性能。在进行细胞成像前,测试了探针JCN的细胞毒性,因为较低的细胞毒性是小分子荧光探针进行活细胞成像必要的前提条件。将10μM探针与HeLa细胞共孵育,每隔10分钟进行成像,结果显示JCN能很好的进入HeLa细胞并发出蓝色荧光,且在30min内荧光强度达到稳定状态。此外,利用探针JCN测试了依托泊苷(etoposide)诱导HeLa细胞凋亡过程,发现在细胞凋亡过程中,细胞内的粘度会增大。
本发明基于二腈乙烯基的双光子荧光粘度探针分子在与其他干扰因素共存的体系中,表现出对粘度的专一性响应。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对HeLa细胞渗透性好,适用于细胞内粘度变化的双光子荧光成像和原位检测,可以原位监测细胞凋亡过程中的粘度变化趋势。
附图说明
图1是10μM探针在不同有机溶剂中的(a)紫外吸收光谱图;(b)荧光发射光谱图。
图2是10μM探针在不同梯度水/甘油混合体系中的(a)荧光发射光谱图;(b)荧光强度(Log I390nm)和粘度(Logη)之间的线性关系图。
图3是10μM探针在不同梯度水/甘油混合体系中的(a)荧光寿命图;(b)荧光寿命(Logτ)和粘度(Logη)之间的线性关系图。
图4是10μM探针在不同梯度水/甘油混合体系中的有效双光子吸收截面图。
图5是10μM探针在不同分析物(Cl-、Br-、Na+、K+、CO3 2-、S2-、HSO3 -、S2O3 2-、SO4 2-、HPO4 2-、H2PO4 -、NO2 -、NO3 -、ClO-、H2O2、Cys、Hcy、GSH、甘油)存在时的荧光发射光谱图。
图6是10μM探针在含30%甘油的水/甘油混合体系中不同pH下的相对荧光强度图。
图7是在不同浓度(0μM、5μM、10μM、15μM、20μM)的探针分子的作用下的HeLa细胞存活率图。
图8是10μM探针在不同时间(0min,10min,20min,30min,40min)培育HeLa细胞的共聚焦荧光成像图。
图9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JCN的合成
将化合物对醛基苯乙炔(1.2g,9.22mmol),双三苯基膦二氯化钯(0.1g,0.14mmol),碘化亚铜(0.5g,2.63mmol)和中间体1(1.5g,3.78mmol)加入到100ml施莱克瓶中,氩气保护,再分别加入三乙胺5ml,四氢呋喃20ml,80℃下搅拌反应24h,将混合物冷却至室温后,通过旋转蒸发仪除去溶剂,将得到的粗产物萃取制样,然后通过柱色谱法(石油醚:乙酸乙酯=4:1作为洗脱剂)纯化粗产物,得到目的产物JCN,0.8g,产率为53.3%。
1H NMR(400MHz,DMSO-d6):δ10.01(s,1H),8.78(d,J=1.8Hz,1H),8.52(s,1H),8.42(m,1H),8.17(m,1H),7.93(d,J=8.5Hz,3H),7.79(m,3H),7.45(d,J=8.5Hz,1H),4.52(d,J=7.0Hz,2H),1.33(m,3H).ESI-MS m/z:{C27H18N3O+,[M+H]+}calcd,400.1439;found,400.1440.
实施例2:荧光探针分子的光谱测试
将本发明双光子荧光探针溶于DMSO中制得2mM的母液,各取15μL母液于3mL不同的溶剂中。首先研究了探针分子JCN在不同溶剂中的光学性能,选择了1,4-二氧六环,乙酸乙酯(EA)、四氢呋喃(THF)、苯甲腈、正己醇、二甲基亚砜(DMSO)、乙二醇、乙腈、甲醇、水、甘油等不同溶剂作为考察对象。观察到JCN的主要紫外吸收峰在330nm和407nm左右(图1a),选择330nm作为激发波长,其在375nm和390nm左右出现了两个主要荧光发射肩峰(图1b),虽然JCN的紫外峰和荧光峰因溶剂的极性不同存在一定的位移,但其在各溶剂中的荧光强度均不高,只有在甘油中才显示出明显的荧光强度,说明探针对粘度响应。进一步测试了JCN在不同水/甘油体系(从1.03cp到1410.00cp)中的荧光响应详细情况(图2a),结果显示其在390nm处的荧光强度随着测试体系黏度的增大逐渐增强,增大了近20倍,利用福特斯-霍夫曼方程对荧光强度(Log I390nm)和粘度(Logη)进行了线性拟合,R2=0.98,具有良好的线性关系(图2b),这表明JCN可用于检测溶液的粘度大小。为了证明JCN能否专一性检测粘度,我们在相同条件下测试了各种干扰分析物对其荧光光谱的影响,包括Cl-、Br-、Na+、K+、CO3 2-、S2-、HSO3 -、S2O3 2-、SO4 2-、HPO4 2-、H2PO4 -、NO2 -、NO3 -、ClO-、H2O2、Cys、Hcy、GSH等。从图5可以看出,只有在甘油中才有明显的荧光发射强度,说明JCN能专一的响应粘度。为了排除pH的影响,测试其pH稳定性(图6),在含30%甘油的水/甘油混合体系中,其pH值在3-10范围内,JCN荧光强度数值变化不大,此实验结果表明pH值对探针JCN的影响较小,其对pH的变化不敏感。以上结果证明探针JCN可以特异性检测粘度且不受外界环境的干扰。
实施例3:荧光探针分子的荧光寿命测试
进一步测试了JCN在不同水和甘油混合溶剂体系中的荧光寿命,荧光寿命随着粘度的增加而不断增加(图3a)。同时对荧光寿命数据与粘度数值进行了线性拟合,R2=0.99,呈现出很好的线性关系(图3b)。
实施例4:荧光探针分子的双光子性能测试
JCN在不同水和甘油混合溶剂体系中(甘油的含量分别为60%,80%和99%),有效双光子吸收截面在820nm出现最大并随着甘油含量的增大,逐渐从14GM增加到87GM(图4)。证明JCN有能力用于细胞内粘度的双光子共聚焦荧光成像。
实施例5: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。JCN在活HeLa细胞中加入各种浓度(0μM、5μM、10μM、15μM、20μM)培养,24小时后测试,结果如图7所示,以上显示JCN的生物毒性很小,可以进行生物应用。
实施例6:荧光探针分子在HeLa细胞中的共聚焦成像
将10μM的JCN加入到HeLa细胞内培养,时间每隔10分钟(0min、10min、20min、30min、40min)进行共聚焦成像(图8),结果显示JCN能够随着时间的不断推移逐渐进入HeLa细胞中而且能够很好的发出蓝色的荧光,并随着时间的推移荧光也逐渐增强,且在30min内达到稳定状态,表明了探针分子JCN能够很好的进入细胞并表现出良好的光学性质。
实施例7:细胞凋亡的共聚焦荧光成像
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:粘度。由于细胞凋亡过程中细胞内粘度会发生变化,因此可以通过检测粘度波动来监测凋亡。因此,我们做了以下实验(图9)。将JCN(10μM,0.5小时)入细胞内孵育,之后将etoposide(50μM)加入细胞内培育0.5小时,分别进行共聚焦荧光成像。通过成像可以发现在蓝色通道(λem=410-460nm)中的荧光强度明显增强。通过以上数据的分析,我们能够清晰的发现,加入依托泊苷(etoposide)培育后,引起了细胞的凋亡,这时细胞内粘度增大(蓝色通道荧光增强)。这说明细胞凋亡会引起细胞内粘度增大。这些数据证明我们通过细胞内粘度的变化来监测细胞凋亡是可行的。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 (6)
2.一种权利要求1所述的基于二腈乙烯基的双光子荧光粘度探针的制备方法,其特征在于包括如下步骤:
步骤1:将化合物9-乙基-6-碘-9H-咔唑-3-甲醛2.0g、丙二腈1.5g、三乙胺2ml和DMF加入反应器中,常温下搅拌,点板监控至反应过程结束;反应结束后分离纯化,得到中间体1;
步骤2:将化合物对醛基苯乙炔1.2g、双三苯基膦二氯化钯0.1g、碘化亚铜0.5g和中间体1 1.5g加入施莱克瓶中,氩气保护,再分别加入三乙胺5ml和四氢呋喃,80℃下搅拌反应24h,将混合物冷却至室温后,通过旋转蒸发仪除去溶剂,将得到的粗产物萃取制样,然后通过柱色谱法纯化粗产物,得到目标产物JCN。
3.根据权利要求2所述的制备方法,其特征在于:
步骤1中,所述分离纯化是将乙酸乙酯加入反应器中充分溶解,然后倒入装有水的烧杯中,搅拌均匀,用分液漏斗萃取,收集有机相,再用乙酸乙酯萃取一次,合并有机相,水洗后旋蒸制样,并通过柱色谱法纯化粗产物,得到中间体1。
4.根据权利要求3所述的制备方法,其特征在于:
步骤1中,通过柱色谱法纯化粗产物时使用的洗脱液为石油醚:乙酸乙酯=5:1,v/v。
5.根据权利要求2所述的制备方法,其特征在于:
步骤2中,通过柱色谱法纯化粗产物时使用的洗脱液为石油醚:乙酸乙酯=4:1,v/v。
6.一种权利要求1所述的基于二腈乙烯基的双光子荧光粘度探针的用途,其特征在于:
是以非治疗或诊断为目的,在定性检测活细胞中的粘度变化时作为检测试剂使用。
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