CN111849460A - 一种基于荧光共振能量转移检测生物硫醇的近红外比率荧光探针及其制备和应用 - Google Patents
一种基于荧光共振能量转移检测生物硫醇的近红外比率荧光探针及其制备和应用 Download PDFInfo
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Abstract
本发明公开了一种基于荧光共振能量转移检测生物硫醇的近红外比率荧光探针，其分子结构包括氟硼二吡咯染料(能量供体)、近红外氧杂蒽染料(能量受体)和二硫基团(链接基团)；结构式如式(I)所示。本发明的探针在高选择性地识别生物硫醇，以GSH为例，探针在GSH的作用下，荧光发射强度在512nm处逐渐增强，在656nm处逐渐减弱，二者强度比值与GSH浓度在一定范围内呈线性关系，并能够在培养的细胞内实现比率成像。
Description
技术领域
本发明涉及一种比率荧光传感及其应用，具体涉及一种基于荧光共振能量转移的用于检测生物硫醇的比率荧光探针的制备与应用，属于有机荧光传感技术领域。
背景技术
生物硫醇，如半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)等，在生理和病理事件中起着重要作用，特别是维持氧化还原平衡和代谢。通常情况下，生物硫醇的异常与多种疾病有关，如GSH浓度的异常与白细胞减少、肝脏损伤、生长缓慢和癌症等。因此，开发高效的方法监测和原位定量细胞中的生物硫醇是非常必要的，这对研究相关疾病具有十分重要的意义。
荧光探针因其高灵敏度、高选择性、高时空分辨率，是生物系统中最有力的检测方法之一(Chem.Soc.Rev.2015,44,6143–6160)。到目前为止,已有许多用于检测生物硫醇的荧光探针的报道。然而，许多基于单一荧光强度变化的荧光探针对硫醇的检测存在系统误差，如受到探针浓度、辐射光波动和细胞环境影响等。比率荧光探针具有通过测量两个不同波长的发射强度变化进行自我校准的能力，以克服上述系统误差。常用的构建探针的方法有基于分子间电荷转移(ICT)、跨键能量转移(TBET)和荧光共振能量转移(FRET)策略。而基于FRET的比率荧光探针一般是由能量供体、连接体和能量受体三部分构成，而且能量供体发射光谱与能量受体吸收光谱要有光谱重叠，才能产生有效的能量转移。探针在与生物硫醇作用后，FRET过程逐渐被阻断，探针内的能量供体和受体的荧光发射逐渐发生变化。因此，产生的荧光强度比值与生物硫醇呈现线性函数关系。经检索，现有比率型荧光探针的荧光发射限制于可见光区域，存在诸如问题，如生物组织穿透性差、细胞和组织荧光背景干扰以及强的光损伤，以及两个发射峰之间的距离较小等等。为了制备出优异性能的比率型荧光探针，需要开发构筑在近红外区域新型FRET类型的荧光探针。
发明内容
本发明的主要目的是克服已有技术的不足，提供一种检测生物硫醇的近红外比率荧光探针。
本发明提供了一种基于荧光共振能量转移的检测生物硫醇的近红外比率荧光探针，所述比率型荧光探针分子结构包括氟硼二吡咯荧光团(能量供体)、近红外氧杂蒽染料(能量受体)和二硫基团(链接基团)；该比率型荧光探针化学结构式如式(I)所示：
本发明还提供了一种所述的近红外比率荧光探针的制备方法，包括以下步骤：
根据已报道方法制备氧杂蒽染料1与肌氨酸叔丁酯盐酸盐在卡特缩合剂(BOP)，三乙胺以及溶剂二氯甲烷中进行酰胺偶联反应，处理得到化合物2；将氟硼二吡咯3与盐酸胱胺在1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI)，N-羟基丁二酰亚胺(NHS)，三乙胺以及溶剂二氯甲烷等进行酰胺偶联反应，处理后得到化合物4；接着将化合物2在二氯甲烷中酸化后，再与化合物4在EDCI和NHS试剂进行酰胺偶联反应，反应结束后经过后处理得到比率荧光探针。
制备化学反应式如下
本发明还提供了一种所述的近红外比率荧光探针在生物硫醇检测和成像方面的应用。
本发明的近红外比率荧光探针在没有生物硫醇存在下，488nm激发探针的氟硼二吡咯能量供体，发生能量转移，探针发射出656nm的能量受体近红外荧光；在生物硫醇存在(GSH)下，探针内的二硫键被切断，能供体和受体单元分离，FRET能量转移过程被阻断，此时488nm激发探针只产生氟硼二吡咯能量供体的512nm荧光，从而实现了比率荧光检测生物硫醇衍生物，如图1所示。
本发明所述的近红外荧光探针，生物硫醇以GSH为例，在488nm激发下，随GSH浓度的增加，在512nm处荧光发射强度逐渐增强，656nm处荧光发射强度逐渐减弱；二者波长的比值相对GSH浓度在一定范围内呈线性关系。可确定本发明所述的荧光可以定量检测GSH浓度，如图2和3所示。
将本发明所述的近红外荧光探针加入到Hela活细胞中为实验组，以及在加探针之前加入N-乙基马来酰亚胺(NEM，巯基清除试剂)和外加GSH的对照组细胞染色，荧光显微镜成像变化明显；实验组中细胞绿色通道荧光较强，FRET的红色通道荧光很弱；NEM处理的对照组细胞绿色通道荧光减弱，红色通道荧光急剧增强。在另外对照组中，加入NEM处理后在加入GSH和探针后，两通道荧光强度与实验组相比没有明显的变化；如图6。
本发明的有益效果为：该比率荧光探针可高选择性检测生物硫醇，随着GSH浓度的增加，其荧光发射强度在512nm处逐渐增强，在656nm处逐渐减弱；二者荧光比值与GSH浓度在一定范围内呈线性关系。该比率荧光探针不仅可以定量检测溶液体系中的GSH，而且能够用于活细胞内的GSH比率成像；
附图说明
图1为本发明比率荧光探针与GSH作用的结构变化。
图2为本发明比率荧光探针与不同浓度GSH作用的荧光发射变化图。
图3为本发明比率荧光探针对不同浓度GSH作用后的荧光比值变化(F512nm/F656nm)谱图。
图4为本发明比率荧光探针对不同物种作用后的荧光比值变化(F512nm/F656nm)谱图。
图5为本发明比率荧光探针对GSH、Hcy及Cys的荧光比值(F512nm/F656nm)随时间变化。
图6为本发明比率荧光探针在Hela细胞的荧光成像图。
具体实施方式
实施例1
称取化合物1(0.59g,1mmol)溶于无水二氯甲烷(20ml)中并加入BOP试剂(0.66g，1.5mmol)，Et3N(2ml)和肌氨酸叔丁酯盐酸盐(0.27g,1.5mmol)。反应在室温下搅拌过夜，反应中加入水(20ml)，用二氯甲烷(20ml)萃取反应混合物，收集有机层，用Na2SO4干燥，过滤后真空蒸发，将得到的物质通过硅胶柱色谱纯化，得到化合物2，质量为0.31g，产率为43％。1HNMR(CDCl3,400MHz)δ:8.14(d,J＝9.2Hz,1H),7.65-7.57(m,3H),7.33-7.30(m,1H),7.09(d,J＝9.6Hz,1H),6.91-6.86(m,2H),6.79-7.76(m,1H),6.49(s,1H),3.99-3.95(m,1H),3.79-3.75(m,1H),3.58-3.52(m,8H),2.97-2.94(m,1H),2.88(m,1H),2.86(s,3H),2.59-3.54(m,1H),1.21-1.27(m,21H)；13C NMR(CDCl3,100MHz)δ:167.3,167.2,164.9,156.6,154.6,153.7,153.3,146.5,134.9,131.5,130.2,129.8,129.6,129.4,128.9,127.5,120.4,114.9,114.3,113.6,112.0,110.1,96.0,81.8,49.3,45.4,45.3,38.5,29.7,27.9,27.8,24.5,12.7,12.4.ESI-MS:C39H48N3O4 +[M-ClO4]+计算值:622.3639,发现值:622.3132。
实施例2
将化合物3(0.17g,0.5mmol)溶于无水二氯甲烷(20ml)中并加入EDCI(0.15g，0.78mmol)，NHS,(0.07g,0.6mmol)和Et3N(0.2ml),混合物搅拌30分钟加入胱胺二盐酸盐(0.18g,0.78mmol)，然后连续搅拌12h之后。加入水(10ml)洗混合物，有机层用硫酸钠干燥，过滤后旋干溶剂再通过硅胶柱色谱分离纯化得到化合物4(0.1g，产率为40％)。1H NMR(CDCl3,400MHz)δ:8.04(d,J＝8.0Hz,2H),7.31(d,J＝7.6Hz,2H),5.94(s,2H),3.71-3.70(m,2H),3.63(s,1H),3.36-3.30(m,2H),3.06-3.04(m,2H),2.92-2.89(m,2H),2.51(s,6H),1.30(m,6H).13C NMR(CDCl3，100MHz)δ:14.62,26.35,26.43,29.68,39.35,46.27,46.31,76.69,77.01,77.33,105.11,121.47,128.18,128.36,130.96,134.14,138.36,140.29,142.89,155.86,166.93。
实施例3
将化合物2(0.14g，0.2mmol)溶于无水二氯甲烷(10ml)中，加入三氟乙酸(1ml)。反应在室温下搅拌3h后旋干溶剂直接进行下一步反应，加入EDCI(57mg，0.3mmol)，NHS(35mg，0.3mmol)，Et3N(0.2ml)和化合物4(0.1g，0.2mmol)。反应在室温下搅拌过夜，加入水(20ml)混合，混合物用二氯甲烷萃取，得到后有机相用Na2SO4干燥，旋干后再通过硅胶柱色谱分离纯化得到探针(48mg，产率为21％)。1H NMR(CDCl3,400MHz)δ:8.07(d,J＝8.4Hz,2H),7.99(d,J＝8.8Hz,1H),7.77(d,J＝8.8Hz,1H),7.54-7.53(m,3H),7.28(d,J＝8.0Hz,2H),7.13-7.11(m,2H),6.71-6.70(m,2H),6.54(s,1H),5.93(s,2H),4.25-4.21(m,1H),3.98-3.94(m,1H),3.80-3.77(m,2H),3.63-3.59(m,4H),3.54-3.50(m,8H),3.06-3.02(m,2H),2.97-2.96(m,2H),3.18(s,3H),2.79-3.76(m,1H),2.74-3.72(m,1H),2.53(s,6H),1.31-1.25(m,18H)；13C NMR(CDCl3,100MHz)δ:169.3,168.6,166.8,156.5,155.4,153.7,146.3,143.3,137.7,135.0,134.9,131.1,129.9,129.6,129.4,128.3,128.1,127.9,121.2,120.3,116.2,115.2,111.7,110.5,95.4,53.9,45.6,45.3,39.9,39.1,38.7,38.2,33.8,29.7,27.9,24.4,22.7,14.6,12.7,12.4.ESI-MS:C59H67N7O4S2BF2 +[M-ClO4]+计算值:1050.4752,发现值:1050.4359.
实施例4
探针对不同浓度的GHS荧光发射谱图：探针加入到EtOH/PBS(pH 7.4,20mM,v/v 1:2)测试溶液中配置成浓度为5μmol/L的溶液，而后滴加不同浓度的GSH水溶液，待平衡后，测定荧光发射光谱，结果见图2，其荧光强度比值F512nm/F656nm变化如图3.
从图2和3所知，加入GSH后，探针在512nm处荧光发射显著增强，而在656nm荧光减弱，因此探针可以作为GSH的比率型荧光探针，并实现对GSH的定量检测。
实施例5
探针对不同物种作用后的荧光强度比值F512nm/F656nm变化：探针加入到EtOH/PBS(pH 7.4,20mM,v/v 1:2)测试溶液中配置成浓度为5μmol/L的溶液，然后加入250μM的不同物种，待平衡后，测定荧光发射光谱，整理结果见图4。
由图4可知，探针的荧光强度比值仅对生物硫醇有很大的变化，其他物种影响很弱。
实施例6
在室温下，探针加入到EtOH/PBS(pH 7.4,20mM,v/v 1:2)测试溶液中配置成浓度为5μmol/L的溶液，然后加入250μM的GSH、Hcy和Cys，记录不同时间的荧光光谱，如图5所示。
结果显示，随着时间的增加，荧光强度比值F512nm/F656nm对GSH、Hcy和Cys均能增强；说明探针可以作为生物硫醇的比率型荧光探针。
实施例7
细胞内荧光成像测试：将Hela细胞转移到小的玻璃瓶中孵化24h后，实验组用该探针(10μM)溶液孵化30分钟，然后PBS洗涤三次进行共聚焦细胞成像检测。对照组一在细胞内先用NEM(1mM)溶液孵化30分钟后，再加探针孵化30分h,接着用PBS洗涤三次进行共聚焦细胞成像检测。对照组二，先用NEM(1mM)溶液孵化30分钟后，PBS洗涤三次后，再加GSH(1mM)和探针(10μM)孵化30分,接着用PBS洗涤三次进行共聚焦细胞成像检测。所用激发波长为488nm，绿色通道收集波长为500-550nm，红色通道收集波长为630-680nm，如见图6。
Claims (8)
1.一种基于荧光共振能量转移检测生物硫醇的近红外比率荧光探针，其特征在于，荧光探针的结构式如下：
2.一种如权利要求1所述的近红外比率荧光探针的制备方法，其特征在于，包括以下步骤：
(1)将氧杂蒽染料1与肌氨酸叔丁酯盐酸盐和卡特缩合剂进行酰胺偶联反应，反应结束后经过后处理得到化合物2；
(2)将氟硼二吡咯3与盐酸胱胺在1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和N-羟基丁二酰亚胺进行酰胺偶联反应，反应结束后经过后处理得到化合物4：
(3)步骤(1)得的化合物2在二氯甲烷中酸化后，再与步骤(2)得到的化合物4在EDCI和NHS试剂进行酰胺偶联反应，反应结束后经过后处理得到近红外比率荧光探针。
3.一种如权利要求1所述的近红外比率荧光探针在生物硫醇检测和成像方面的应用。
4.根据权利要求3所述的近红外比率荧光探针在生物硫醇检测和成像方面的应用，其特征在于，所述的生物硫醇为半胱氨酸、同型半胱氨酸或谷胱甘肽。
5.根据权利要求3或4所述的近红外比率荧光探针在生物硫醇检测和成像方面的应用，其特征在于，所述的荧光探针用于生物硫醇的定性检测，具体方法如下：
将所述的荧光探针配置成测试溶液，然后加入待测样品，在488nm激发下，测定512nm和656nm处荧光发射强度变化，若512nm处荧光强度变强，656nm处荧光强度变弱，则确定待测样品中含有生物硫醇。
6.根据权利要求3或4所述的近红外比率荧光探针在生物硫醇检测和成像方面的应用，其特征在于，所述的荧光探针用于生物硫醇的定量检测，具体方法如下：
(1)将所述的荧光探针配置成测试溶液，然后加入待测样品，在488nm激发下，测定512nm和656nm处荧光发射强度；
(2)计算得到荧光强度比值F512nm/F656nm，根据荧光强度比值F512nm/F656nm得到待测样品中的生物硫醇的含量。
7.根据权利要求6所述的近红外比率荧光探针在生物硫醇检测和成像方面的应用，其特征在于，所述测试溶液的配制方法如下：
将所述荧光探针加入到EtOH/PBS溶液中，配置成浓度为5μmol/L的测试溶液。
8.根据权利要求3或4所述的近红外比率荧光探针在生物硫醇检测和成像方面的应用，其特征在于，所述的荧光探针用于细胞内荧光成像测试，具体方法如下：将待测细胞用含有荧光探针的溶液进行孵化，然后PBS洗涤三次，进行共聚焦细胞成像检测；
共聚焦细胞成像检测时，激发波长为488nm，绿色通道收集波长为500-550nm，红色通道收集波长为630-680nm。
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