CN116217512A - 一种用于检测粘度的半菁染料荧光探针及其制备方法、检测方法以及在溶液及生物体系中的应用 - Google Patents
一种用于检测粘度的半菁染料荧光探针及其制备方法、检测方法以及在溶液及生物体系中的应用 Download PDFInfo
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Abstract
一种用于检测粘度的半菁染料荧光探针及其制备方法、检测方法以及在溶液及生物体系中的应用,半菁染料荧光探针的结构式为:其中,X=S、O、Se、C(CH3)2;R1=H、Ph、CH3、OH、COOH、CH2CH3;R2=CH3、CH2CH3、(CH2)2OH、CH2C6H5、(CH2)3SO3 ‑;R3=H、CH3、Ph、OH、COOH、OCH3;R4=OH、NH2、OCH3、SCH3;R5=H、F、NO2、OCH3、Ph。上述荧光探针通过荧光的强弱变化在检测溶液、细胞中粘度具有很好的应用。本发明的优点是:粘度荧光探针合成方法和纯化处理简单;能够实现对粘度的高灵敏性检测和优良的抗干扰能力。本发明是一种成本低且容易实现,合成方法简单,易于修饰,具有高灵敏的粘度特异性检测荧光探针,其在细胞、组织以及活体小鼠等生物分子检测领域中具有广阔的应用前景。
Description
技术领域
本发明涉及有机小分子荧光探针领域,本发明涉及一种用于检测粘度的半菁染料荧光探针及其制备方法、检测方法以及在溶液及生物体系中的应用。
背景技术
细胞微环境参数的异常变化会导致正常细胞功能障碍,并最终导致多种特定疾病的发生。细胞内的粘度同样作为微环境的一个关键参数参与着各种生物过程,它会影响与生物分子的相互作用、信号和质量传输以及反应性代谢物的扩散。例如,蛋白质的聚集是从低粘度的可溶性蛋白质低聚物的形成开始,逐渐演变成高粘度的不可溶性蛋白质聚集物的过程。异常粘度总是与心血管疾病、阿尔茨海默病和肿瘤等疾病相关。因此,检测细胞中粘度的波动对于解释疾病的不同发病机制至关重要。
尽管目前已有较多粘度响应的荧光探针被报道,但是仍有诸多缺陷,比如合成方法繁琐、潜在干扰多、荧光背景高和单通道荧光发射等。本发明提供了一系列合成方法更加简便,响应效果更加灵敏,易于修饰,实现目标细胞器定位的半菁染料型粘度荧光探针。通过引入修饰位点还可实现对不同细胞器的多通道荧光标记。
发明内容
发明目的:针对现有粘度探针所存在的不足,本发明提供了一种能够在溶液、细胞、组织甚至生物体内检测粘度的半菁染料荧光探针,并提供了荧光探针的合成方法、检测方法及应用。
技术方案:本发明为实现上述目的采用如下技术方案。
本发明的第一个发明目的是:提供一种检测粘度的半菁染料荧光探针,其结构通式为:
其中,X=S、O、Se、C(CH3)2;R1=H、Ph、CH3、OH、COOH、CH2CH3;R2=CH3、CH2CH3、(CH2)2OH、CH2C6H5、(CH2)3SO3 -;R3=H、CH3、Ph、OH、COOH、OCH3;R4=OH、NH2、OCH3、SCH3;R5=H、F、NO2、OCH3、Ph。
优选的,结构通式中取代基:X=S、C(CH3)2;R1=H、CH2CH3;R2=CH3、CH2CH3、(CH2)3SO3 -;R3=H、Ph、OCH3;R4=OH、OCH3;R5=H、OCH3、Ph。
进一步优选的,X=S,R1=H;R2=CH2CH3;R3=H;R4=OH;R5=H。
本发明的第二个发明目的是:提供如式I所示的半菁染料荧光探针的制备方法,以探针I为例,包括如下步骤:
S1:将2-甲基苯并噻唑和碘乙烷按1∶1-5投入于包裹锡纸的厚壁耐压管中,130-175℃回流反应6-11h,反应结束后用适量甲醇溶解,滴入乙醚中沉淀,离心后得到固体即为化合物1;
优选方案:2-甲基苯并噻唑与碘乙烷按摩尔比优选1∶1.5,反应温度优选170℃,回流时间优选10h;
S2:将化合物1和3,4-二甲氧基苯甲醛按照摩尔比1∶0.8-3投入三口烧瓶中,超干甲醇作为溶剂,三乙胺作为催化剂,在N2保护下于70-90℃回流反应6-10h。反应结束后用适量甲醇溶解,滴入乙醚中沉淀得到荧光探针I。
优选方案:化合物1和3,4-二甲氧基苯甲醛的摩尔比优选1∶1;反应温度优选85℃,回流时间优选8h。
本发明的合成路线如下:
本发明的机理如下:
扭曲分子内电荷转移(Twisted Intramolecular Charge Transfer,TICT)机制是指在分子光激发条件下发生的一种电子转移过程。如上述结构所示的半菁染料是一种单双键交替结构,该染料在稀溶液下单键会呈现自由转动,容易形成非平面结构,导致探针激发态的非辐射衰减和荧光发射的减弱。随着环境粘度的增大,限制了染料激发态的转动,阻止了TICT的形成,共轭体系下∏电子云会增多,因此在荧光光谱上会显示出荧光增强的现象。总之,粘度探针响应机制是由于分子的旋转会使得基态和激发态的能量有所变化,从而在光谱上表现出荧光变化。这种响应机制有利于设计出具有“开-关”响应的高灵敏型粘度响应探针,通过粘度的增加来抑制半菁染料TICT机制的发生能显著提高荧光强度以及光稳定性。
本发明的第三个发明目的是:提供如式I所示的半菁染料荧光探针在溶液和细胞中粘度检测的应用。
本发明的第四个发明目的是:提供如式I所示的半菁染料荧光探针在溶液和细胞中粘度的检测方法,包括如下步骤:
S1:准确称取一定质量的荧光探针固体溶解于二甲基亚砜中制得1mM的母液;准确量取一定体积的甘油/水或甘油/甲醇混匀,制得不同比例的甘油/水或甘油/甲醇的母液,采用数字式粘度计测其准确粘度值(η);
S2:准确吸取一定量的荧光探针母液释于甘油/水或甘油/甲醇混合溶液中并混匀,使得样品总体积为800μL,荧光探针浓度为10μM,甘油/水或甘油/甲醇的体积比为梯度变化,范围为0-100%;
S3:测试样品的吸收光谱和荧光光谱,其中,测试吸收光谱前采用不同比例的甘油/水或甘油/甲醇的母液校准基线,采集波长范围为250-800nm;测试荧光光谱时,激发波长采用413nm,发射波长采集范围为450-750nm;
S4:读取荧光峰处的峰值强度(I),以logη为横坐标,log I为纵坐标绘制散点图,在线性范围内拟合直线方程得到标准曲线;
S5:待测样本如上处理后,测试样本溶液中荧光探针的荧光光谱,并读取峰值,将峰值强度代入拟合直线方程中计算得出样本中粘度。
本发明的有益效果:
(1)利用TICT机制设计合成了一系列能够对粘度具有“开-关”响应的半菁染料荧光探针。
(2)本发明利用半菁染料作为粘度荧光探针,其合成方法以及纯化过程简单。
(3)本发明所涉及的荧光探针对粘度具有灵敏的响应性、优异的抗干扰能力,并且能够在活细胞中监测粘度的变化。通过对取代基团的简单修饰能达到不同细胞器和微环境指标的特异性识别,在分子检测领域具有广阔的应用价值。
附图说明
图1为荧光探针I的1H NMR谱图;
图2为荧光探针I在不同粘度的溶液中的荧光光谱图;
图3为荧光探针I检测不同粘度的溶液的线性关系;
图4为荧光探针I在不同干扰物中的荧光强度柱状图;
图5为荧光探针I的细胞共定位成像;
图6为荧光探针I在不同粘度细胞中的成像;
具体实施方式
下面结合具体实施例和附图对本申请做进一步说明,但本申请并不受限于下述实施例。所述方法如无特别说明均为常规方法。
实施例1荧光探针I的合成:
(1)中间产物的合成:
称取2-甲基苯并噻唑1.5341g(10mmol)和碘乙烷2.3409g(15mmol)置于包裹锡纸的厚壁耐压管中,设置油浴锅温度170℃加热反应10h。反应结束冷却至室温后用适量甲醇溶解产物,滴入乙醚中沉淀,洗涤数次,离心后烘干得到固体即为化合物1,产率85.26%。1HNMR(400MHz,DMSO)δ8.47(dd,J=8.2,0.6Hz,0H),8.36(d,J=8.4Hz,1H),7.90(ddd,J=8.5,7.3,1.2Hz,1H),7.87-7.77(m,1H),4.78(q,J=7.3Hz,2H),3.27-3.19(m,1H),1.47(t,J=7.3Hz,2H).
(2)终产物探针的合成,即探针I的合成如下:
称取0.3030g(1mmol)的化合物1和0.1650g(1mmol)的3,4-二甲氧基苯甲醛置于100mL三口烧瓶中,通入N2排除瓶内空气。加入20mL的甲醇作溶剂,15滴三乙胺做催化剂,85℃回流反应8h。反应结束冷却至室温后用适量甲醇溶解产物,滴入乙醚中沉淀,洗涤数次,并通过薄层色谱板粗略监测产物纯度,离心后干燥得到固体即为荧光探针I,产率为15.24%。1H NMR(400MHz,DMSO)δ8.44(d,J=7.9Hz,1H),8.29(d,J=8.4Hz,1H),8.21(d,J=15.7Hz,1H),7.94-7.83(m,1H),7.79(t,J=7.6Hz,1H),7.70(d,J=6.1Hz,1H),7.16(d,J=8.9Hz,1H),4.99(q,J=7.0Hz,2H),3.90(d,J=9.3Hz,3H),1.48(t,J=7.1Hz,2H).
实施例2荧光探针II的合成:
(1)中间产物的合成,同实施例1。
(2)终产物探针的合成,即探针II的合成如下:
称取0.3025g(1mmol)的化合物1与0.1832g(1mmol)丁香醛置于100mL三口烧瓶中,通入N2排除瓶内空气。加入20mL超干甲醇作溶剂,滴入15滴三乙胺做催化剂,90℃回流6h。反应结束冷却至室温后用适量甲醇溶解产物,滴入乙醚中沉淀,洗涤数次,并通过薄层色谱板粗略监测产物纯度,离心后干燥得到固体即为荧光探针II,产率为11.08%。1H NMR(400MHz,DMSO-d6,):δ(ppm)=9.73(s,1H),8.43(d,J=8.0Hz,1H),8.27(d,J=8.4Hz,1H),8.18(d,J=15.5Hz,1H),7.90-7.74(m,3H),7.44(s,2H),4.99(q,J=6.8Hz,2H),3.91(s,6H),1.48(t,J=7.1Hz,3H).
实施例3荧光探针III的合成:
(1)中间产物的合成:
称取1.4920g(10mmol)2-甲基苯并噻唑和0.9770g(8mmol)1,3-丙磺酸内酯混合在厚壁耐压管中,设置油浴锅温度175℃加热反应6h。反应结束冷却至室温后用适量甲醇溶解产物,滴入乙醚中沉淀,洗涤数次,离心后烘干得到固体即为化合物2,产率80.60%。1H NMR(400MHz,DMSO-d6,):δ(ppm)=8.43(dd,J=11.4,4.3Hz,2H),7.89(ddd,J=8.5,7.3,1.1Hz,1H),7.80(dd,J=11.8,4.5Hz,1H),4.98-4.83(m,2H),3.22-3.16(m,3H),2.64(t,J=6.5Hz,2H),2.22-2.05(m,2H).
(2)终产物探针的合成,即探针III的合成如下:
称取0.2742g(10mmol)化合物2和0.1927g(11mmol)丁香醛置于100mL三口烧瓶中,通入N2排除瓶内空气。加入20mL甲醇做溶剂,15滴三乙胺作催化剂。70℃回流10h。反应结束冷却至室温后用适量甲醇溶解产物,滴入乙醚中沉淀,洗涤数次,并通过薄层色谱板粗略监测产物纯度,离心后干燥得到固体即为荧光探针III,产率为93.98%。1H NMR(400MHz,DMSO-d6,):δ(ppm)=9.68(s,1H),8.37(d,J=7.8Hz,1H),8.30(d,J=8.4Hz,1H),8.23-8.10(m,2H),7.82(dd,J=11.6,4.1Hz,1H),7.73(t,J=7.7Hz,1H),7.50(s,2H),5.09(t,J=7.6Hz,2H),3.91(s,6H),2.73-2.65(m,2H),2.24(d,J=6.6Hz,2H).
实施例4荧光探针I对溶液中粘度的响应:
室温下,配制浓度为1mM的实例1中所得探针I的二甲基亚砜溶液,作为测试的染料母液待用;配制不同粘度比例(甘油与水或甲醇的体积比分别为0%、20%、40%、60%、80%、99%)的溶液体系,其中荧光探针浓度设置为10μM。设置激发波长为413nm,激发狭缝和发射狭缝均为10nm,进行荧光光谱测试。如图2所示,随着溶液中粘度的增加,在528nm处的荧光强度逐渐增加;如图3所示,以logη为横坐标,log I528mm(I528nm为荧光峰值强度)为纵坐标,建立在528nm处荧光强度与粘度变化的标准曲线。由图3可知,荧光强度与粘度存在良好的线性关系,线性方程为y=-0.00735+1.00654x;说明荧光探针I能够对粘度变化进行检测。
实施例5荧光探针I的抗干扰能力:
选用了16种可能对探针产生影响的不同阴阳离子和生物分子的潜在干扰物(1.空白对照;2.NaCl;3.KCl;4.KI;5.NaHCO3;6.KNO3;7.Na2HPO4;8.Na2CO3;9.Na2SO4;10.CaCl2;11.CuCl2;12.FeCl2;13.MgSO4;14.Cys;15.GSH;16.Glucose;17.ATP;18.Glycerol),将溶液体系中的干扰物浓度设定为100μM。测试了10μM探针在pH=7.2的Britton-Robinson缓冲溶液中与不同种类干扰物共孵育的荧光光谱。如图4所示,除了在甘油体系当中以外,荧光探针在其他干扰物当中的荧光光谱并没有明显变化,说明其他干扰物的存在对探针的结构不会产生影响,体现了探针优异的抗干扰能力。
实施例6荧光探针I的细胞共定位实验:
为了考察荧光探针I对细胞器的定位效果,我们开展了细胞共定位成像实验。如图5所示,用探针I和商业的线粒体染料(MTDR)对HeLa细胞进行共染色,用激光共聚焦显微镜进行细胞的荧光成像。可以明显的观察到细胞在绿色通道(探针I)和红色通道(MTDR)中分别显示出较强的荧光。将两个通道的图像叠加后发现图像复合程度良好。以上结果表明,探针I能够靶向细胞中的线粒体。
实施例7荧光探针I在不同粘度的细胞中的成像:
考察荧光探针在不同粘度的活细胞中的成像效果。离子载体可以诱导细胞超微结构改变或使其肿胀,使得细胞当中的粘度增加,因此本发明使用制霉菌素(nystain)作为离子载体来改变活细胞内的粘度,并用荧光探针监测细胞内的粘度变化情况。如图6所示,仅用探针I孵育的HeLa细胞只表现出较弱的荧光。使用低剂量(10mM)的制霉菌素刺激20min后再用荧光探针I孵育30min,整个细胞在绿色通道的荧光强度增强。结果表明,制霉菌素的加入使HeLa细胞的粘度增加,导致探针的荧光强度增强。因此可以认为荧光探针I在实时检测活细胞粘度变化方面具有实用性。
实施例8荧光探针I在溶液及细胞生物分子领域中应用的检测方法,包括如下步骤:
S1:准确称取一定质量的荧光探针固体溶解于二甲基亚砜中制得1mM的母液;准确量取一定体积的甘油/水或甘油/甲醇混匀,制得不同比例的甘油/水或甘油/甲醇的母液,采用数字式粘度计测其准确粘度值(η);
S2:准确吸取一定量的荧光探针母液释于甘油/水或甘油/甲醇混合溶液中并混匀,使得样品总体积为800μL,荧光探针浓度为10μM,甘油/水或甘油/甲醇的体积比为梯度变化,范围为0-100%;
S3:测试样品的吸收光谱和荧光光谱,其中,测试吸收光谱前采用不同比例的甘油/水或甘油/甲醇的母液校准基线,采集波长范围为250-800nm;测试荧光光谱时,激发波长采用413nm,发射波长采集范围为450-750nm;
S4:读取荧光峰处的峰值强度(I),以logη为横坐标,log I为纵坐标绘制散点图,在线性范围内拟合直线方程得到标准曲线,其中标准曲线拟合方程为:y=-0.00735+1.00654x。
S5:待测样本如上处理后,测试样本溶液中荧光探针的荧光光谱并读取峰值,将峰值强度代入拟合直线方程中计算得出样本中粘度。
所述生物体系可以为细胞、组织以及活体小鼠。
以上描述是对本发明申请的解释,不是对发明申请的限定,本发明所限定的范围参见权利要求。对于熟悉本领域的人员而言,可容易地实现另外的修改,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节。
Claims (10)
2.如权利要求1所述的半菁染料荧光探针,其特征在于,优选X=S、C(CH3)2;R1=H、CH2CH3;R2=CH3、CH2CH3、(CH2)3SO3 -;R3=H、Ph、OCH3;R4=OH、OCH3;R5=H、OCH3、Ph。
3.如权利要求2所述的半菁染料荧光探针,其特征在于,取代基进一步优选为:X=S;R1=H;R2=CH2CH3;R3=H;R4=OH;R5=H。
5.如权利要求4所述的制备方法,其特征在于,S1步骤中,2-甲基苯并噻唑与碘乙烷按摩尔比优选1∶1.5,反应温度优选170℃,回流时间优选10h;S2步骤中,化合物1和3,4-二甲氧基苯甲醛的摩尔比优选1∶1;反应温度优选85℃,回流时间优选8h。
6.如权利要求1所述的用于检测粘度的半菁染料荧光探针在溶液及生物体系中检测粘度的应用。
7.根据权利要求6所述的用于检测粘度的半菁染料荧光探针在溶液及生物体系中检测粘度的应用,其特征在于,检测的具体方法为荧光检测或活细胞成像检测。
8.根据权利要求7所述的用于检测粘度的半菁染料荧光探针在溶液及生物体系中检测粘度的应用,其特征在于,以荧光探针I为例,应用于溶液中荧光检测,其激发波长为413nm;应用于活细胞成像检测时的激发波长为405nm。
9.一种如权利要求1所述的半菁染料荧光探针在溶液及生物体系检测领域中用于检测粘度的检测方法,其特征在于,包括如下步骤:
S1:准确称取一定质量的荧光探针固体溶解于二甲基亚砜中制得1mM的母液;准确量取一定体积的甘油/水或甘油/甲醇混匀,制得不同比例的甘油/水或甘油/甲醇的母液,采用数字式粘度计测其准确粘度值(η);
S2:准确吸取一定量的荧光探针母液释于甘油/水或甘油/甲醇混合溶液中并混匀,使得样品总体积为800μL,荧光探针浓度为10μM,甘油/水或甘油/甲醇的体积比为梯度变化,范围为0-100%;
S3:测试样品的吸收光谱和荧光光谱,其中,测试吸收光谱前采用不同比例的甘油/水或甘油/甲醇的母液校准基线,采集波长范围为250-800nm;测试荧光光谱时,激发波长采用413nm,发射波长采集范围为450-750nm;
S4:读取荧光峰处的峰值强度(I),以logη为横坐标,logI为纵坐标绘制散点图,在线性范围内拟合直线方程得到标准曲线;
S5:待测样本如上处理后,测试样本溶液中荧光探针的荧光光谱,并读取峰值,将峰值强度代入拟合直线方程中计算得出样本中粘度。
10.根据权利要求9所述的检测方法,其特征在于,所述生物体系可以为细胞、组织以及活体试验动物。
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