CN116606220A - 一种单组分有机余辉化合物及其生物检测的应用 - Google Patents
一种单组分有机余辉化合物及其生物检测的应用 Download PDFInfo
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- C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
- C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- C07C2602/04—One of the condensed rings being a six-membered aromatic ring
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Abstract
本发明公开了一种单组分有机余辉化合物及其生物检测的应用,是波长可调控的仿生型有机余辉化合物及其水相中生物检测的应用,属于发光成像及诊断检测领域。本发明提供的单组分有机余辉化合物具有在生理环境中高发光效率的优点,其发射峰在580‑720nm之间,具有强的组织穿透能力。该余辉化合物具有式I所示的分子结构通式,含有富电子基取代的双键基团是其必要的分子结构特征。该种单组分余辉化合物在水相中能观察到不同波长的余辉发光,可用于发光成像或发光诊断标记物。
Description
技术领域
本发明属于发光成像及诊断检测领域,具体涉及一种不同发光波长小分子有机余辉化合物及其在余辉成像中的使用方法。
背景技术
余辉发光是指发光材料在激发光移除后持续发光的一种现象,因为避免了实时光激发带来的光散射、背景荧光的干扰,受到了广大科研学者的青睐。目前,无机余辉发光量子点已在防伪、油墨、安全指示、装饰等领域广泛应用。但受限于无机量子点,尤其是其中重金属离子潜在的生物安全性隐患,在生物成像以及发光检测更倾向于采用高生物相容性的有机余辉材料。
现阶段有机余辉材料的报道主要分为两大类:其一是氧气淬灭型余辉发光材料,主要由磷光或热致延迟荧光材料通过物理包裹,如主客体配位或微晶包裹形成的余辉发光纳米颗粒,这类材料在充分溶解状态下与溶解氧接触会导致自身余辉发光性质的丧失(Chem.Eur.J.,2022,28,e2022008);其二是耗氧型余辉发光材料,主要由光敏剂、储能单元、发光单元的多元掺杂形成余辉发光纳米颗粒或余辉发光微球,余辉发光的产生依赖光敏剂在光照下产生的单线态氧是这类材料的特征(J.Am.Chem.Soc.,2022,144,3429)。然而,这两类传统余辉发光材料都需要制备纳米颗粒,并非水溶液中均相发光,批次间发光效果很难实现一致。
针对传统有机余辉发光材料上述问题,2020年报道了水相中单一小分子余辉发光化合物(Angew.Chem.Int.Ed.2020,59,9059),实现了分散态下单组分小分子化合物不依赖与单线态氧的余辉发光,中国发明专利(CN 110804009 A)。在该发明中提出新一代小分子有机余辉化合物,采用光照模拟了萤光素酶、Mg2+,ATP的催化小分子化合物与氧气反应高效生成1,2-二氧杂环丁烷结构,随后该结构快速分解,从而在水溶液中释放出高强度余辉发光。
相较于多组分有机余辉纳米颗粒,本发明提供的余辉发光化合物能够避免纳米颗粒的制备过程,直接在溶液态使用。同时本发明提供的余辉发光化合物发光过程不依赖单线态氧氧化产生余辉发光,其自身能在pH大于5.0的环境中在光照下与氧气反应,因此可以免除光敏剂的使用,实现单组分分散态余辉发光。此外,本发明提供的余辉发光化合物具有优异的拓展性,通过吸电子基团A的替换,构建了多种发光波长与发光寿命的小分子有机余辉发光化合物,并且成功用于水溶液、细胞、活体余辉发光成像。
发明内容
本发明的目的是提供一种小分子有机余辉化合物,其分子结构特征包含推拉电子体系与富电子基取代的双键基团。余辉化合物在pH值5.0以上微环境中,光照下迅速发生电子转移,形成双自由基,进一步与氧气加成生成1,2-二氧杂环丁烷高能不稳定结构,随后该结构快速分解产生余辉发光。通过调控吸电子基团A,能够获得不同发光波长的有机余辉化合物。
本发明设计有机余辉化合物的分子结构特征包含推拉电子体系与富电子基取代的双键基团,一种单组分有机余辉化合物,其结构通式如下:
式I中吸电子基团A为至少包含一个氰基的生色团,X为卤素原子;其中,甲氧基(富电子基)取代双键基团是必要的结构,且该结构处于吸电子A的邻位或对位,共同组成推拉电子体系。
进一步的,所述吸电子基团A为如下含有氰基结构中的任意一种:
R1为氰基、乙酰基、羧基甲酯、羧基乙酯、羧基中的任意一种;R2为甲基、异丙基、叔丁基、环丙烷基中的任意一种;
X为卤素原子F、Cl、Br的一种。
在优化选取中,吸电子基团A选自如下结构的任意一种:
其中,R1为氰基、乙酰基、羧基甲酯、羧基乙酯、羧基中的任意一种。
本发明还提出了上述单组分有机余辉化合物的生物检测应用,包括应用于与各类商用发光增强剂、表面活性剂、环糊精、脱脂奶粉、血清蛋白等复配,能显著提升化合物的发光强度。
余辉发光化合物不需要添加光敏剂,也不需要与光敏剂、嵌段聚合物混合形成纳米颗粒;化合物单独使用或与上述的增强剂复配使用皆可。
该类余辉化合物能够在水相溶液、活细胞、组织及动物活体中使用,均具有明显的余辉发光特性。
本发明还提出了单组分有机余辉化合物的应用方法,其使用方法如下:
操作条件:(1)将余辉底物置于pH大于5.0的溶液或凝胶中;(2)光源的光照时间为1-10秒,光源为商品化的LED光源(100mW·cm-2)。在使用中必当且仅当光照操作在化合物处于pH大于5.0的环境中才能有效的产生余辉发光。该发光具体过程描述为,余辉化合物在pH值5.0以上微环境中,通过光照生成1,2-二氧杂环丁烷高能不稳定结构,随后该结构快速分解产生余辉发光。
附图说明
图1.Rubine-1(详见实施例2)在Tris-HCl溶液(含10% HSA)中的紫外吸收(1×10-5mol·L-1)、荧光(1×10-5mol·L-1)及余辉发光图谱(1×10-4mol·L-1);
图2.Rubine-2(详见实施例2)在Tris-HCl溶液(含10% HSA)中的紫外吸收(1×10-5mol·L-1)、荧光(1×10-5mol·L-1)及余辉发光图谱(1×10-4mol·L-1);
图3.Rubine-3(详见实施例2)在Tris-HCl溶液(含10% HSA)中的紫外吸收(1×10-5mol·L-1)、荧光(1×10-5mol·L-1)及余辉发光图谱(1×10-4mol·L-1);
图4.Rubine-4(详见实施例2)在Tris-HCl溶液(含10% HSA)中的紫外吸收(1×10-5mol·L-1)、荧光(1×10-5mol·L-1)及余辉发光图谱(1×10-4mol·L-1);
图5.Rubine-5(详见实施例2)在Tris-HCl溶液(含10% HSA)中的紫外吸收(1×10-5mol·L-1)及荧光图谱(1×10-5mol·L-1);
图6.Rubine-1、Rubine-2、Rubine-3、Rubine-4、Rubine-5光物理参数汇总;其中,λabs为化合物的最大吸收峰、εabs为化合物最大吸收峰处摩尔消光系数、λem为化合物的最大发射峰、ΦFL为化合物的荧光量子效率、λCL为化合物的余辉发光波长,测试体系为Tris-HCl溶液(含10% HSA)、温度25℃;
图7.Rubine-1、Rubine-2、Rubine-3、Rubine-4在Tris-HCl溶液(含10% HSA)中光激活余辉发光成像,余辉化合物浓度为1×10-4mol·L-1,荣耀30Pro拍摄;
图8.Rubine-2的余辉发光动力学测量,测试体系为Tris-HCl溶液(含10% HSA)、测试浓度1×10-5mol·L-1,激发光强100mW·cm-2(5s);其中,上方为Imaging Quant4000系统采集图像,下方是图像中光强度的定量;
图9.Rubine-2在A549细胞内的共聚焦荧光成像;
图10.Rubine-2在A549皮下瘤模型小鼠上活体余辉成像的应用,测试浓度1×10- 5mol·L-1,激发光强400mW·cm-2(10s)。
具体实施方式
相关描述短语的定义:
小分子:在本发明专利中小分子是指分子量小于700的一类有机化合物。
分散态:在表面活性剂、环糊精或蛋白等添加剂作用下,余辉化合物以单个分子的形式均匀分散在水溶液中,不会产生明显的两个或多个余辉发光分子π-π堆积而荧光红移或者淬灭的现象。
双自由基:在本发明专利中是在光照下,处于激发态的余辉发光分子其供电子部分一个电子转移至吸电子部分,形成单分子内的电荷分离态。吸电子基团:在本发明专利中是指通过乙烯基连接在酚羟基邻位具有明显吸电子能力的基团,包含氰基是这类基团的共有特征。
增强剂:在本发明专利中是指在余辉发光体系中加入表面活性剂、环糊精、蛋白等物质,与余辉发光底物通过疏水作用等发生结合,提升发光强度。
本发明旨在提供一种小分子有机余辉化合物及其使用方法。可以用于抗原或抗体标记用于免疫分析,另一方面可以作为余辉造影剂对小鼠等进行活体发光造影。在潜在的应用中亦可以对化合物的羟基进行修饰,制备金属离子、硫醇、活性氧、酶等各类激活型余辉发光探针。此外,光照下化合物产生电荷分离态是引发反应关键,因此需要这类化合物具有推拉电子结构。
在使用方法中需强调的是,化合物的酚羟基需要去质子化才能保持光激活性能,在使用时确保使用环境的pH值能够使化合物的酚羟基部分或全部去质子化是十分必要的。另外,这类化合物疏水性较强,分子间的π-π堆积不利于后续的光化学反应,与添加剂连用能够有效的解决分子聚集,提升发光强度。
作为多种发射波长余辉发光化合物的示例,化合物结构如下:
以Rubine-1为例,人血清白蛋白做添加剂,余辉发光的激活过程如下:取10微升的1×10-3mol·L-1Rubine-1二甲基亚砜溶液,加入890微升Tris缓冲液与100微升人血清白蛋白(40mg/mL),并将溶液充分混合均匀;随后取200微升混合液(1×10-5mol·L-1)置于黑色96孔板中,使用100mW·cm-2的白光光照5秒,光照完毕立即使用ImageQuant LAS 4000系统记录不同时刻的发光强度或图像。值得注意的是为了避免余辉发光底物的分解及降低发光溶液的背景噪声,溶液在光激活前应严格避光。另外,也可以将最终浓度为1×10-5mol·L- 1Rubine-1溶液添加在水凝胶或者涂在琼脂凝胶表面,在使用100mW·cm-2的白光光照5秒亦可以观察到明显的发光信号。
以下是具体实施例,下述实施例意图例证而不是限制本发明。
实施例1:
降冰片醛基中间体的合成
1)降冰片醛基前体的合成
在100mL干燥的三口瓶中,加入磷脂中间体(10.0g,25.3mmol)与35mL超干N,N-二甲基甲酰胺,冰浴下加入氢化钠(0.67g,27.9mmol)搅拌混合均匀。随后逐滴滴加2-降冰片(3.1g,27.9mmol)的THF溶液,在室温下反应12h。TCL监测反应进程。反应完毕,反应液中加入100mL饱和碳酸氢钠溶液,混合液用正己烷(150mL×3)萃取,有机相用无水Na2SO4干燥,柱色谱分离,得到白色固体(4.9g,产率:51%)。1H NMR(400MHz,CDCl3,ppm):δ7.13-7.08(m,1H,-Ph-H),6.91-6.84(m,2H,-Ph-H),3.30-3.34(d,3H,-O-CH3),2.41-2.28(s,2H,-Norcamphor-H),2.07-1.90(s,1H,-Norcamphor-H),1.52-1.19(m,7H,-Norcamphor-H),1.04(d,9H,-Si-C-(CH3)3),0.24-0.22(d,6H,-Si-CH3).Mass spectrometry(ESI-MS,m/z):[M+H]+calcd for C21H32ClO2Si,349.1860;found,349.1868.
2)降冰片醛基前体去TBS保护
在100mL干燥的单口瓶中加入上述产物(3.0g,7.9mmol),用30mL THF溶解。再加入四丁基氟化铵(1.0M in THF,8.7mL,8.7mmol),冰浴下搅拌2h。TCL监测反应进程。反应完成后,用乙酸乙酯(150mL)稀释反应液,并用1M的HCl(100mL)洗涤。无水Na2SO4干燥有机相,减压浓缩除去有机溶剂。柱层析(PE:EtOAc=85:15)纯化得到乳白色固体(2.0g,产率:95%)。1H NMR(400MHz,CDCl3,ppm):δ7.19-7.14(m,1H,-Ph-H),7.02-6.97(m,1H,-Ph-H),6.88-6.84(m,1H,-Ph-H),3.35-3.31(d,3H,-O-CH3),2.42-2.30(m,2H,-Norcamphor-H),2.08-1.93(m,1H,-Norcamphor-H),1.59-1.22(m,7H,-Norcamphor-H).Mass spectrometry(ESI-MS,m/z):[M–H]-calcd for C16H20ClO2,263.0839;found,263.0842.
3)降冰片醛基中间体的合成
在250mL干燥的双口瓶中,依次加入MgCl2(1.75g,18.4mmol)、多聚甲醛(2.47g,82.3mmol)、Et3N(2.73g,27.0mmol)、去保护中间体(3.25g,12.3mmol)与150mL乙腈,搅拌混合均匀。随后在氮气保护下,将反应混合物在80℃回流下加热过夜。点板(PE∶EtOAc=95∶10)监控反应进程。反应完毕,将反应液冷却致室温,随后用DCM(100mL)稀释反应液。抽滤,滤液用盐水洗涤,经Na2SO4干燥并减压浓缩除去有机溶剂。粗产物通过硅胶柱色谱柱纯化(PE∶EtOAc=95∶5),得到浅黄色固体产物(1.80g,产率:60%)。1H NMR(400MHz,CDCl3,ppm):δ11.65(s,1H,-Ar-OH),9.91-9.90(d,J=5.6Hz,1H,-CHO),7.51-7.48(m,1H,-Ph-H),7.03-6.99(m,1H,-Ph-H),3.38-3.34(d,3H,-O-CH3),2.44-2.34(m,2H,-Norcamphor-H),2.10-1.96(m,1H,-Norcamphor-H),1.56-1.27(m,7H,-Norcamphor-H).Mass spectrometry(ESI-MS,m/z):[M-H]-calcd for C16H16ClO3,291.0788;found,291.0794.
实施例2:
余辉发光化合物的合成
1)Rubine-1的合成
在100mL干燥的双口瓶中加入2-(2,3-二氢-1H-茚-1-亚基)丙二腈(246mg,1.37mmol),降冰片醛基中间体(200mg,0.68mmol),乙腈(30mL),哌啶(0.5mL)。将混合物回流11h,冷却后减压浓缩除去有机溶剂。粗固体通过硅胶柱色谱纯化(PE∶EtOAc=65∶35),得到黄褐色固体(79mg,产率:25%)。Rubine-1:1H NMR(400MHz,CDCl3,ppm):δ8.42-8.40(d,J=8.0Hz,1H),7.85(s,1H),7.68-7.66(d,J=7.6Hz,1H),7.50-7.39(m,3H),6.97-6.95(d,J=7.6Hz,1H),3.76(s,2H),3.38-3.34(d,3H),2.47-2.34(m,2H),2.11-1.99(m,1H),1.55-1.25(m,7H).Mass spectrometry(ESI-MS,m/z):[M-H]-calcd for C28H22ClN2O2,453.1370;found,453.1372.
2)Rubine-2的合成
在100mL干燥的双口瓶中加入2-(2,3-二氢-3-氧代-1H-茚-1-亚基)丙二腈(266mg,1.37mmol),降冰片醛基中间体(200mg,0.68mmol),乙腈(25mL),哌啶(0.5mL)。将混合物回流6h,冷却后减压浓缩除去有机溶剂。粗固体通过硅胶柱色谱纯化(PE∶EtOAc=65∶35),得到黄褐色固体(61mg,产率:19%)。Rubine-2:1H NMR(400MHz,CDCl3,ppm):δ8.69-8.67(d,J=7.6Hz,1H),8.32(s,1H),8.01-7.99(d,J=7.6Hz,1H),7.92-7.83(m,2H),7.71-7.69(d,J=8.0Hz,1H),7.01-6.99(d,J=8.0Hz,1H),3.39-3.35(d,3H),2.46-2.34(m,2H),2.11-1.99(m,1H),1.57-1.28(m,7H).Mass spectrometry(ESI-MS,m/z):[M-H]-calcd forC28H20ClN2O3,467.1162;found,467.119968.
3)Rubine-3的合成
在100mL干燥的双口瓶中加入4-(二氰亚甲基)-2,6-二甲基-4H-吡喃(235mg,1.37mmol),降冰片醛基中间体(200mg,0.68mmol),乙腈(30mL),哌啶(0.5mL)。将混合物回流6h,冷却后减压浓缩除去有机溶剂。粗固体通过硅胶柱色谱纯化(PE∶EtOAc=65∶35),得到黄褐色固体(100mg,产率:33%)。Rubine-3:1H NMR(400MHz,CDCl3,ppm):δ7.69-7.63(d-d,J1=16.0Hz,J2=5.8Hz,1H),7.41-7.37(t,J=7.2Hz,2H),6.96-6.91(m,2H),6.71(s,1H),6.72(s,1H),3.37-3.33(d,2H),2.46-2.34(m,5H),2.10-1.98(m,1H),1.43-1.25(m,7H).Mass spectrometry(ESI-MS,m/z):[M-H]-calcd for C26H22ClN2O3,445.1319;found,445.1318.
4)Rubine-4的合成
在100mL干燥的单口瓶中依次加入降冰片醛基中间体(200mg,0.68mmol)、异氟尔睛(255mg,1.37mmol)、哌啶(0.5mL)和适量乙腈。在氩气保护下,加热回流6h,反应温度90℃。反应结束后得到有橙红色荧光的红色液体。反应液用通过减压浓缩除去溶剂,采用柱层析(DCM:MeOH=98:2)纯化得到产品,产品为橙色固体(210mg,产率:67%)。1H NMR(400MHz,CDCl3,ppm):δ7.45-7.43(d,J=8.0Hz,1H),7.39-7.35(d,J=16.4Hz,1H),7.12-7.08(d,J=16.4Hz,1H),6.90-6.88(d,J=8.0Hz,1H),6.85(s,1H),3.37-3.33(d,2H),2.61(s,2H),2.50(s,2H),2.46-2.34(m,2H),2.10-1.98(m,1H),1.57-1.28(m,7H),1.09(s,6H).Massspectrometry(ESI-MS,m/z):[M-H]-calcd for C26H22ClN2O3,445.1319;found,445.1318.
5)Rubine-5的合成
在100mL干燥的双口瓶中加入1,3-双(二氰基亚甲基)茚满(331mg,1.37mmol),降冰片醛基中间体(200mg,0.67mmol),乙腈(25mL),哌啶(0.5mL)。将混合物回流1h,冷却后减压浓缩除去有机溶剂。粗固体通过硅胶柱色谱纯化(PE∶EtOAc=65∶35),得到蓝色固体(37mg,产率:10%)。Rubine-1:1H NMR(400MHz,CDCl3,ppm):δ8.72-8.70(m,2H),8.40(s,1H),8.11-8.08(m,2H),7.71-7.69(d,J=8.0Hz,1H),7.12-7.10(d,J=8.0Hz,1H),3.38-3.34(d,3H),2.44-2.32(m,2H),2.09-1.79(m,1H),1.55-1.28(m,7H).Mass spectrometry(ESI-MS,m/z):[M-H]-calcd for C31H20ClN4O2,515.1275;found,515.1279.
实施例3:
余辉化合物Rubine-1的紫外吸收、荧光、余辉发光光谱。取实施例2制备的Rubine-1溶于分析纯二甲基亚砜中,制成1.0×10-2M的储备液。然后取Tris缓冲溶液(1.8mL)与人血清白蛋白溶液(0.2mL)制备混合溶剂2mL。随后取2μL Rubine-1储备液加入到已制备的混合溶剂中,混合均匀后转移至光学石英比色皿(10×10mm)中测试其吸收与荧光光谱。如图1所示,其最大吸收波长位于463nm,以该波长激发得到最大发射波长为582nm的荧光光谱。同理,在混合溶剂中加入20μL Rubine-1储备液加入到已制备的混合溶剂中,混合均匀后转移至光学石英比色皿(10×10mm)中测试其余辉发光光谱,在100mWcm-2的白光光照5s后的到发射峰值为589nm的余辉发光光谱。
实施例4:
余辉化合物Rubine-2的紫外吸收、荧光、余辉发光光谱。取实施例2制备的Rubine-2溶于分析纯二甲基亚砜中,制成1.0×10-2M的储备液。然后取Tris缓冲溶液(1.8mL)与人血清白蛋白溶液(0.2mL)制备混合溶剂2mL。随后取2μL Rubine-2储备液加入到已制备的混合溶剂中,混合均匀后转移至光学石英比色皿(10×10mm)中测试其吸收与荧光光谱。如图2所示,其最大吸收波长位于475nm,以该波长激发得到最大发射波长为618nm的荧光光谱。同理,在混合溶剂中加入20μL Rubine-2储备液加入到已制备的混合溶剂中,混合均匀后转移至光学石英比色皿(10×10mm)中测试其余辉发光光谱,在100mWcm-2的白光光照5s后的到发射峰值为635nm的余辉发光光谱。
实施例5:
余辉化合物Rubine-3的紫外吸收、荧光、余辉发光光谱。取实施例2制备的Rubine-3溶于分析纯二甲基亚砜中,制成1.0×10-2M的储备液。然后取Tris缓冲溶液(1.8mL)与人血清白蛋白溶液(0.2mL)制备混合溶剂2mL。随后取2μL Rubine-3储备液加入到已制备的混合溶剂中,混合均匀后转移至光学石英比色皿(10×10mm)中测试其吸收与荧光光谱。如图3所示,其最大吸收波长位于494nm,以该波长激发得到最大发射波长为621nm的荧光光谱。同理,在混合溶剂中加入20μL Rubine-3储备液加入到已制备的混合溶剂中,混合均匀后转移至光学石英比色皿(10×10mm)中测试其余辉发光光谱,在100mWcm-2的白光光照5s后的到发射峰值为638nm的余辉发光光谱。
实施例6:
余辉化合物Rubine-4的紫外吸收、荧光、余辉发光光谱。取实施例2制备的Rubine-4溶于分析纯二甲基亚砜中,制成1.0×10-2M的储备液。然后取Tris缓冲溶液(1.8mL)与人血清白蛋白溶液(0.2mL)制备混合溶剂2mL。随后取2μL Rubine-4储备液加入到已制备的混合溶剂中,混合均匀后转移至光学石英比色皿(10×10mm)中测试其吸收与荧光光谱。如图4所示,其最大吸收波长为430/543nm,以540激发得到最大发射波长为653nm的荧光光谱。同理,在混合溶剂中加入20μL Rubine-4储备液加入到已制备的混合溶剂中,混合均匀后转移至光学石英比色皿(10×10mm)中测试其余辉发光光谱,在100mWcm-2的白光光照5s后的到发射峰值为693nm的余辉发光光谱。
实施例7:
余辉化合物Rubine-5的紫外吸收、荧光、余辉发光光谱。取实施例2制备的Rubine-5溶于分析纯二甲基亚砜中,制成1.0×10-2M的储备液。然后取Tris缓冲溶液(1.8mL)与人血清白蛋白溶液(0.2mL)制备混合溶剂2mL。随后取2μL Rubine-5储备液加入到已制备的混合溶剂中,混合均匀后转移至光学石英比色皿(10×10mm)中测试其吸收与荧光光谱。如图5所示,其最大吸收波长为610nm,以518激发得到最大发射波长为670nm的荧光光谱。
实施例8:
余辉化合物Rubine-1、Rubine-2、Rubine-3、Rubine-4、Rubine-5光物理参数汇总。如图6所示,λabs表示化合物的最大吸收峰、εabs表示化合物最大吸收峰处摩尔消光系数、λem表示化合物的最大发射峰、ΦFL表示化合物的荧光量子效率、λCL表示化合物的余辉发光波长,测试体系为Tris-HCl溶液(含10% HSA)、温度25℃;测试仪器与实施例3-8均为安捷伦紫外可见吸收光谱仪Cary 500spectrophotometer与安捷伦荧光光谱仪Cary Eclipsefluorescence spectrophotometer。
实施例9:
余辉化合物Rubine-1、Rubine-2、Rubine-3、Rubine-4余辉发光照片。如图7所示,不同发光波长的Rubine系列余辉发光化合物;测试体系为Tris缓冲液(包含10% HSA),化合物浓度1.0×10-4M,在500mWcm-2的白光光照5s后手机拍摄得到。
实施例10:
余辉化合物Rubine-2的余辉发光动力学。取实施例2制备的Rubine-2溶于分析纯二甲基亚砜中,制成1.0×10-2M的储备液。然后取Tris缓冲溶液(0.9mL)与人血清白蛋白溶液(0.1mL)制备混合溶剂1mL。随后取1μL Rubine-2储备液加入到已制备的混合溶剂中,混合均匀后转移至黑色96孔板(200μL/孔)中测试其余辉发光动力学,其中余辉发光信号采用Imaging Quant 4000系统采集,曝光时间1min。如图8所示,在光照前余辉发光化合物几乎没有发光信号;在100mWcm-2的白光光照5s后,发光信号立即达到最高(1.43*108RLU),并随时间逐渐衰减,发光时间持续约1小时。
实施例11:
Rubine-2在A549细胞内的共聚焦荧光成像。人非小细胞肺癌细胞(humanadenocarcinoma alveolar basal epithelial cells,A549 cell)购买自上海细胞生物研究所。F-12K细胞培养基含有10%胎牛血清(FBS,Biological Industry,Kibbutz BeitHaemek,Israel)和1%双抗(10,000U mL-1penicillin and 10mg/ml streptomycin,Solarbio life Science,Beijing,China)。培养条件为37℃,含5% CO2的湿润气体氛围。
将A549细胞以1×105细胞/孔的密度转移到含有1.5mL完全培养基的玻璃培养皿中,待细胞充分贴壁后,加入含有1μM Rubine-2的F-12K培养基,避光孵育2h;孵育完成后使用PBS清洗(1mL×3),随后使用莱卡共聚焦激光显微镜Leica TCS SP8(63×油镜)成像。激发488nm,荧光610–650nm。如图9所示,细胞质中显示出明显的红色荧光,表明Rubine-2具备良好的细胞膜渗透性。
实施例12:
Rubine-2在活体余辉成像中的应用。本发明中所有活体实验均遵守实验室动物饲养和使用的规章制度,并得到华东理工大学伦理委员会批准。实验用荷瘤裸鼠购自上海华东师范大学,饲养在无菌室中层流通风橱内的无菌鼠笼中,使用高压蒸汽处理过的食物和水进行喂食。
首先将20微升10μM Rubine-2的Tris-HCl溶液原位注射在肿瘤区域,注射15分钟后,对肿瘤区域进行10秒光照(光强:400mW·cm-2),随后立即采用Perkin Elmer In-VivoProfessional Imaging System成像系统记录小鼠体内余辉发光信号。如图10所示,光照前小鼠肿瘤区域几乎无余辉发光信号;而在光激活后小鼠肿瘤区域显示出远高于其余区域的余辉发光信号,表明Rubine-1具备出色的活体余辉成像能力。
Claims (8)
1.一种单组分有机余辉化合物,其特征在于,该有机余辉化合物的分子结构特征包含推拉电子体系与富电子基取代的双键基团,其结构通式如下:
式I中吸电子基团A为至少包含一个氰基的生色团,X为卤素原子;甲氧基(富电子基)取代双键基团是必要的结构,且该结构处于吸电子A的邻位或对位,共同组成推拉电子体系。
2.根据权利要求1所述的一种单组分有机余辉化合物,其特征在于,
吸电子基团A为如下含有氰基结构中的任意一种:
R1为氰基、乙酰基、羧基甲酯、羧基乙酯或羧基中的任意一种;R2为甲基、异丙基、叔丁基或环丙烷基中的任意一种。
3.根据权利要求1所述的一种单组分有机余辉化合物,其特征在于,X为卤素原子F、Cl或Br的一种。
4.根据权利要求2所述的一种单组分有机余辉化合物,其特征在于,
吸电子基团A选自如下结构的任意一种:
其中,R1为氰基、乙酰基、羧基甲酯、羧基乙酯或羧基中的任意一种。
5.根据权利要求1所述一种单组分有机余辉化合物的生物检测应用,其特征在于,该余辉发光化合物与各类商用发光增强剂、表面活性剂、环糊精、脱脂奶粉或血清蛋白复配,用于提升化合物的发光强度。
6.根据权利要求1所述一种单组分有机余辉化合物的生物检测应用,其特征在于,所述余辉发光化合物不需要添加光敏剂,也不需要与光敏剂、嵌段聚合物混合形成纳米颗粒;通过化合物单独使用。
7.根据权利要求5或6所述的应用,其特征在于,所述余辉发光化合物在水相溶液、活细胞、组织或动物实验中的余辉发光应用。
8.根据权利要求1所述的一种单组分有机余辉化合物的应用方法,其特征在于,使用中包括如下操作条件:(1)将余辉底物置于pH大于5.0的溶液或凝胶中;(2)光源的光照时间为1-10秒,光源为商品化的LED光源(100mW·cm-2)。
余辉化合物在pH值5.0以上微环境中,通过光照生成1,2-二氧杂环丁烷高能不稳定结构,随后该结构快速分解产生余辉发光。
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