CN114409856B - 一类检测氧气的荧光/磷光发光寿命聚合物探针及其应用 - Google Patents
一类检测氧气的荧光/磷光发光寿命聚合物探针及其应用 Download PDFInfo
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 81
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- 239000001301 oxygen Substances 0.000 title claims abstract description 81
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Abstract
本发明公开了一类检测氧气的比率型荧光/磷光发光寿命聚合物探针及其应用,该类聚合物探针通过自由基聚合反应将荧光参比单元和铱(III)配合物单元以及水溶性单元连接在一起,以实现细胞内氧气检测。其中聚合物探针P1由吩噁嗪衍生物PO1和磷光铱(III)配合物Ir1以及PEG950构成,PO1是参比,对氧气没有响应;Ir1能够对氧气响应,其发光强度会随着氧气含量的增强而减弱;PEG950赋予聚合物良好的水溶性和生物相容性。本发明公开的用于检测氧气的荧光/磷光发光寿命聚合物的发光强度以及发射寿命会随着氧气含量的增加而减少,具有良好的水溶性和生物相容性,为实现细胞内氧气含量检测提供了新的思路,在生物成像与传感方面具有重要的应用前景。
Description
技术领域
本发明属于生物成像与检测领域,具体涉及一类用于检测氧气的荧光/磷光发光寿命聚合物探针及其在细胞中检测氧气的应用。
背景技术
近年来,随着光学技术的发展,光学探针已广泛用于细胞内各种物质传感及成像分析,如细胞内各种离子、气体分子、活性氧、蛋白质等的检测。光学氧气探针是通过探针分子与被检测物氧气发生特异性相互作用,引起探针分子的光学性质发生变化,从而达到检测的目的。
目前,根据构筑方式的不同,关于光学氧气探针的研究主要分为基于小分子的光学氧气探针、基于量子点或纳米晶的光学氧气探针以及基于聚合物的光学氧气探针。基于小分子的光学氧气探针是将荧光分子和磷光分子通过化学键连接起来,它们具有对生物体细胞亲和力强、易于化学修饰等优点,但大多数有机分子水溶性较差,需要将其先负载到磷脂膜或经过繁琐的合成来改变其水溶性,基于量子点的比率式探针往往具有一定的毒性,而相较于基于小分子和基于量子的光学氧气探针,聚合物氧气探针在光稳定性、水溶性等方面具有相对突出的优点。
磷光过渡金属配合物是一类非常重要的光电材料,金属中心和配体之间的电荷转移和能量迁移展现出特殊的光电性能,具有高效的三线态磷光发射、较长的寿命、大的斯托克斯位移等优点,广泛应用于发光二极管和生物医学领域。其中,磷光金属铱配合物出色的光物理性质,如较高的量子效率、可调的发射波长、较大的斯托克斯位移,良好的光化学稳定性等,在生物传感以及细胞成像领域都具有良好的应用前景。同时,利用Ir1的长发射寿命的优势,可以通过使用光致发光寿命成像和时控发光成像技术有效消除背景荧光干扰,通过将收集到的信号通过时间分辨技术加以区分,能改善生物成像中的信噪比,提高比率法检测的精准度。
目前,关于以铱配合物为检测位点,水溶性聚合物为基体材料的用于检测氧气的生物探针的报道还较为少见,现有技术中光学氧气探针的水溶性、生物相容性、响应性、分析性能等有待进一步构建提升,因此,设计合成出应用比率型发光寿命聚合物探针是有必要的。
发明内容
本发明的目的在于提供一类利用发光寿命变化实现细胞内检测氧气含量的荧光/磷光发光寿命聚合物探针的设计新思路。
本发明的目的是通过以下技术方案来实现:
本发明提供一类利用发光寿命变化实现细胞内检测氧气含量的荧光/磷光发光寿命聚合物探针,具有如下结构通式:
其中a+b+c=1;
通式中A为聚合物的参比单元,为下列结构中的任意一种:
其中,n可独立地选择含有1至7个碳原子的直链、支链或者环状烷基(醚)链;
通式中B为一类可增加聚合物水溶性的基团单体,为下列结构中的任意一种:
n=17~19;
通式中C为一类磷光金属配合物单体,为下列结构中的任意一种:
其中,所述磷光金属配合物结构通式中,
中的任意一种;
中的任意一种;
n可独立地选择含有1至7个碳原子的直链、支链或者环状烷基(醚)链;
中的任意一种。
进一步地,本发明的可用于细胞内检测氧气含量的荧光/磷光发光寿命聚合物探针的制备是通过自由基聚合反应将各单元连接在一起的,通过观察各单元在细胞内与氧气作用后发光寿命的变化不同以达到检测的目的。
进一步地,所述聚合物探针可通过自由基聚合反应将荧光参比单元A和铱(III)配合物单元以及可增加聚合物水溶性的基团单体B连接在一起制备成探针,检测灵敏度高,相互之间并不影响,实现比率检测。
进一步地,所述聚合物探针可用于对氧气的特异性检测。
进一步地,所述聚合物探针可用于特异性检测细胞内源性或外源性氧气含量。
进一步地,所述聚合物探针可通过铱(III)配合物对细胞中氧气含量变化检测。
进一步地,所述聚合物探针可通过选择合适的配合物来提高氧气检测效率。
进一步地,所述聚合物探针可应用于细胞成像和传感领域。
本发明的有益效果是:本发明的用于检测氧气的聚合物探针通过对氧气探针的组装进行研究,构建了一类具有良好的水溶性、生物相容性以及毒性小的光学探针,可用于内源性/外源性氧气含量的检测,是很好的生物细胞探针;其次,本发明的聚合物探针结合比率法和时间分辨技术,在氧气检测过程中实现了不依赖发光强度,不受背景荧光干扰,而依赖发光寿命的比率型探针,能有效提升比率法检测精准度;本发明聚合物探针所采用的配合物Ir1对氧气具有良好的响应性,铱(III)配合物对氧气检测发光、寿命具有明显的差异,而荧光参比单元和水溶性单元发光、寿命无明显的差异,避免了利用发光波长检测时因波长信号重叠而造成的干扰,各响应端发光寿命的变化能较好的区分,且磷光寿命和荧光寿命的比值可以进一步校准误差,能有效提高探针的准确性。
附图说明
图1为本发明测试例1中探针参比单元单体在氮气、空气以及氧气下的发射光谱和其在氧气和氮气条件下550nm处发光寿命衰减曲线。
图2为本发明测试例2中探针配合物单体在不同氧气含量下的发射光谱和其在550nm处发光寿命衰减曲线。
图3为本发明测试例3中水溶性聚合物探针P1在不同氧气含量下的发射光谱和其在550nm处发光寿命衰减曲线。
图4为本发明测试例3中水溶性聚合物探针P1在不同氧气含量下的荧光/磷光寿命占比。
图5为本发明测试例4中水溶性聚合物探针P1细胞毒性实验结果。
图6为本发明测试例5中水溶性聚合物探针P1在不同氧气浓度下,固定HeLa细胞中的光致发光成像、光致发光寿命成像和寿命分布图。
具体实施方式
为了相关技术领域人员更好的理解本发明专利的内容,下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实例。
本发明中使用的化学试剂皆为市购。
以下述具体可用于内源性/外源性氧气含量的检测的探针P1为例:
上述聚合物分子的合成步骤为:
首先合成作为参比的有机荧光吩噁嗪衍生物,再合成O2响应端的磷光金属配合物单体,最后通过自由基聚合的方式将参比有机荧光小分子和O2响应的磷光金属配合物单体以及增加聚合物水溶性以及生物相容性的PEG同时聚合制备成聚合物探针P1。
先合成对氧气响应不敏感且氧气含量变化对其发光寿命不影响的具有短发光寿命的荧光小分子作为水溶性聚合物探针的参比PO1,再合成对O2响应且具有长发光寿命的磷光金属配合物单体Ir1用作对氧气响应,其寿命随氧气含量的变化而变化,最后通过聚合反应将各响应端与可增加聚合物水溶性的基团单体聚合在一起制备成探针P1。水溶性聚合物探针P1的结构如下所示:
其中,化学结构式为具水溶性的基团单体,其中n=17~19、化学结构式/>为具有短发光寿命的参比有机荧光小分子单体PO1、化学结构式为O2响应端的具有长发光寿命的磷光金属配合物单体Ir1。
实施例1:参比PO1的制备
将酚噁嗪(550mg,3mmol)溶解在DMSO溶液(5mL)中,随后加入3-溴丙烯(600mg,5mmol),然后将氢氧化钾(336mg,6mmol)溶解在5mL水中,加入到上述溶液中,在室温下搅拌过夜。待反应完成后,将反应液倒入水中(50mL),然后用二氯甲烷萃取,收集有机相并用无水硫酸镁干燥,最后将减压蒸馏得到的粗产物通过硅胶柱层析法分离纯化,洗脱剂为石油醚/乙酸乙酯(50:1,v/v)得到白色粉末状化合物475mg(产率71%);
在0℃氮气氛围下,将三氯氧磷(POCl3)(230mg,1.5mmol)溶解在无水DMF(2mL)中搅拌30min。然后将上步化合物(223mg,1mmol)溶解在1,2-二氯乙烷(2mL)中,加入到上述溶液中,在85℃下搅拌6h。待反应完成后,将反应液倒入冰水(20mL)中并用0.1M的NaOH水溶液调节至中性,随后用二氯甲烷萃取,收集有机相并用无水硫酸镁干燥,最后将减压蒸馏后得到的粗产物通过硅胶柱层析法分离纯化,洗脱剂为石油醚/乙酸乙酯(50:1,v/v),得到黄色粉末状化合物201mg(产率80%);
1H NMR(400MHz,CDCl3)δ=9.70(s,1H),7.31-7.28(m,1H),7.12(d,J=1.9Hz,1H),6.83-6.68(m,3H),6.51–6.47(m,2H),5.92–5.83(m,1H),5.32–5.26(m,2H),4.22-4.20(m,2H).13C NMR(100MHz,CDCl3)δ=189.83,145.36,144.82,139.62,131.82,130.19,129.79,128.72,123.99,122.74,117.38,115.61,114.16,112.84,111.39,47.68。
实施例2:氧气响应配合物Ir1的制备
在氮气氛围下,将2-(2,4-二氟苯基)吡啶(382mg,2mmol)和IrCl3·3H2O(316mg,1mmol)溶解在9mL除去氧气的2-乙氧基乙醇/去离子水的混合溶液中(3:1,v/v),110℃搅拌12h。待反应结束后,冷却至室温,加入10mL去离子水,此时有大量绿色沉淀析出,抽滤并用乙醇洗涤后干燥。得到绿色2-(2,4-二氟苯基)吡啶二氯桥365mg(产率60%)。
在氮气氛围下,将化合物1(76mg,0.4mmol)和铱(III)2-(2,4-二氟苯基)吡啶二氯桥联二聚体(243mg,0.2mmol)溶解在15mL甲醇/二氯甲烷(1:3,v/v)混合溶液中,避光回流8h。待反应完成后,将反应液冷却至室温,随后加入KPF6(147mg,0.8mmol)继续搅拌12h。然后除去溶剂,加入去离子水(30mL)并用二氯甲烷萃取,收集有机相用无水硫酸镁干燥。除去溶剂后的粗产物通过硅胶柱层析法分离纯化,洗脱剂为二氯甲烷/甲醇(40:1,v/v),得到绿色固体96mg(产率78%)。
将上步配合物(90mg,0.1mmol)溶解在无水二氯甲烷(5mL)中,并加入无水K2CO3(117mg,1.1mmol)。然后将丙烯酰氯(45mg,0.5mmol)溶解在无水二氯甲烷(5mL)中,缓慢加入到上述溶液中,混合物在冰浴、氮气氛围下搅拌2h,然后在常温下搅拌10h。待反应完成后,过滤除去K2CO3,然后旋转蒸发除去有机溶剂,粗产物通过硅胶柱层析法分离纯化,洗脱剂为二氯甲烷/甲醇(80:1,v/v),得到绿色固体74mg(产率90%);
1H NMR(400MHz,CDCl3)δ=8.59(s,1H),8.49(s,1H),8.30-8.28(m,2H),7.86(d,J=5.7Hz,1H),7.83-7.77(m,2H),7.75(d,J=5.6Hz,1H),7.52(t,J=6.2Hz,2H),7.39(d,J=5.6Hz,1H),7.27(d,J=5.6Hz,1H),7.14-7.07(m,2H),6.58-6.46(m,3H),6.23-6.17(m,1H),5.95(d,J=10.5Hz,1H),5.69-5.64(m,2H),5.50(s,2H),2.64(s,3H).13C NMR(100MHz,CDCl3)δ=165.57,155.74,154.91,153.24,150.70,149.91,149.23,148.95,148.77,139.07,132.58,131.28,129.34,128.03,127.49,127.40,126.68,125.83,123.94,123.80,123.70,123.58,123.50,114.08,113.95,113.80,99.43,99.17,98.91,63.65,29.72,21.52,16.18,16.11,14.13.MALDI-TOF MS:m/z=827.358[M-PF6 -]+。
实施例3:聚合物P1的制备
其中a、b、c≥55;n=17~19;
将化合物PO1(10mg,0.04mmol)、mPEG950(950mg,1mmol)、配合物IrG2(49mg,0.06mmol)和2,2-偶氮二异丁腈(AIBN,3mg,0.02mmol)溶解在无水四氢呋喃(THF,2.0mL)中,在氮气氛围、80℃条件下,搅拌48小时。待反应完成后,将反应液冷却至室温,除去THF后将粗产物分别在甲醇和CH2Cl2中透析12小时而纯化,得到黄绿色油状产物GP1。GPC(THF,聚苯乙烯标准):Mn=14482,PDI=1.11。
测试例1:参比PO1在氮气、空气以及氧气下的发射光谱和其在氧气和氮气条件下550nm处发光寿命测试
测试浓度为10μM,测试溶剂为甲醇/PBS(1:9,v/v),激发波长为405nm。如图1所示,参比小分子荧光PO1在氮气、氧气以及空气中的发射光谱没有明显的变化,由寿命测试结果可知,其在氮气和氧气条件下的寿命无明显的变化。光谱数据和寿命数据均说明了参比小分子荧光PO1对氧气不敏感,符合PO1的参比条件。
测试例2:对氧气敏感的配合物Ir1在不同氧气含量下的发射光谱和其在550nm处发光寿命测试
测试浓度为10μM,测试溶剂为甲醇/PBS(1:9,v/v),激发波长为405nm。如图2所示,对氧气敏感的配合物Ir1在氧气含量由0%变化到100%的过程中,其发光强度在不断降低,与之相对应的寿命值也由原来的931ns降低到251ns。光谱数据和寿命数据均说明了配合物Ir1对氧气具有良好的响应性。
测试例3:对氧气敏感的水溶性聚合物探针P1在不同氧气含量下的发射光谱和其在550nm处发光寿命测试,在不同氧气含量下的荧光/磷光寿命占比测试
测试浓度为200μg/mL,测试溶剂为甲醇/PBS(1:9,v/v),激发波长为405nm。如图3、4所示对氧气敏感的聚合物P1在氧气含量由0%变化到100%的过程中,其总发光强度在不断降低,参比小分子的寿命几乎不变,而磷光部分在总的发光寿命中的占比则随着氧气的增加由81.79%下降到了52.0%。光谱数据和寿命数据均说明了配合物Ir1对氧气具有良好的响应性,且磷光寿命和荧光寿命的比值可以进一步校准误差,提高了探针的准确性。
测试例4:聚合物探针P1的MTT细胞毒性实验
实验中使用MTT分析法分析聚合物探针P1的细胞毒性。将HeLa细胞接种到生长于培养基(100μL)中的96孔平底微孔板中(10,000个细胞/孔),并在37℃、5%CO2氛围下孵育24h。然后在生长培养基/DMSO(99:1,v/v)的混合物中,将浓度为0-600μg/mL的P1加入孔中。含有无细胞生长培养基的孔用作空白对照,将微孔板在37℃,5%CO2氛围下孵育24h。然后将含有MTT的PBS溶液(10μL,5mg/mL)加入每个孔中,并再孵育4h。之后,向每个孔中加入DMSO(150μL),并将微孔板进一步孵育1h,最后,用微孔板分光光度计(TECAN SUNRISE)测量溶液在570nm处的吸光度。通过以下公式计算得出细胞存活率:
细胞存活率(%)=(实验组OD570平均值/对照组OD570平均值)×100%
如图5可以得出,P1同HeLa细胞共同孵育浓度在0-400μg/mL范围内,细胞的存活率均在90%以上,说明P1在该浓度范围内对细胞的生理活性影响较小、毒副作用较小。
测试例5:对氧气敏感的水溶性聚合物探针P1在不同氧气浓度下,固定HeLa细胞中的光致发光成像、光致发光寿命成像和寿命分布图测试
将P1(200μg/mL)孵育到预先固定有HeLa细胞的共聚焦培养皿中,放置在活细胞工作站中,通过改变工作站中通入混合气体中氧气含量以营造氧气浓度为0、5%、15%、20%和40%环境,保持气体流通30min后进行共聚焦光致发光成像和寿命成像。如图6所示,P1在HeLa细胞内分布均匀,随着细胞所处环境氧气浓度的增加,细胞内P1的发光逐渐减弱,说明氧气逐步猝灭了P1的磷光部分,而光致发光寿命成像中可以观察到绿色像素点的增加,也说明了细胞内P1的磷光被氧气猝灭,磷光寿命缩短。随着细胞所处环境氧气浓度从0加到40%,从发光寿命成像分析可知,细胞内荧光寿命几乎没有变化,而磷光寿命则从109ns缩短到64ns。
以上所述仅为本发明的较佳实施例,并不以上述实施方式为限,但凡本领域普通技术人员根据本发明所揭示内容所作的等效修饰、等同替换和改进等,皆应纳入权利要求书中记载的保护范围。
Claims (7)
1.一类检测氧气的荧光/磷光发光寿命聚合物探针,其特征在于,所述聚合物探针化学结构式如下所示:
其中a+b+c=1;n=17~19。
2.如权利要求1所述的一类检测氧气的荧光/磷光发光寿命聚合物探针,其特征在于,所述聚合物探针通过自由基聚合反应将具有短发光寿命的参比单元和磷光金属配合物单体/>以及具水溶性的基团单体/>连接在一起制备成探针,相互之间不影响,实现比率检测。
3.如权利要求1所述的一类检测氧气的荧光/磷光发光寿命聚合物探针,其特征在于,所述聚合物探针通过磷光金属配合物单体对氧气进行变化检测,检测细胞中氧气含量。
4.如权利要求1-3任一项所述的一类检测氧气的荧光/磷光发光寿命聚合物探针对氧气的特异性检测的应用,其应用为非疾病的诊断或治疗目的。
5.如权利要求1-3任一项所述的一类检测氧气的荧光/磷光发光寿命聚合物探针特异性检测细胞内源性或外源性氧气含量的应用,其应用为非疾病的诊断或治疗目的。
6.如权利要求1-3任一项所述的一类检测氧气的荧光/磷光发光寿命聚合物探针通过选择合适的配合物来提高氧气检测效率的应用,其应用为非疾病的诊断或治疗目的。
7.如权利要求1-3任一项所述的一类检测氧气的荧光/磷光发光寿命聚合物探针在细胞成像和传感领域的应用,其应用为非疾病的诊断或治疗目的。
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