CN115181068A - Tpi衍生物荧光探针及其在制备铜离子检测试剂中的应用 - Google Patents
Tpi衍生物荧光探针及其在制备铜离子检测试剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种TPI衍生物荧光探针及其在制备铜离子检测试剂中的应用,属于生物荧光探针技术领域。该TPI衍生物荧光探针的化学名称为2‑氨基‑3‑(((E)‑4‑(4,5‑二苯基‑1H‑咪唑‑2‑基)亚苄基)氨基)马来腈,分子式为C26H18N6。在Cu2+存在的情况下,该TPI衍生物荧光探针的C=N键会提供孤对电子进行配位,得到另一种新的化合物,由此产生荧光的变化。本发明的TPI衍生物荧光探针丰富了Cu2+类荧光探针的种类,为有机分析和光化学提供了新型的探针分子,可广泛应用于荧光分析或检测领域。
Description
技术领域
本发明属于生物荧光探针技术领域,具体涉及一种TPI衍生物荧光探针及其在制备铜离子检测试剂中的应用。
背景技术
铜是人体内含量第三的微量元素,参与了生命体内多种生理过程,在细胞呼吸、神经递质生物合成和酶活性中起着十分重要的作用。铜离子的含量与人体健康息息相关,缺乏时会导致贫血、动脉异常、心血管疾病等,而高浓度时会引起各种神经退行性疾病,如阿尔兹海默症、帕金森病等,因此,美国环境保护署(EPA)将饮用水中Cu2+离子的可接受浓度定为小于20μM。由于铜离子在生命系统中的积极和消极影响,十分有必要开发能在真实环境和活细胞中快速、高选择性检测Cu2+的有效方法。
传统的分析检测方法有色谱、光谱和电化学分析,但因为设备昂贵、预处理繁杂、耗时长,不适合实时实地检测,而荧光分析方法具有操作简单、响应速度快、高选择性、高灵敏度、无损成像等优点,被广泛用于铜离子检测领域。目前,一些基于碳量子点(CDs)、金属有机框架(MOFs)、贵金属纳米材料的荧光探针已被用于Cu2+检测。但鉴于实际样品和细胞中Cu2+的极低含量,一些已报道的荧光探针的检测限无法达到要求,所以开发一种极具高选择性、灵敏性和环境友好性的微量Cu2+检测荧光探针是非常重要的。
有机荧光小分子作为一类荧光材料,制备简单,生物毒性低,荧光性能优异,是理想的荧光探针,被广泛应用于生物分子检测、荧光活体成像、癌症早期诊断、药物代谢追踪,为相关疾病的早期诊断提供了重要信息。研究表明在生命体内Cu2+和胺基、咪唑基、羧基、硫基团具有极强的结合力,且荧光比色检测能够提供肉眼目测,结果清晰直观。所以,在满足上述两个要求下,可以实现在生理条件下对Cu2+进行实时、高效、准确的定量检测。
但是,基于比色法和荧光法检测Cu2+的高灵敏度探针仍十分缺乏,这类探针亟待开发。
发明内容
针对目前可用于检测Cu2+的比色型荧光探针缺乏的现状,本发明的目的之一是提供一种TPI衍生物荧光探针,其结构式如式Ⅰ所示:
该荧光探针的化学名称为2-氨基-3-(((E)-4-(4,5-二苯基-1H-咪唑-2-基)亚苄基)氨基)马来腈,分子式为C26H18N6。
该荧光探针的合成路线如下式所示:
具体制备步骤如下:
第一步,制备4-(4,5-二苯基-1H-咪唑-2-基)苯甲醛;
将苯偶酰、对苯二甲醛、醋酸铵溶于醋酸中,加热回流,用薄层色谱法(TLC)监测进程,待反应完全后冷却至室温,将反应液倒入冰水中,静置1小时,抽滤,用纯水对沉淀进行洗涤,干燥,粗产物通过柱层析分离得4-(4,5-二苯基-1H-咪唑-2-基)苯甲醛(化合物Ⅱ)。
其中,苯偶酰和对苯二甲醛的物质的量比为0.5-1;加热回流温度120-130℃。
第二步,4-(4,5-二苯基-1H-咪唑-2-基)苯甲醛和2,3-二氨基马来腈在醋酸催化下反应,制得所述荧光探针(化合物Ⅰ)。
将4-(4,5-二苯基-1H-咪唑-2-基)苯甲醛和2,3-二氨基马来腈溶解在乙醇中,加入催化剂量的醋酸,加热回流,TLC点板跟踪至反应结束,抽滤后得到黄色固体2-氨基-3-(((E)-4-(4,5-二苯基-1H-咪唑-2-基)亚苄基)氨基)马来腈(化合物Ⅰ)。
所述有机溶剂选自甲苯、乙腈、二氯乙烷、二氯甲烷、N,N-二甲基甲酰胺、四氯化碳、正己烷、四氢呋喃、甲醇或乙醇中的一种。优选乙醇。
上述制备方法中,反应结束后的后处理方式并无特别的限定,本领域技术人员可以依据物料的理化性质,结合公知常识的分离手段,采用常规的有机分离手段来实现目标产物的分离。优选的技术方案为层析分离。所述层析分离进一步优选二氯甲烷和乙酸乙酯的混合溶剂作为柱层析洗脱剂,更进一步优选,洗脱剂中二氯甲烷和乙酸乙酯的体积比为20:1。
上述制备方法中,通过薄层色谱法(TLC)监测反应终点,反应时间无特别的限定。
本发明的目的之二是提供了上述TPI衍生物荧光探针在制备铜离子检测试剂中的应用。
本发明的目的之三是提供了上述TPI衍生物荧光探针在制备细胞成像试剂中的应用。
本发明借助4-(4,5-二苯基-1H-咪唑-2-基)苯甲醛和2,3-二氨基马来腈通过席夫碱反应形成C=N双键,提供可以用于Cu2+检测的响应位点。
根据相关报道文献推测本发明的探针TPIA与Cu2+发生作用时可能的反应机制是:在Cu2+存在的情况下,TPIA的C=N键会提供孤对电子进行配位,得到另一种新的化合物,由此产生荧光的变化。如图1所示。
本发明提供了一种新的TPI衍生物荧光探针TPIA,丰富了Cu2+类荧光探针的种类,为有机分析和光化学提供了新型的探针分子,可广泛应用于荧光分析或检测领域。一方面,该新型荧光分子探针实现了对Cu2+的高灵敏检测,其检测限为0.067μM。另一方面,该荧光分子探针可以实现在HeLa细胞中对Cu2+的检测和成像。
附图说明
图1为探针分子TPIA检测Cu2+的机理图。
图2为实施例2中TPIA在MeCN溶液中紫外及荧光图。
图3为实施例3中TPIA在MeCN溶液中对不同浓度Cu2+的荧光图。
图4为实施例4中TPIA在MeCN溶液中对其他不同类型干扰物的荧光图。
图5为实施例5中TPIA在MeCN溶液中对Cu2+检测动力学图。
图6为实施例6中TPIA在HeLa细胞中对Cu2+的成像图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
下述实施例中所用的材料、试剂和未注明具体条件的实验方法,如无特殊说明,均为按照本领域常规条件。
实施例1
TPI衍生物荧光探针的合成
第一步、化合物4-(4,5-二苯基-1H-咪唑-2-基)苯甲醛的制备
将苯偶酰(0.627g,2.98mmol)、对苯二甲醛(0.537g,4mmol)和醋酸铵(3.45g,44.76mmol)溶于50ml醋酸,120℃回流5h。反应完全后,冷却至室温,倒入冰水(50ml),静置1h,抽滤得粗产物,干燥。粗产物用乙酸乙酯:二氯甲烷=1:20(v/v)洗脱得到化合物4-(4,5-二苯基-1H-咪唑-2-基)苯甲醛。产率:42%,0.4071g;纯度99%以上。
1H NMR(400MHz,DMSO-d6)δ13.02(s,1H),10.03(s,1H),8.30(d,J=8.3Hz,2H),8.02(d,J=8.5Hz,2H),7.54(dd,J=13.7,7.0Hz,4H),7.47(t,J=7.3Hz,2H),7.41(t,J=7.1Hz,1H),7.32(t,J=7.4Hz,2H),7.25(t,J=7.3Hz,1H).
第二步、探针分子2-氨基-3-(((E)-4-(4,5-二苯基-1H-咪唑-2-基)亚苄基)氨基)马来腈的制备
称取化合物Ⅱ(0.100g,0.3084mmol)、2,3-二氨基马来腈(0.033g,0.3084mmol)溶于乙醇,加入催化剂量HOAc,80℃回流6h,反应完成后,冷却至室温,析出黄色固体,抽滤,并用乙醇洗涤,干燥得到探针TPIA。产率:62.5%,0.08 0g,纯度99%以上。
1H NMR(400MHz,DMSO-d6)δ12.92(s,1H),8.30(s,1H),8.20–8.11(m,4H),8.02(s,2H),7.59–7.50(m,4H),7.39(s,6H).
实施例2
探针分子TPIA的紫外吸收光谱及荧光光谱性质测试。
测试仪器:PE 950s型紫外光谱仪,日立F7100型分子荧光光谱仪。
实验方法为:将实施例1制得的探针分子TPIA溶解于MeCN溶液中得到1mM的探针母液,4℃冰箱保存,避光。实验测定中用MeCN将溶液稀释成0.01mM的标准液进行测试。
测量时移取3mL探针的MeCN溶液到1cm比色皿中先后进行紫外吸收光谱及荧光光谱测试,如图2所示。结果表明:探针TPIA最强紫外吸收峰出现在390nm左右,荧光发射峰出现在570nm左右。
实施例3
TPIA的MeCN溶液对Cu2+的定量分析。
测试仪器:日立F7100型分子荧光光谱仪。
实验方法为:将实施例1制得的探针分子TPIA溶解于MeCN中得到1mM的探针母液,4℃冰箱保存。Cu2+用二次水配置成0.01M母液,实验测定中将探针溶液稀释成10μM的标准溶液进行测试。
采用标准加入法测试探针分子对Cu2+的荧光响应,移取1mL的探针母液(10μM)至比色皿中,每次加入1μL的Cu2+检测荧光强度变化,Cu2+含量加到25μM不再继续加入,如图3所示,随着Cu2+含量的增加,在460nm处的荧光峰强度不断增强,而在570nm处的荧光峰强度不断减弱,因此,该探针对Cu2+有较高的灵敏度,可用于生物体内微量Cu2+的检测。
实施例4
探针分子TPIA在对其他不同类型干扰物的荧光图。
测试仪器:日立F7100型分子荧光光谱仪。
实验方法为:将实施例1制得的探针分子TPIA溶解于MeCN中得到1mM的探针母液,4℃冰箱保存。取ZnSO4、AgNO3、Co(NO3)2、NiCl2、FeCl3、PbCl2、NaNO3、MnCl2、KCl、CaCl2、MgCl2用二次水配置成0.01M母液。实验测定中将探针溶液稀释成10μM的标准溶液进行测试。
测量时移取1mL探针的MeCN溶液到1cm比色皿中分别滴加100μM的ZnSO4、AgNO3、Co(NO3)2、NiCl2、FeCl3、PbCl2、NaNO3、MnCl2、KCl、CaCl2、MgCl2进行荧光测试。结果如图4所示。结果表明:探针TPIA对Cu2+表现出比率荧光现象,而对于一些生物体内常见的金属阳离子和阴离子几乎没有什么变化,进一步说明探针TPIA具有优异的选择性,能够应用于生物体内。
实施例5
探针分子TPIA在Cu2+存在下的动力学实验图。
测试仪器:日立F7100型分子荧光光谱仪。
实验方法为:将实施例1制得的探针分子TPIA溶解于MeCN中得到1mM的探针母液,4℃冰箱保存。Cu2+用二次水配置成0.01M母液,实验测定中将探针溶液稀释成10μM的标准溶液进行测试。
移取1mL的探针母液(10μM)至比色皿中,设置荧光激发波长为390nm,分别测试探针,探针+Cu2+溶液在不同的时间(0秒、10秒、30秒、60秒、120秒、200秒、300秒、400秒、500秒)荧光强度的变化,如图5所示。实验结果表明,开始探针溶液的荧光强度随时间的增加而增强,探针+Cu2+在200秒内荧光强度达到最高值,后荧光强度趋于稳定,说明该探针响应迅速且稳定性较好。
实施例6
探针分子TPIA在HeLa细胞中对Cu2+的成像研究。
实验方法为:将实施例1制得的探针分子TPIA溶解于MeCN中得到1mM的探针母液,4℃冰箱保存。实验测定中用MeCN将溶液稀释成0.01mM的标准液进行测试。
为了证明探针在生物系统的实际应用,在共聚焦荧光显微镜下对细胞进行了在不同pH值下的生物荧光成像实验。将HeLa细胞接到培养皿中在37℃条件下培养24h,然后将TPIA标准液(10μM)加入到培养皿中,加入不同浓度的Cu2+(0、2、8μM)继续孵育半小时后进行荧光成像,如图6所示。实验结果表明探针分子TPIA在随着Cu2+浓度增加,荧光在不断增强。这些结果表明,探针TPIA可以作为检测细胞内Cu2+的荧光标签进入细胞,因而具有在生物体内检测Cu2+的潜力。
需要说明的是,以上内容仅仅说明了本发明的技术思想,不能以此限定本发明的保护范围,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰均落入本发明权利要求书的保护范围之内。
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