CN112574246B - Zn2+比率荧光探针、制备及应用 - Google Patents
Zn2+比率荧光探针、制备及应用 Download PDFInfo
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- CN112574246B CN112574246B CN202011471554.1A CN202011471554A CN112574246B CN 112574246 B CN112574246 B CN 112574246B CN 202011471554 A CN202011471554 A CN 202011471554A CN 112574246 B CN112574246 B CN 112574246B
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- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/0803—Compounds with Si-C or Si-Si linkages
- C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
- C07F7/0812—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring
- C07F7/0816—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring said ring comprising Si as a ring atom
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- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/0803—Compounds with Si-C or Si-Si linkages
- C07F7/0825—Preparations of compounds not comprising Si-Si or Si-cyano linkages
- C07F7/083—Syntheses without formation of a Si-C bond
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Abstract
Description
技术领域
本发明涉及荧光探针领域,具体涉及一种Zn2+比率荧光探针、制备及应用。
背景技术
在人体中,锌是继铁之后的第二富集的过渡金属,总含量大约为2 g,90%的锌离子与金属蛋白紧密结合在一起,具不完全统计锌离子存在于3000多种蛋白中,具有催化、结构、调节功能,参与诸如基因转录、金属酶调控、神经信号传输等许多生命过程;而松散结合或者游离的锌离子存在各种生物组织中,比如大脑、视网膜、胰腺和肠等。锌是生物体内重要的微量元素,具有重要的生理功能,锌离子新陈代谢的紊乱会造成很多疾病,例如胚胎发育不良、Alzheimer氏病、前列腺癌、糖尿病、肌萎缩性(脊髓)侧索硬化、癫痛症和帕金森氏病等。
鉴于高敏感性、可视化、生物兼容性、无辐射、实时检测等特点,荧光技术已经是生物学、医学领域不可或缺的研究手段。迄今,已有许多锌离子荧光探针被报道,并成功用于活体细胞中锌离子的影像,如日本东京大学的Nagano教授课题组、美国麻省理工学院的Lippard教授课题组以及我国华东理工大学的钱旭红教授课题组所报道的系列锌离子荧光探针。到目前为止,最常见的锌离子荧光探针属于识别型(recognition-based)探针,这类探针通常由荧光团(信号单元)、连接臂和锌离子螯合基团(识别位点)三部分组成,探针由于光诱导的电荷转移(PeT)效应没有荧光,与锌离子作用后引起荧光信号增强。然而,最常使用的锌离子配体,即双(2-吡啶甲基)胺(DPA)、N,N,N',-三(2-吡啶甲基)乙二胺(TPEN)、N,N-二(2-吡啶甲基)乙二胺(DPEN),尽管与Zn2+有强的配位作用,但也容易受到其他离子(如,Cd2+)的干扰,从而造成假信号;此外,基于荧光关-开型探针在定量检测细胞内Zn2+浓度方面易受仪器效率、探针浓度、细胞微环境的影响;另外,在影像细胞内的Zn2+时,一分子探针会螯合一分子Zn2+,有可能会引起细胞功能的紊乱。
本发明开发了一种反应型(reaction-based)Zn2+比率荧光探针,Zn2+与探针作用后可催化探针发生水解反应生成新的荧光团,该探针能通过两个发射峰的自校正而避免这些干扰因素,因此在定量检测方面更具优势。
发明内容
本发明提出了一种Zn2+比率荧光探针、制备及应用,该探针是由硅吡啰红酮染料与N,N,-二(2-吡啶甲基)乙二胺(DPEN,一种Zn2+配体)经化学偶联制得,探针本身的最大发射峰在611 nm处,向探针中加入Zn2+后,Zn2+会与探针的DPEN部分络合,导致硅吡咯红染料9号碳的碳正性增强,络合物会进一步发生水解,最终生成硅吡啰红酮染料,最大发射峰蓝移至511 nm处,从而实现了对Zn2+的比率检测。
实现本发明的技术方案是:
Zn2+比率荧光探针,结构式如下:
所述的Zn2+比率荧光探针的制备方法,步骤如下:在0 ℃、氮气环境中,将硅吡啰红酮溶于CH3CN中,搅拌10 min后逐滴加入三氟甲烷磺酸酐,反应液继续搅拌反应10 min,随后加入N,N-二(2-吡啶甲基)乙二胺,反应混合物室温搅拌过夜;薄层色谱检测反应结束后,减压去除溶剂、柱色谱分离得荧光探针。
所述硅吡啰红酮的制备步骤如下:氮气保护下,将4,4’-亚甲基双(N,N-二甲基苯胺)溶于超干四氢呋喃(约200 mL)中,反应液控温至-78℃后,向上述溶液中逐滴加入正丁基锂的正己烷溶液,并在此温度下搅拌2小时;随后缓慢加入二氯二甲基硅烷,反应液缓慢升至室温并搅拌2小时;将盐酸水溶液小心加入到反应液中,有机相经萃取、洗涤、干燥后得到中间体;将中间体粗溶于丙酮中,反应液控温至-15℃后,缓慢加入高锰酸钾粉末,反应液缓慢升至室温并搅拌2小时,反应液经过滤、干燥、柱色谱分离后得到黄色固体。
所述4,4’-亚甲基双(N,N-二甲基苯胺)、n-BuLi和二氯二甲基硅烷的摩尔比为1:4:1.8。
所述硅吡啰红酮、三氟甲烷磺酸酐和4,4’-亚甲基双(N,N-二甲基苯胺)的摩尔比为1:4:10。
本发明还提供一种荧光探针在锌离子的比率检测中的应用。
检测机理如下:
该探针是锌离子的比率荧光探针(探针的发射峰由620 nm蓝移到511 nm),能通过两个发射峰内在的校正,避免探针浓度、仪器效率等因素造成的检测错误。
本发明的有益效果是:
(1)探针选择性好,不受Cd2+及其他阴阳离子干扰;
(2)利用水解原理实现比率检测,可以定量检测锌离子;
(3)可以实现在活体中Zn2+的比率检测。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为探针的1H NMR图(CDCl3, 600 MHz)。
图2为探针的13C NMR图(CDCl3, 150 MHz)。
图3为探针的HRMS图。
图4为探针在PBS缓冲溶液(10 mM, pH 7.4)中处理60 min的(A)紫外可见光谱图和(B)荧光光谱图;lex = 460 nm。
图5为Job-Plot图,Zn2+与探针的配比为1:1。
图6为探针在PBS缓冲溶液(10 mM, pH 7.4)中与不同浓度的Zn2+作用后的荧光强度随时间的变化图,(A) 0.5 equiv., (B) 1 equiv., (C) 10 equiv., (D) 50 equiv;狭缝,5/10 nm;λex = 460 nm。
图7为荧光滴定图,(A)为随着Zn2+用量的增加,探针(2μM)荧光光谱的变化;(B)为518 nm与610 nm处荧光强度的比值与Zn2+用量的线性关系图。
图8(A1-A2)为探针(2μM)经100μM的不同物质处理后的荧光光谱变化;(B1-B2)为探针(2μM)经100μM的不同物质处理,继续经Zn2+(100μM)处理后的荧光光谱变化。其中,(1)只有探针;(2)Na+;(3)K+;(4)Mg2+;(5)Ca2+;(6)Al2+;(7)Ba2+;(8)Pd2+;(9)Fe2+;(10)Fe3+;(11)Cr2+;(12)Sn2+;(13)Mn2+;(14)Ni2+;(15)Co2+;(16)Cu2+;(17)Cd2+;(18)Zn2+。
图9为探针(0-100μM)对A549细胞的毒性试验。
图10为A549细胞经探针(2μM)处理不同时间的共聚焦成像图。绿色通道:激发波长为405 nm,收集波长为410-480 nm;红色通道:激发波长为488 nm,收集波长为500-700 nm。
图11为探针(2μM)影像A549细胞中外源性及内源性Zn2+的共聚焦成像图;(i)为细胞仅孵化探针(2 μM,10 min);(ii)为细胞经Zn2+/pyrithione(200 μM)预处理30 min后,继续孵化探针(2 μM,10 min);(iii)为细胞经Zn2+/pyrithione(200 μM)以及TPEN(200 μM)预处理30 min后,继续孵化探针(2 μM,10 min)(iv)为细胞经H2O2(500 μM)预处理6 h后,继续孵化探针(2 μM,10 min);(v)为细胞经H2O2(500 μM)以及TPEN(50 μM)预处理6 h后,继续孵化探针(2 μM,10 min);绿色通道:激发波长为405 nm,收集波长为410-480 nm;红色通道:激发波长为488 nm,收集波长为500-700 nm。
图12中(A)为裸鼠经腹腔注射探针(10μM, 50μL)的活体成像图;(B)为(A)中裸鼠继续经腹腔注射Zn2+(100μM, 50μL)后活体成像图;绿色通道(430 nm/535 nm),红色通道(470 nm/600 nm)。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
荧光探针的制备方法,步骤如下:
(1)硅吡啰红酮制备
氮气保护下,将4,4’-亚甲基双(N,N-二甲基苯胺)(6.00 g, 14.6 mmol)溶于超干四氢呋喃(约200 mL)中,反应液控温至-78℃后,向上述溶液中逐滴加入正丁基锂(正己烷溶液,24.3 mL, 58.3 mmol),在此温度下搅拌2小时;随后缓慢加入二氯二甲基硅烷(3.2mL, 26.28 mmol),反应液缓慢升至室温并搅拌2小时;将盐酸水溶液小心加入到反应液中,有机相经萃取、洗涤、干燥后得到中间体;将中间体粗溶于丙酮中,反应液控温至-15℃后,缓慢加入高锰酸钾粉末(5.75g),反应液缓慢升至室温并搅拌2小时,反应液经过滤、干燥、柱色谱分离(CH2Cl2)后得到黄色固体(1.65 g, 产率为34.7%);
1H NMR (600 Hz, CDCl3) δ8.44 (d, J = 9.0 Hz, 2H), 6.87(d, J = 9.0 Hz,2H), 6.83(s, 2H), 3.11 (s, 12H), 0.49 (s, 6H); 13C NMR (150 MHz, CDCl3) δ185.3, 151.4, 140.5, 131.6, 129.7, 114.3, 113.2, 40.1, 0.97; ESI-MS[M+H]+:calcd for 325.1736, Found 325.1734.
(2)探针的制备
在0 ℃、氮气环境中,将硅吡啰红酮(0.1 g, 0.3 mmol)溶于CH3CN(10 mL)中,搅拌10 min后逐滴加入三氟甲烷磺酸酐(200μL, 1.2 mmol),反应液继续搅拌反应10 min,随后加入N,N-二(2-吡啶甲基)乙二胺(0.726 g, 3 mmol),反应混合物室温搅拌过夜;薄层色谱检测反应结束后,减压去除溶剂、柱色谱分离(MeOH/CH2Cl2 = 20/1)得荧光探针(80 mg,产率为44.3%)。
1H NMR (600 Hz, CD3Cl) δ 8.15 (d, J = 3.6 Hz, 2H), 8.03 (d, J = 8.4Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.58 (m, 2H), 7.38 (d, J = 7.8 Hz, 2H),7.10 (m, 2H), 6.91 (d, J = 3.0 Hz, 2H), 6.82 (m, 2H), 4.08 (t, J = 6.0 Hz,2H), 4.03 (s, 4H), 3.11 (s, 12H), 2.98 (t, J = 6.0 Hz, 2H), 0.45 (s, 6H);13CNMR (150 MHz, CD3Cl) δ 171.7, 157.9, 151.8, 151.7, 148.8, 141.9, 139.4,137.2, 131.9, 130.3, 127.9, 125.2, 123.6, 122.6, 120.3, 119.8, 116.1, 115.3,112.8, 111.8, 58.9, 50.6, 47.8, 40.1, 39.9, -2.1; ESI-MS: [M]+ calcd for549.3156, Found 549.3126.
性能测试
溶液配制
将探针用乙腈配成2 mM的储液,随后用20 mM的PBS(pH 7.4)稀释至相应浓度;阴阳离子分别来自其对应的钠盐或者硫酸盐,并用去离子水配成20 mM的储液。
细胞培养和荧光成像
HeLa细胞培养在37℃、含5%的二氧化碳的培养箱中,培养基为DMEM(高糖)并包含10%的胎牛血清、100 U /mL青霉素G钠和100 μg/ mL链霉素;进行细胞影像实验前,预先将细胞置于30 mm的玻底细胞培养皿上,静置12小时待细胞贴壁,用磷酸缓冲液(PBS)洗涤细胞3次,然后使用Ceiss LMS 880共聚焦显微镜进行荧光成像。绿色通道:激发波长为405nm,收集波长为410-480 nm;红色通道:激发波长为488 nm,收集波长为500-700 nm。
细胞毒性试验
采用CCK-8细胞增殖实验研究探针的细胞毒性。简单地说,首先将贴壁生长的Hela细胞消化为细胞悬液,细胞以每孔8.0×103个细胞的密度接种于96孔板中,用100 μL的DMEM培养基孵育过夜至细胞贴壁。然后将探针的储液(2 mM)加入到上述培养基中,终浓度保持在0 ~ 10 μM,对照及测试组均重复6次,细胞培养24 h后,弃去旧培养基,PBS洗涤2次,更换含10% CCK-8的新鲜培养基,孵育0.5 h,用iMark TM MicroplateAbsorbance Reader测定450 nm处的吸光度。
活细胞中Zn2+成像研究
在影像细胞中外加Zn2+的实验中,HeLa细胞首先用Zn2+和羟基吡啶硫酮(Pyrithione,Zn2+载体)混合液(200 μM)预处理20分钟(或者同时加入200 μM的TPEN),然后用探针(2 μM)处理10分钟,PBS洗涤三次后,进行荧光影像;在影像细胞中内生Zn2+的实验中,HeLa细胞用500 μM的H2O2预处理6小时(或者同时加入50 μM的TPEN),然后用探针(2 μM)处理10分钟,PBS洗涤三次后,进行荧光影像。
活体中Zn2+成像研究
所有的动物实验都是按照山西大学伦理委员会颁布的相关法律和指南进行的,BALB/c雄性裸鼠(6-8周龄)购自北京维通利华实验动物技术有限公司。探针(10 μM)经腹腔注射到裸鼠体内后,立即影像;随后继续经腹腔注射Zn2+(100 μM),1 h后影像;活体动物成像是在Bruker多模式活体成像系统中进行的,选择430 nm的激发滤光片和535 nm的发射滤光片(绿色通道)及470 nm的激发滤光片和600 nm的发射滤光片(红色通道)。
试验结果
探针的稳定性
由于探针在水中具有良好的溶解性,故体外测试体系均选取纯PBS缓冲体系。首先在PBS(20 mM, pH 7.4)中研究探针的稳定性,如图4所示,在PBS(20 mM, pH 7.4)中,探针(2 μM)的最大吸收和最大发射峰值分别为460 nm和610 nm,在该体系中动态检测1 h后,探针的吸收峰和发射峰均未发生明显变化,说明该探针在PBS体系中具有较强的稳定性。
探针与Zn2+的反应性能研究
在相同条件下,进一步研究了探针与不同浓度Zn2+反应前后荧光光谱的变化,如图5所示,探针(2 μM)的最大发射峰值为610 nm,向该溶液中分别加入1 μM(图6A)、2 μM(图6B)、20 μM(图6C)和100 μM(图6D)的Zn2+后,610 nm处的发射峰迅速下降,518 nm处出现新的发射峰,该发射峰的荧光强度逐渐上升且在1 h后趋于稳定,说明探针与Zn2+的螯合速度非常快,而进一步的水解速度较慢。根据Job-Plot分析法可知,探针与Zn2+的螯合比为1:1(图5)。荧光滴定实验表明,Zn2+的浓度与I518 nm/I610 nm呈良好的线性关系,检测限低至5.36μM(图7)。选择性实验表明,仅有Zn2+可以引起探针显著的比率变化,而顺磁性的Ni2+、Co2+、Cu2+和Cd2+尽管会导致探针荧光猝灭,但不会在580 nm处出现新峰,向该溶液中继续加入Zn2 +后,也不会引起荧光光谱的明显变化,说明这几种离子可以和探针发生配位导致荧光猝灭,但不能发生随后的水解反应;其他阳离子不与探针发生反应,向该溶液中继续加入Zn2+后,同样会引起荧光光谱明显的比率变化,说明这些离子的存在不会影响探针对Zn2+的传感(图8)。
细胞中Zn2+的影像研究
在进行细胞成像之前,我们首先评估了探针的细胞毒性,CCK-8细胞增殖实验研究结果显示,该探针在0-60 μM的浓度范围内具有较低的细胞毒性,细胞存活率大于85%,说明该探针具有可以忽略的细胞毒性(图9)。随后,用2 μM探针孵化HeLa细胞以测试其渗透细胞的能力,如图9所示,由于具有合适的亲水亲油性,该探针可以迅速透过细胞膜,红色通道的荧光强度在约10分钟时可达到饱和(图10)。
接下来,在HeLa细胞中检测探针影像外源性及内源性Zn2+的能力。如图11所示,当预先孵化探针(2 μM)的HeLa细胞分别用405 nm和488 nm激发时,在红色通道具有强的荧光信号,绿色通道的荧光信号几乎可以忽略;当HeLa细胞预先孵化Zn2+和羟基吡啶硫酮(Pyrithione,Zn2+载体)混合液(200 μM),再孵化探针(2 μM)时,绿色通道荧光强度明显增强,红色通道荧光强度明显升高;当HeLa细胞预先孵化Zn2+和羟基吡啶硫酮(Pyrithione,Zn2+载体)混合液(200 μM)以及200 μM的Zn2+螯合剂(TPEN),再孵化探针(2 μM)时,不会引起绿色通道荧光的明显增加,说明绿色和红色通道的信号变化是由Zn2+引起的。
文献报道H2O2诱导细胞凋亡后可释放出Zn2+,接下来,我们模拟该过程以验证探针是否可以影像内源性Zn2+,如图11(iv-v)所示,当HeLa细胞预先被H2O2诱导6 h,继续孵化探针后,可以明显观察到绿色通道荧光增强以及红色通道荧光减弱;当HeLa细胞预先被H2O2以及TPEA处理6 h,再继续孵化探针后,绿色及红色通道的荧光强度均未观察到明显的改变。
上述结果表明,该探针具有良好的细胞膜通透性,并且可以影像细胞内源性及外源性的Zn2+。
活体中Zn2+的影像研究
为了验证探针的实际应用价值,我们在裸鼠腹腔内验证了探针对Zn2+的影像能力。如图12所示,首先,探针(10 μM)经腹腔注射到裸鼠体内,红色通道有明显的荧光信号,绿色通道仅有微弱的荧光信号;随后继续经腹腔注射Zn2+(100 μM),1 h后的影像结果显示,红色通道的荧光信号明显降低,绿色通道表现出微弱的荧光增强,可能的原因是绿色通道采用的激发光波长以及产物的发射波长均较短,组织的穿透性差。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
2.权利要求1所述的Zn2+比率荧光探针的制备方法,其特征在于步骤如下:在0 ℃、氮气环境中,将硅吡啰红酮溶于CH3CN中,搅拌10min后滴加三氟甲烷磺酸酐,搅拌反应10min加入N,N,-二(2-吡啶甲基)乙二胺,反应混合物室温搅拌过夜;薄层色谱检测反应结束后,减压去除溶剂、柱色谱分离得荧光探针。
3.根据权利要求2所述的Zn2+比率荧光探针的制备方法,其特征在于,所述步骤(1)中硅吡啰红酮的制备步骤如下:氮气保护下,将4,4’-亚甲基双(N,N-二甲基苯胺)溶于超干四氢呋喃中,反应液控温至-78℃后,向上述溶液中逐滴加入正丁基锂的正己烷溶液,在此温度下搅拌2小时;随后缓慢加入二氯二甲基硅烷,反应液缓慢升至室温并搅拌2小时;将盐酸水溶液加入到反应液中,有机相经萃取、洗涤、干燥后得到中间体;将中间体溶于丙酮中,反应液控温至-15℃后,缓慢加入高锰酸钾粉末,反应液缓慢升至室温并搅拌2小时,反应液经过滤、干燥、柱色谱分离后得到黄色固体。
4.根据权利要求3所述的Zn2+比率荧光探针的制备方法,其特征在于:所述4,4’-亚甲基双(N,N-二甲基苯胺)、n-BuLi和二氯二甲基硅烷的摩尔比为1:4:1.8。
7.根据权利要求2所述的Zn2+比率荧光探针的制备方法,其特征在于:所述硅吡啰红酮、三氟甲烷磺酸酐和N,N,-二(2-吡啶甲基)乙二胺的摩尔比为1:4:10。
8.权利要求2-7任一项制备方法制备得到的荧光探针在锌离子的比率检测中的应用。
9.根据权利要求8所述的应用,其特征在于:荧光探针在检测锌离子时,发射峰由620nm蓝移到511nm。
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