CN114426534A - 一种检测铜离子的可逆荧光探针及其制备方法 - Google Patents
一种检测铜离子的可逆荧光探针及其制备方法 Download PDFInfo
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- CN114426534A CN114426534A CN202111532544.9A CN202111532544A CN114426534A CN 114426534 A CN114426534 A CN 114426534A CN 202111532544 A CN202111532544 A CN 202111532544A CN 114426534 A CN114426534 A CN 114426534A
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- copper ions
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- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims abstract description 8
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims abstract description 8
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 claims abstract description 4
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
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- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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Abstract
本发明公开了一种检测铜离子的可逆荧光探针及其制备方法,所述制备方法为:先基于含喹啉基团的化合物制得中间产物6‑(杂氮环丁烷)喹啉‑2‑羧酸甲酯,再将中间产物6‑(杂氮环丁烷)喹啉‑2‑羧酸甲酯与水合肼反应得到目标产物。本发明可逆荧光探针选择性高,只对铜离子响应,对其它金属离子无响应;同时对铜离子具有高的敏感性,在体外和体内均能瞬间和Cu2+反应,对Cu2+检出限低至0.101μM;另外本发明可逆荧光探针通过荧光猝灭机制检测Cu2+,然后在溶液中加入H2S以增加荧光,这种带有“on‑off‑on”型荧光识别系统的探针在Cu2+和H2S的实时识别过程中表现出优异的抗干扰性能,并且还显示出对活细胞中Cu2+和H2S的敏感性和选择性。
Description
技术领域
本发明涉及一种检测铜离子的可逆荧光探针,还涉及上述可逆荧光探针的制备方法。
技术背景
铜作为生命不可缺少的元素,在生物体的各种生理过程中发挥着重要作用。脑内铜的稳态与多种疾病有关,缺铜会引起贫血、动脉异常等疾病,但过量接触铜会引起神经退行性疾病,如阿尔茨海默病、威尔逊病、帕金森、肌萎缩侧索硬化症、门克斯病。随着工业的不断发展,铜被广泛应用于各种工业生产中。同时,它也给环境和人类生命健康带来了潜在危害。小分子荧光探针用于检测环境中的铜含量具有选择性好、毒性低、容易制备等优点,荧光探针已成为生物和环境问题检测的重要诊断工具。
近年来,关于铜离子荧光探针的报道有很多,其中一些淬灭型荧光探针已经成功应用到生物领域。但是大部分探针都存在对特异性物质选择性不高、响应时间长、溶解度低的问题。此外,有些探针只能应用在非水溶剂中,这就限制了它在环境和生物体这样的水相环境中的应用。
发明内容
发明目的:本发明目的之一是提供一种能在细胞中应用、高选择性、高灵敏度、能够快速识别生物体和环境中铜离子的荧光探针;本发明另一目的是提供上述检测铜离子的可逆荧光探针的制备方法。
技术方案:本发明所述的检测铜离子的可逆荧光探针,所述化合物的结构式为:
上述检测铜离子的可逆荧光探针的制备方法,所述制备方法为:先基于含喹啉基团的化合物制得中间产物6-(杂氮环丁烷)喹啉-2-羧酸甲酯,再将中间产物6-(杂氮环丁烷)喹啉-2-羧酸甲酯与水合肼反应得到目标产物。
其中,所述中间产物6-(杂氮环丁烷)喹啉-2-羧酸甲酯采用如下方法制备而成,包括如下步骤:
(1)往6-溴-2-甲基喹啉的吡啶溶液中加入二氧化硒,在高温加热过夜后,旋干溶剂,将所得混合物先用水洗,除去残留吡啶,再用甲醇洗,洗去杂质,烘干得到6-溴喹啉-2-羧酸;
(2)冰浴下,将6-溴喹啉-2-羧酸溶解于有机溶剂中,再往其中滴加氯化亚砜,混合物回流过夜,将反应后冷却至室温的悬浮液倒入饱和碳酸氢钠水溶液中,将所得混合物用有机溶剂萃取,萃取后的有机萃取液用无水硫酸钠干燥并过滤,然后真空蒸发除去溶剂,得到6-溴喹啉-2-羧酸甲酯;
(3)在氩气下,将6-溴喹啉-2-羧酸甲酯、碳酸铯、醋酸钯和BINAP(1,1'-联萘-2,2'-双二苯膦)混合,将混合物在高温下搅拌过夜,反应后冷却至室温,用乙酸乙酯和水萃取反应混合物;合并有机萃取液;有机萃取液用无水硫酸钠干燥并过滤,然后真空蒸发除去溶剂,得到6-(杂氮环丁烷)喹啉-2-羧酸甲酯。
其中,所述中间产物6-(杂氮环丁烷)喹啉-2-羧酸甲酯与水合肼的反应温度为80~82℃,反应后冷却至室温,将混合物倒入冰中,过滤固体即可。
其中,所述中间产物6-(杂氮环丁烷)喹啉-2-羧酸甲酯与水合肼的摩尔比为0.75:40。
其中,步骤(1)中,6-溴-2-甲基喹啉与二氧化硒的反应摩尔比为0.45:1,反应温度为100~105℃。
其中,步骤(2)中,6-溴喹啉-2-羧酸与氯化亚砜的反应摩尔比为5:6。
其中,步骤(3)中,对于每mmol 6-溴喹啉-2-羧酸甲酯,加入144mg Cs2CO3、8mg Pd(OAc)2和24mg BINAP与6-溴喹啉-2-羧酸甲酯进行反应。
本发明的反应方程式为:
本发明化合物检测铜离子的机理为:本发明化合物以喹啉环为骨架,并利用末端酰肼基团和铜离子螯合引起荧光猝灭,从而实现对铜离子的检测,由于酰肼上的N原子通过和铜离子鳌合形成多元环,减弱喹啉醛荧光团的推挽电子效应,从而导致荧光猝灭;在硫化氢存在下,通过硫化氢与铜离子重新结合生成硫化铜沉淀,从而导致荧光恢复。
有益效果:相比于现有技术,本发明具有的显著效果为:(1)本发明可逆荧光探针选择性高,只对铜离子响应,对其它金属离子无响应;(2)本发明可逆荧光探针对铜离子具有高的敏感性,在体外和体内均能瞬间和Cu2+反应,对Cu2+检出限低至0.101μM;(3)本发明可逆荧光探针通过荧光猝灭机制检测Cu2+,然后在溶液中加入H2S以增加荧光,这种带有“on-off-on”型荧光识别系统的探针在Cu2+和H2S的实时识别过程中表现出优异的抗干扰性能,并且还显示出对活细胞中Cu2+和H2S的敏感性和选择性;(4)本发明可逆荧光探针斯托克斯位移大(斯托克斯位移指探针激发和发射波长的差值,位移越大,抗干扰效果越好,能避免激发光对发射信号的干扰,且对生物样品损伤小,样品穿透性强),脂溶性好。
附图说明
图1为CuP-1、CuP-2和CuP-3本身和加入Cu2+后的激发、发射光谱以及对不同金属离子的选择性的光谱图;
图2为本发明CuP-1在体外检测不同浓度Cu2+和H2S的荧光光谱强度变化示意图;
图3为在长波长紫外光下,在PBS缓冲液中添加各种金属离子后,CuP-1的荧光强度变化拍摄的照片;
图4为在长波长紫外光下,CuP-1相对于不同浓度(0-10μM)Cu2+的荧光强度变化拍摄的照片;
图5为CuP-1与Cu2+(0-3μM)浓度之间的线性相关性示意图;
图6为含有Cu2+的CuP-1与NaHS(0-40μM)浓度之间的线性相关性示意图;
图7为不存在(黑色条)HS-和存在(红色条)HS-(50μM)情况下,添加Cu2+和不同阴离子(50μM)后,CuP-1(5μM)的荧光发射光谱示意图;
图8为存在或不存在Cu2+(5μM)和NaHS(100μM)、Na2S4(100μM)、Cys(200μM)、NAC(200μM)、Hcy(50μM)、GSH(5mM)的情况下,CuP-1(5μM)的荧光光谱示意图;
图9为在长波长紫外光下,含有Cu2+的CuP-1相对于不同硫醇类物质的荧光强度变化拍摄的照片;
图10为在不同pH值下,CuP-1对Cu2+的荧光响应示意图;
图11为CuP-1在HepG2细胞的细胞毒性;
图12A为CuP-1在HT22细胞中加入不同浓度铜离子和H2S处理后的荧光成像;
图12B为图2A的定量分析数据示意图;
图13为实施例1制得的CuP-1的1H NMR谱图;
图14为实施例1制得的CuP-1的13CNMR谱图;
图15为实施例1制得的CuP-1的质谱图(HR-MS);
图16为实施例2制得的CuP-2的1H NMR谱图;
图17为实施例2制得的CuP-2的13CNMR谱图;
图18为实施例2制得的CuP-2的质谱图(HR-MS);
图19为实施例3制得的CuP-3的1H NMR谱图;
图20为实施例3制得的CuP-3的13CNMR谱图;
图21为实施例3制得的CuP-3的质谱图(HR-MS)。
具体实施方式
以下结合具体实施例对本发明的技术方案做进一步说明。
实施例1
本发明检测铜离子的可逆荧光探针的合成方法,包括如下步骤:
(1)向6-溴-2-甲基喹啉(100mg,0.45mmol)的吡啶(5mL)溶液中加入二氧化硒(110mg,1.0mmol),在100℃加热过夜后,浓缩滤液(旋干溶剂);将所得混合物先用水洗,除去残留吡啶,再用甲醇洗,洗去杂质,烘干得到6-溴喹啉-2-羧酸;
(2)将6-溴喹啉-2-羧酸(10.0mmol)溶解在无水甲醇中并置于冰浴中,然后往其中滴加SOCl2(0.92mL,12.0mmol),将得到的混合物回流过夜,直到TLC分析检测不到起始材料;将冷却至室温的悬浮液小心倒入饱和NaHCO3水溶液中,所得混合物用CH2Cl2萃取两次,合并有机萃取液,用无水Na2SO4干燥并过滤,然后通过真空蒸发除去溶剂,所得残渣经柱层析纯化得到相应的化合物3:6-溴喹啉-2-羧酸甲酯;
(3)在氩气下,将6-溴喹啉-2-羧酸甲酯(1.0mmol)、Cs2CO3(144mg,1.50mmol)、Pd(OAc)2(8mg,3mol%)和BINAP(24mg,4mol%)混合置于Schlenk管中,反应过程中,Pd盐是催化剂,加快反应进行,BINAP用来活化Pd盐,碱也是催化剂,用于活化底物;将Schlenk管抽真空并用氩气冲洗,然后通过隔膜通过注射器往Schlenk管中加入无水甲苯(10mL)和氮杂环丁烷(114mg,2.0mmol),用螺旋盖密封Schlenk管,并将混合物在100℃下搅拌过夜,直到在TLC分析中检测不到起始材料;反应后冷却至室温,用乙酸乙酯和水萃取反应混合物;合并有机萃取液,用无水Na2SO4干燥并过滤,然后通过真空蒸发除去溶剂;所得残留物通过柱色谱纯化得到化合物4:6-(杂氮环丁烷)喹啉-2-羧酸甲酯;
(4)将6-(杂氮环丁烷)喹啉-2-羧酸甲酯(0.75mmol)、无水乙醇(5mL)和质量百分数为98%的水合肼(40mmol)混合,混合溶液在80℃下搅拌4小时并通过TLC监测;反应后冷却至室温,将混合物倒入冰中,过滤固体,得到CuP-1。得到的化合物CuP-1结构通过1H(图13)和13C NMR(图14)光谱以及质谱(HR-MS)(图15)充分表征。1H NMR(400MHz,DMSO-d6)δ9.77(s,1H),8.18(d,J=14.8Hz,1H),7.91(d,J=15.0Hz,2H),7.13(d,J=15.9Hz,1H),6.68(s,1H),4.56(s,2H),3.99(t,J=35.5Hz,4H),2.43-2.33(m,2H).13C NMR(101MHz,CDCl3)δ165.59,150.58,144.50,140.84,134.42,131.15,130.60,119.15,118.47,102.75,52.12,52.12,16.66.ESI:m/z cald.for C13H14N4O,242.1[M+H]+,found 243.1.实施例2
一种荧光探针的合成方法,包括如下步骤:
(1)向6-溴-2-甲基喹啉(100mg,0.45mmol)的吡啶(5mL)溶液中加入二氧化硒(110mg,1.0mmol),在100℃加热过夜后,浓缩滤液;将所得混合物先用水洗,除去残留吡啶,再用甲醇洗,洗去杂质,烘干得到6-溴喹啉-2-羧酸;
(2)将6-溴喹啉-2-羧酸(10.0mmol)溶解在无水甲醇中并置于冰浴中,然后往其中滴加SOCl2(0.92mL,12.0mmol),将得到的混合物回流过夜,直到TLC分析检测不到起始材料;将冷却至室温的悬浮液小心倒入饱和NaHCO3水溶液中,所得混合物用CH2Cl2萃取两次,合并有机萃取液,用无水Na2SO4干燥并过滤,然后通过真空蒸发除去溶剂,所得残渣经柱层析纯化得到相应的化合物3:6-溴喹啉-2-羧酸甲酯;
(3)在氩气下,将6-溴喹啉-2-羧酸甲酯(1.0mmol)、Cs2CO3(144mg,1.50mmol)、Pd(OAc)2(8mg,3mol%)和BINAP(24mg,4mol%)混合置于Schlenk管中;将Schlenk管抽真空并用氩气冲洗,然后通过隔膜通过注射器往Schlenk管中加入无水甲苯(10mL)和氮杂环丁烷(114mg,2.0mmol),用螺旋盖密封Schlenk管,并将混合物在100℃下搅拌过夜,直到在TLC分析中检测不到起始材料;反应后冷却至室温,用乙酸乙酯和水萃取反应混合物;合并有机萃取液,用无水Na2SO4干燥并过滤,然后通过真空蒸发除去溶剂;所得残留物通过柱色谱纯化得到化合物4:6-(杂氮环丁烷)喹啉-2-羧酸甲酯;
(4)将6-(杂氮环丁烷)喹啉-2-羧酸甲酯(1.0mmol)溶解在10mL甲醇中,并用1.0mL10eqNaOH溶液处理;搅拌24小时后,蒸发挥发物;向水相中滴加盐酸酸化至pH<1,过滤沉淀,用水洗涤,干燥,得到化合物5:6-(杂氮环丁烷)喹啉-2-羧酸;
(5)往6-(杂氮环丁烷)喹啉-2-羧酸(1.0mmol)的DMF(10mL)溶液中加入EDC·HCl(1.1mmol)、HOBt(1.1mmol)和三乙胺(1.1mmol),然后再加入2-肼基吡啶(1.0mmol),混合物在氩气气氛下,常温下搅拌反应24小时;反应后将混合物料倒入H2O(150mL)中,并用乙酸乙酯(30mL×3)萃取;合并有机相,分别用饱和NaHCO3(30mL×2)和饱和NaCl(30mL×1)洗涤,再用Na2SO4干燥;将溶液真空浓缩并通过硅胶柱色谱纯化,得到化合物CuP-2。CuP-2结构通过1H(图16)和13C NMR(图17)光谱以及质谱(HR-MS)(图18)充分表征。1H NMR(400MHz,DMSO-d6)δ10.42(s,1H),8.56(s,1H),8.19(s,1H),8.06(s,1H),7.87(d,J=34.5Hz,2H),7.51(s,1H),7.30(s,1H),6.88–6.51(m,3H),4.00(d,J=7.2Hz,2H),3.80(s,1H),3.29(s,2H),2.39(s,1H),2.09(s,1H).13C NMR(101MHz,CDCl3)δ164.38,159.39,150.68,147.97,144.16,140.84,138.12,134.43,131.39,130.75,119.39,118.53,116.38,107.21,102.69,52.08,29.72,16.65.ESI:m/zcald.for C18H17N5O,319.2[M+H]+,found 320.2.
制备化合物CuP-2的化学反应方程式为:
实施例3
一种荧光探针的合成方法,包括如下步骤:
(1)向6-溴-2-甲基喹啉(100mg,0.45mmol)的吡啶(5mL)溶液中加入二氧化硒(110mg,1.0mmol),在100℃加热过夜后,浓缩滤液;将所得混合物先用水洗,除去残留吡啶,再用甲醇洗,洗去杂质,烘干得到6-溴喹啉-2-羧酸;
(2)将6-溴喹啉-2-羧酸(10.0mmol)溶解在无水甲醇中并置于冰浴中,然后往其中滴加SOCl2(0.92mL,12.0mmol),将得到的混合物回流过夜,直到TLC分析检测不到起始材料;将冷却至室温的悬浮液小心倒入饱和NaHCO3水溶液中,所得混合物用CH2Cl2萃取两次,合并有机萃取液,用无水Na2SO4干燥并过滤,然后通过真空蒸发除去溶剂,所得残渣经柱层析纯化得到相应的化合物3:6-溴喹啉-2-羧酸甲酯;
(3)在氩气下,将6-溴喹啉-2-羧酸甲酯(1.0mmol)、Cs2CO3(144mg,1.50mmol)、Pd(OAc)2(8mg,3mol%)和BINAP(24mg,4mol%)混合置于Schlenk管中;将Schlenk管抽真空并用氩气冲洗,然后通过隔膜通过注射器往Schlenk管中加入无水甲苯(10mL)和氮杂环丁烷(114mg,2.0mmol),用螺旋盖密封Schlenk管,并将混合物在100℃下搅拌过夜,直到在TLC分析中检测不到起始材料;反应后冷却至室温,用乙酸乙酯和水萃取反应混合物;合并有机萃取液,用无水Na2SO4干燥并过滤,然后通过真空蒸发除去溶剂;所得残留物通过柱色谱纯化得到化合物4:6-(杂氮环丁烷)喹啉-2-羧酸甲酯;
(4)将6-(杂氮环丁烷)喹啉-2-羧酸甲酯(1.0mmol)溶解在10mL甲醇中,并用1.0mL10eqNaOH溶液处理。搅拌24小时后,蒸发挥发物。向水相中滴加盐酸酸化至pH<1。过滤沉淀,用水洗涤,干燥,得到化合物5:6-(杂氮环丁烷)喹啉-2-羧酸;
(5)往6-(杂氮环丁烷)喹啉-2-羧酸(1.0mmol)的DMF(10mL)溶液中加入EDC·HCl(1.1mmol)、HOBt(1-羟基苯并三唑)(1.1mmol)和三乙胺(2mmol),然后再加入8-氨基喹啉(1.2mmol),混合物在氩气气氛下,常温下搅拌反应24小时;反应后将混合物料倒入H2O(150mL)中,并用乙酸乙酯(30mL×3)萃取;合并有机相,分别用饱和NaHCO3(30mL×2)和饱和NaCl(30mL×1)洗涤,再用Na2SO4干燥;将溶液真空浓缩并通过硅胶柱色谱纯化,得到化合物CuP-3。CuP-3结构通过1H(图19)和13C NMR(图20)光谱以及质谱(HR-MS)(图21)充分表征。1HNMR(400MHz,DMSO-d6)δ12.24(s,1H),9.08(d,J=8.9Hz,1H),8.91(d,J=6.6Hz,1H),8.48(d,J=8.9Hz,1H),8.29(d,J=6.1Hz,1H),8.18(d,J=8.4Hz,1H),8.03(d,J=6.4Hz,1H),7.77-7.65(m,3H),7.22(d,J=8.4Hz,1H),6.74(s,1H),4.04(t,J=12.3Hz,4H),2.43-2.37(m,2H).13C NMR(101MHz,CDCl3)δ163.61,150.63,148.65,146.02,140.93,139.48,136.19,134.90,134.58,131.27,131.13,128.18,127.38,121.64,121.58,119.32,118.26,116.63,102.77,52.13,29.73,16.68.ESI:m/z cald.for C22H18N4O,354.2[M+H]+,found 355.2.
制备化合物CuP-3的化学反应方程式为:
对实施例1~3制得的CuP-1、CuP-2、CuP-3的激发、发射光谱以及对不同金属离子的选择性进行测试:
为了检查三种化合物对Cu2+的响应,首先通过荧光分光光度法分别测量Cu2+不存在和Cu2+存在时的激发光谱和发射光谱,三个探针在浓度为20mM的HEPES缓冲液(pH7.4,0.5%DMSO)中表现出强荧光。将Cu2+(2.0当量)引入三种化合物的溶液中会导致几乎完全的荧光猝灭,这表明这些探针可以通过荧光“开-关”行为识别Cu2+,如图1a、1b和1c所示。
测试CuP-1、CuP-2和CuP-3在包含各种金属离子(Na+、K+、Ag+、Zn2+、Al3+、Cr3+、Pb2+、Fe3+、Co2+、Ca2+、Fe2+、Cd2+、Mg2+、Mn2+、Cu+、Cu2+)中对Cu2+的特异性选择性:如图1d、1e和1f所示,在20mM HEPES缓冲液中使用相同浓度的CuP-1、CuP-2和CuP-3,只有CuP-1表现出对Cu+和Cu2+的特异性反应,如图3所示。CuP-2和CuP-3对Cu+和Cu2+均没有特异性响应,CuP-2同时还对Zn2+、Co2+、Fe2+、Cd2+表现出响应;CuP-3同时还对Co2+、Fe2+和Cd2+表现出响应。
表1为三种探针的光谱特性,通过表1可知,相比于CuP-2和CuP-3,CuP-1具有优异的光学性能。
表1
CuP-1对Cu2+浓度依赖响应:
CuP-1对Cu2+的响应呈浓度依赖性模式,540nm处发射强度随着Cu2+浓度的增加而逐渐降低,并在1.0equiv时达到饱和状态。将Cu2+添加到CuP-1溶液中,如图2a和2c所示,使用紫外光可以清楚地看到荧光强度的变化,如图4所示。探针可以在540nm的荧光强度和Cu2+浓度(0-3μM)之间建立线性关系(R2=0.98523),如图5所示,检测限为0.101μM(3σ/slope)。
CuP-1的荧光量子产率在纯甲醇中测定,以罗丹明B(10μM,Φ=0.89,λex=495nm)作为参考,量子产率使用如下公式I计算:
Φu=[(AsFun2)/(AuFsn02)]Φs;
其中,As和Au是参考和样品溶液在参考激发波长下的吸光度,Fs和Fu是相应的积分荧光强度,n和n0是样品的溶剂折射率;所有荧光测量都是针对吸光度接近0.05的稀释溶液获得的,用上式计算CuP-1在PBS中的荧光量子产率,量子产率:Φ=0.3331。荧光量子产率越高,发射的荧光越强,将有助于提高检测的灵敏度,使检测极限维持在较低浓度的水平,说明探针的灵敏度高,荧光强度强。
通过摇瓶法测定CuP-1在正辛醇/磷酸盐缓冲体系中的亲脂性:
将CuP-1溶解在NaCl溶液(0.9%w/v,用正辛醇饱和)中,然后加入等体积的辛醇溶液(用0.9%NaCl饱和,w/v),并用振荡器以150rpm室温下旋转9小时,使样品均匀分布在两相中。样品在8000rpm下离心5min,用紫外-可见光测量样品在两相中的吸光度;LogPo/w定义为有机相和水相中样品浓度的对数比,确定Log Po/w为2.44;良好的亲脂性代表探针更容易通过脂质双分子层进入细胞内发挥作用。
CuP-1与Cu2+对H2S浓度依赖响应:
如图2b和2d所示,将NaHS(0-80μM)加入到含有Cu2+(5μM)的CuP-1溶液中后,荧光迅速恢复,因此该探针可用于实时监测生物样品中的硫化氢。此外,CuP-1与Cu2+在540nm处的荧光强度和NaHS浓度(0-40μM)之间建立线性关系,R2=0.9904,如图6所示。
CuP-1对不同阴离子和氨基酸的抗干扰能力:
高选择性是荧光传感器的重要性能,在生理条件下研究在其他阴离子存在下,CuP-1与Cu2+对硫化氢的选择性荧光响应。发现其他阴离子和氨基酸的存在和不存在几乎没有差异,例如Cl-、Br-、CH3COO-、NO2 -、NO3 -、HPO4 2-、S2O5 2-、ClO-、SO4 2-、Thr、Trp、H2O2、Lys、Ile,不会导致荧光强度的显着变化和相应的荧光恢复,但CuP-1可通过在Cu2+溶液中添加NaHS和Na2S4来再生,如图7所示。
CuP-1对pH在4-10之间和Cu2+响应的荧光强度变化:
在不同pH值的HEPES缓冲液(20mM,0.5%DMSO)中,CuP-1(5μM)对Cu2+(5μM)的荧光响应,发现探针在6.0-10.0的pH范围内对Cu2+具有良好的选择性响应,如图10所示,这是因为探针含有-NH2,在酸性条件下容易和酸反应成盐,不与铜离子鳌合。
CuP-1的细胞毒性:
进行MTT测定以评估CuP-1的细胞毒性。HepG2细胞置于96孔板中,37℃、5%CO2气氛培养12h,然后用CuP-1(浓度分别为10、20、30、40、50和100μM)处理细胞并孵育24小时;然后用5μL5mg/mL MTT处理细胞并再孵育4小时,然后弃去上清液后,再加入150μLDMSO,通过MTT进行分析,处理后HepG2细胞存活率仍超过90%,如图11所示。
在HT-22细胞中,检测CuP-1对外源性Cu2+和NaHS是否可在活细胞中成像:使用共聚焦荧光显微镜对细胞进行活细胞成像,对CuP-1是否可显示活细胞中的Cu2+和NaHS进行评估。
如图12A和12B所示,HT-22细胞与10μM CuP-1在37℃培养基中孵育30分钟,在细胞中观察到强烈的荧光。接着,向细胞中分别加入50、100和200μMCu2+后,再孵育2小时,细胞荧光迅速淬灭,几乎没有荧光。然而,在此基础上再分别加入100、200μM NaHS孵育2小时后,细胞的荧光可以快速恢复。因此,这些结果表明,CuP-1具有用于检测活细胞中铜离子的能力,并且它也是一个很好的监测系统,可用于观察活细胞中是否存在硫化物。
Claims (8)
2.权利要求1所述的检测铜离子的可逆荧光探针的制备方法,其特征在于,所述制备方法为:先基于含喹啉基团的化合物制得中间产物6-(杂氮环丁烷)喹啉-2-羧酸甲酯,再将中间产物6-(杂氮环丁烷)喹啉-2-羧酸甲酯与水合肼反应得到目标产物。
3.根据权利要求2所述的检测铜离子的可逆荧光探针的制备方法,其特征在于,所述中间产物6-(杂氮环丁烷)喹啉-2-羧酸甲酯采用如下方法制备而成,包括如下步骤:
(1)往6-溴-2-甲基喹啉的吡啶溶液中加入二氧化硒,在高温加热过夜后,旋干溶剂,将所得混合物先用水洗,除去残留吡啶,再用甲醇洗,洗去杂质,烘干得到6-溴喹啉-2-羧酸;
(2)冰浴下,将6-溴喹啉-2-羧酸溶解于有机溶剂中,再往其中滴加氯化亚砜,混合物回流过夜,将反应后冷却至室温的悬浮液倒入饱和碳酸氢钠水溶液中,将所得混合物用有机溶剂萃取,萃取后的有机萃取液用无水硫酸钠干燥并过滤,然后真空蒸发除去溶剂,得到6-溴喹啉-2-羧酸甲酯;
(3)在氩气下,将6-溴喹啉-2-羧酸甲酯、碳酸铯、醋酸钯和BINAP混合,将混合物在高温下搅拌过夜,反应后冷却至室温,用乙酸乙酯和水萃取反应混合物;合并有机萃取液;有机萃取液用无水硫酸钠干燥并过滤,然后真空蒸发除去溶剂,得到6-(杂氮环丁烷)喹啉-2-羧酸甲酯。
4.根据权利要求2所述的检测铜离子的可逆荧光探针的制备方法,其特征在于:所述中间产物6-(杂氮环丁烷)喹啉-2-羧酸甲酯与水合肼的反应温度为80~82℃,反应后冷却至室温,将混合物倒入冰中,过滤固体即可。
5.根据权利要求2所述的检测铜离子的可逆荧光探针的制备方法,其特征在于:所述中间产物6-(杂氮环丁烷)喹啉-2-羧酸甲酯与水合肼的摩尔比为0.75:40。
6.根据权利要求3所述的检测铜离子的可逆荧光探针的制备方法,其特征在于:步骤(1)中,6-溴-2-甲基喹啉与二氧化硒的反应摩尔比为0.45:1,反应温度为100~105℃。
7.根据权利要求3所述的检测铜离子的可逆荧光探针的制备方法,其特征在于:步骤(2)中,6-溴喹啉-2-羧酸与氯化亚砜的反应摩尔比为5:6。
8.根据权利要求3所述的检测铜离子的可逆荧光探针的制备方法,其特征在于:步骤(3)中,对于每mmol 6-溴喹啉-2-羧酸甲酯,加入144mg Cs2CO3、8mg Pd(OAc)2和24mg BINAP与6-溴喹啉-2-羧酸甲酯进行反应。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105315264A (zh) * | 2015-11-24 | 2016-02-10 | 山西大同大学 | 一种n′-(喹啉-2-亚甲基)-7-二乙胺基香豆素-3-甲酰肼及其制备方法和应用 |
CN106198522A (zh) * | 2016-07-05 | 2016-12-07 | 河南城建学院 | 一种铜离子快速检测试剂盒及其检测方法 |
CN113248430A (zh) * | 2021-05-21 | 2021-08-13 | 西北师范大学 | 一种基于喹啉衍生物的荧光传感器及其合成和在检测铝离子中的应用 |
-
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105315264A (zh) * | 2015-11-24 | 2016-02-10 | 山西大同大学 | 一种n′-(喹啉-2-亚甲基)-7-二乙胺基香豆素-3-甲酰肼及其制备方法和应用 |
CN106198522A (zh) * | 2016-07-05 | 2016-12-07 | 河南城建学院 | 一种铜离子快速检测试剂盒及其检测方法 |
CN113248430A (zh) * | 2021-05-21 | 2021-08-13 | 西北师范大学 | 一种基于喹啉衍生物的荧光传感器及其合成和在检测铝离子中的应用 |
Non-Patent Citations (1)
Title |
---|
LI, QIAN: "Carbon dots-quinoline derivative nanocomposite: facile synthesis and application as a "turn-off" fluorescent chemosensor for detection of Cu2+ ions in tap water", 《RSC ADVANCES》 * |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN115947720A (zh) * | 2022-12-07 | 2023-04-11 | 南京师范大学 | β3肾上腺素能受体锚定型探针的设计及其制备方法与应用 |
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