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CN110128414B - 一种基于半花菁染料的缺氧荧光探针的制备和应用 - Google Patents

一种基于半花菁染料的缺氧荧光探针的制备和应用 Download PDF

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CN110128414B
CN110128414B CN201910409555.4A CN201910409555A CN110128414B CN 110128414 B CN110128414 B CN 110128414B CN 201910409555 A CN201910409555 A CN 201910409555A CN 110128414 B CN110128414 B CN 110128414B
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李春艳
田杨
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Abstract

本发明涉及了一种基于半花菁染料的缺氧荧光探针的制备和应用，该荧光探针的结构式为：
Figure DDA0002062366390000011
本发明提供了以半花菁荧光染料、亚硝酸钠、苯酚等为原料合成该荧光探针的制备方法；该荧光探针是一种具有近红外发射、高选择性的缺氧荧光探针；由于Na2S2O4已被证实可以用来模拟生物样品中的缺氧还原环境，所以我们选择Na2S2O4来代替体外的缺氧环境。首先，该荧光探针对Na2S2O4表现出较高的信噪比，探针与Na2S2O4反应之后荧光显著增强；其次，该荧光探针对Na2S2O4表现出很好的选择性，不受其他离子以及还原性物质的干扰；此外，该荧光探针已成功应用于细胞成像研究，可以检测细胞内的缺氧状况。

Description

一种基于半花菁染料的缺氧荧光探针的制备和应用
技术领域
本发明属于荧光探针技术领域，具体涉及一种基于半花菁染料的缺氧荧光探针的制备和应用。
背景技术
缺氧是恶性肿瘤发生、发展过程中普遍存在的现象，其产生主要与肿瘤无限制生长、氧耗增加及肿瘤组织血管发育不良等有关，也是临床多种疾病共有的病理过程(A.L.Harris,Nat.Rev.Cancer.,2002,2,38-47)。缺氧环境下的肿瘤细胞易发生转移，并且能增加对放疗、化疗的抗拒性，从而降低了治疗效果(L.J.Walker,R.B.Craig,A.L.Harris,I.D.Hickson,Nucl Acids Res,1994,22,4884-4889；R.E.Durand,Int J Radiat OncolBiol Phys,1991,20,253-258；R.E.Durand,In Vivo,1994,8,691-702)。因此，设计有效的方法去准确监控缺氧的发生和发展规律就显得非常重要。
近年来，荧光探针因其在活体样品中的高灵敏度、实时检测和高时空分辨率成像等突出优点而备受关注(H.Kobayashi,M.Ogawa,R.Alford,P.L.Choyke,Y.Urano,Chem.Rev,2010,110,2620-2640.)。到目前为止，已经开发了一些检测缺氧的荧光探针，用于实时监测细胞或者活体内的缺氧状况(L.J.O’Connor,I.N.Mistry,S.L.Collins,L.K.Folkes,G.Brown,S.J.Conway,E.M.Hammond,ACS Cent.Sci.,2017,3,20-30；J.Zhang,H.W.Liu,X.X.Hu,J.Li,L.H.Liang,X.B.Zhang,et al,Anal Chem.,2015,87,11832-11839；K.K.Kiyose,K.Hanaoka,D.Oushiki,T.Nakamura,M.Kajimura,M.Suematsu,et al,J AmChem Soc.,2010,132,15846-15848；W.Piao,S.Tsuda,Y.Tanaka,S.Maeda,F.Liu,S.Takahashi,et al,Angew Chem Int Ed,2013,52,13028-13032)。但是，这些缺氧荧光探针存在着不足之处：(1)荧光探针的发射波长短，容易受到自身背景荧光的干扰，不利于深层组织成像检测。(2)探针的识别基团为醌基或硝基，由于光致电子转移机制(PET)的存在，容易受到pH或极性等条件的影响。因此设计一种长波长发射，对缺氧具有高敏感性的荧光探针是至关重要的。
半花菁染料在荧光探针技术领域中已被广泛应用，它具有大的摩尔吸光系数、高荧光量子产率等优势，最重要的是具有近红外发射性能。近红外发射能够穿透更深的组织，不易受到生物自体荧光的干扰，对生物成像更有利。据报道，利用半花菁荧光探针已经成功检测了许多目标物，如：ALP、CO、H2O2等(S.J.Li,C.Y.Li,Y.F.Li,J.J Fei,P,Wu,B,Yang,J.Ou-Yang,S.X.Nie,Anal.Chem.2017,89,6854-6860；S.J.Li,D.Y.Zhou,Y.F.Li,B.Yang,J.Ou-Yang,J.Jie,J.Liu；C.Y.Li,Talanta 2018,188,691-700；L.Yuan,W.Lin,S.Zhao,W.Gao,B.Chen,L.He,S Zhu,J.Am.Chem.Soc.,2012,134,13510-13523)。但是，现在还没有基于半花菁染料的荧光探针来检测缺氧。因此，设计合成一种半花菁染料的近红外荧光探针，作为检测生物样品中缺氧程度的有效工具，是非常有必要的。
发明内容
根据所提出的要求，本发明人对此进行了深入研究，在付出了大量创造性劳动后，提供了一种基于半花菁的缺氧荧光探针。
本发明的技术方案是，一种基于半花菁染料的缺氧荧光探针，其结构式如下：
Figure BDA0002062366370000021
一种基于半花菁染料的缺氧荧光探针的制备方法。步骤如下：
在100mL圆底烧瓶中，加入10mL含有1当量Cy-NH2的体积比为1:1的CH3CN/CH2Cl2混合溶液，在-5-5℃和氮气保护下，滴加3.5当量的浓盐酸后，搅拌均匀。接着，将含有1当量NaNO2的蒸馏水溶液缓慢滴加到上述反应液中，继续搅拌20-30min。然后，加入含有2当量氨基磺酸的水溶液，继续搅拌5-10min。随后，将含有6当量苯酚的乙腈溶液加到上述反应液中并调节pH为6.0-6.5，同样条件下，继续搅拌1.5-2h。反应完成后，加水稀释并用CH2Cl2萃取，收集有机层，用盐水洗涤，无水Na2SO4干燥，过滤并浓缩。粗产品用体积比为20:1的CH2Cl2/CH3OH洗脱剂进行柱层析，得到绿色固体产物Cy-AP(产率48％)，即为所述的荧光探针。
本发明的有益效果是，一种基于半花菁染料的缺氧荧光探针的良好的光谱响应性能。Na2S2O4由于它的强还原性已经被用来模拟肿瘤细胞内因为缺氧而存在的还原环境，所以我们选择Na2S2O4作为检测缺氧程度的检测物。首先，研究该探针的荧光光谱性质。加入Na2S2O4之前，荧光探针没有近红外的荧光发射峰；加入Na2S2O4之后，在725nm处出现了近红外发射峰。并且随着Na2S2O4浓度的增大，探针的近红外荧光强度不断增强。接着，研究探针的紫外吸收光谱。在没有加入Na2S2O4时，探针在450nm处有一个较高的吸收峰，在650nm处有一个较小的吸收峰；加入Na2S2O4后，450nm处的吸收峰逐渐减小，在680nm附近出现新的强吸收峰。然后，研究探针的选择性。考察探针与各种离子(Na+,K+,Ca2+,Mg2+,Fe2+,ClO-,OH-,Br-,I-,F-)和一些氨基酸(Lysine,Tryptophan,Leucine,Glycine,Alanine)以及检测物(Na2S2O4)的荧光响应情况。结果发现，只有Na2S2O4能引起荧光光谱的改变，其他检测物对探针的荧光光谱没有明显的改变。最后，该荧光探针响应比较迅速，响应时间在15分钟以内。
一种基于半花菁染料的缺氧荧光探针的应用。细胞经过常氧环境培养后，加入荧光探针，几乎没有观察到荧光的产生。细胞经过缺氧环境培养后，加入探针，可以看到荧光明显增强。而且，随着缺氧程度的加深，荧光强度逐渐增强。这些结果说明荧光探针Cy-AP能监控细胞内缺氧程度的变化，这为监控人体内相关病理过程中的缺氧状况提供了一种可靠的手段。
附图说明
图1为荧光探针的合成路线。
图2为荧光探针与不同浓度的Na2S2O4作用后的荧光光谱图。
横坐标为波长，纵坐标为荧光强度。荧光探针的浓度均为10μM，Na2S2O4浓度分别为：0,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0mM。发射波长为725nm对应的激发波长为680nm。
图3为荧光探针与Na2S2O4作用前后的紫外可见吸收光谱图。
横坐标为波长，纵坐标为吸光度。荧光探针的浓度均为10μM，Na2S2O4浓度为5.0mM。
图4为荧光探针的选择性图。
荧光探针的浓度均为10μM，1-16分析物为别是：1,Na+；2,K+；3,Ca2+；4,Mg2+；5,Fe2+；6,ClO-；7,OH-；8,Br-；9,I-；10,F-；11,Lysine；12,Tryptophan；13,Leucine；14,Glycine；15,Alanine；16,Na2S2O4。
图5为荧光探针与Na2S2O4作用后荧光强度随时间变化的关系曲线图。
图6为细胞毒性实验图。横坐标为荧光探针的浓度，纵坐标为细胞的存活率。
图7为细胞缺氧荧光成像图。四种细胞(HepG2 cells,HCT116 cells,HeLa cells,MCF-7 cells)在不同缺氧环境中培养1h，然后探针染色0.5h。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不限于此。
实施例1：
荧光探针的合成
合成路线如图1。在100mL圆底烧瓶中，加入10mL含有Cy-NH2(0.17g,0.3mmol)的CH3CN/CH2Cl2(1:1)混合溶液，在0℃和氮气保护下，加入浓盐酸(0.05mL,1mmol)，搅拌均匀。接着，将含有NaNO2(0.02g,0.3mmol)的蒸馏水溶液缓慢滴加到上述反应液中，继续搅拌30min。然后，加入氨基磺酸(0.06g,0.6mmol)的水溶液，继续搅拌10min。随后，将含有苯酚(0.16mL,1.8mmol)的CH3CN溶液加到反应液中，并调节pH为6.0，同样条件下，继续搅拌1.5h。反应结束后，加水稀释，用CH2Cl2萃取。收集有机层，用盐水洗涤，无水Na2SO4干燥，过滤并浓缩。得到的粗产品用体积比为20:1的CH3OH/CH2Cl2洗脱液进行柱层析，得到深绿色固体Cy-AP(0.08g)，产率48％，即为荧光探针。1H NMR(400MHz,DMSO):δ10.62(s,1H),8.71(d,J＝15.2Hz,1H),8.41(d,J＝8.4Hz,1H),8.22-8.14(m,2H),7.98(d,J＝8.8Hz,1H),7.86-7.65(m,6H),7.41(s,1H),6.98(d,J＝8.8Hz,2H).6.71(d,J＝15.6Hz,1H),4.60(d,J＝7.2Hz,2H),1.84(s,2H),1.43(t,J＝6.4Hz,4H),1.191(s,6H),0.80(t,J＝4.8Hz,3H).13CNMR(100MHz,DMSO):δ179.7,158.9,153.8,153.3,145.8,145.0,139.0,137.3,133.1,131.8,131.4,130.1,128.8,128.7,127.5,127.0,125.9,123.9,123.3,119.7,116.7,115.2,113.0,109.4,106.4,53.0,40.8,29.3,27.3,24.1,22.6,20.3,13.7.MS(TOF):552.5.
实施例2：
荧光探针和Na2S2O4溶液配制
探针溶液的制备：称取一定量探针固体溶解在甲醇中，配成1×10-4M的探针溶液。Na2S2O4溶液的配制(现用现配)：称取一定量的Na2S2O4固体溶于蒸馏水中，配成1×10-1M的检测溶液。将1.0×10-1M的Na2S2O4溶液逐渐稀释，得到1.0×10-1-1.0×10-3M的Na2S2O4溶液。将1.0mL探针的备用溶液和1.0mL的Na2S2O4溶液加入到10mL的容量瓶中，用缓冲溶液定容后，得到浓度为1.0×10-5M的荧光探针和1.0×10-2-1.0×10-4M的Na2S2O4混合待测溶液。
实施例3：
荧光探针与Na2S2O4作用的荧光光谱的测定
图2为荧光探针与Na2S2O4作用的荧光光谱，荧光探针的浓度为10μM，Na2S2O4的浓度依次为0,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0mM。实验所用激发波长为680nm，发射波长范围为705～800nm。狭缝宽度为10.0nm/10.0nm，所用的荧光测定仪器为日立F4600荧光分光光度计。从图2可以看出，加入Na2S2O4之前，由于偶氮键的淬灭作用，荧光探针没有荧光发射峰；加入Na2S2O4之后，在近红外区(725nm)出现了近红外荧光发射峰。这是因为探针分子被Na2S2O4还原，导致偶氮键的断裂，释放出半花菁荧光团，从而产生近红外荧光。并且随着Na2S2O4浓度的增大，探针分子的近红外荧光强度不断增强。
实施例4：
荧光探针与Na2S2O4作用的紫外可见吸收光谱的测定
图3为荧光探针与Na2S2O4作用后的紫外可见吸收光谱图，荧光探针的浓度为10μM，Na2S2O4的浓度为5.0mM。紫外可见吸收光谱测定用的仪器为安捷伦Cary60紫外可见分光光度计。从图3中可以看出，在没有加入Na2S2O4时，探针在450nm处有较高的吸收峰，在650nm处有较小的吸收峰；加入Na2S2O4后，450nm处的吸收峰逐渐消失，在680nm处出现新的强吸收峰。
实施例5：
荧光探针对Na2S2O4测定的选择性
图4为荧光探针对Na2S2O4测定的选择性图。考察在浓度为10μM的荧光探针溶液中加入Na2S2O4(5.0mM)及各种离子(Na+,K+,Ca2+,Mg2+,Fe2+,ClO-,OH-,Br-,I-,F-)，以及一些氨基酸(Lysine,Tryptophan,Leucine,Glycine,Alanine)的荧光响应情况。从图4中可以看出，只有Na2S2O4能引起荧光光谱的改变，其他检测物对探针的荧光光谱没有明显的影响。这些结果表明，荧光探针对Na2S2O4有较好的选择性。
实施例6：
荧光探针与Na2S2O4作用的响应时间的测定
我们研究了荧光探针对Na2S2O4的响应时间，其结果如图5。从图中可以看出，该探针对Na2S2O4的响应时间为15min，这能够满足在实际样品中进行实时监测时对响应时间的要求。从图5我们还可以看出，荧光强度达到最大值后，随着时间的增加，荧光强度不再发生变化，这表明此荧光探针光稳定性较好。
实施例7：
荧光探针在活细胞中的应用
首先，我们做了细胞毒性实验，如图6所示。当加入0～30μM Cy-AP后，四种细胞(HepG2 cells,HCT116 cells,HeLa cells,MCF-7 cells)的存活率均在90％以上，因此可以说明，该荧光探针毒性较小。然后，我们研究荧光探针在活细胞中的应用，选择HepG2cells,HCT116 cells,HeLa cells,MCF-7 cells四种肿瘤细胞进行共聚焦显微成像，结果如图7所示。经过常氧培养(20％O2)的细胞，几乎没有观察到荧光的产生。四种细胞预先不同程度缺氧(10％O2,5％O2,1％O2)培养1h，然后用探针染色0.5h后，荧光明显增强。并且随着缺氧程度的升高，荧光强度逐渐增大。这些结果说明荧光探针能监控细胞内缺氧程度的变化，这为监控人体内相关病理过程中的缺氧状况提供了一种可靠的手段。

Claims (3)

1.一种基于半花菁染料的缺氧荧光探针，即Cy-AP，其结构如下：
Figure FDA0003510293710000011
2.根据权利要求1所述的一种基于半花菁染料的缺氧荧光探针的制备方法，其特征在于，反应步骤如下：
在100mL圆底烧瓶中，加入10mL含有1当量Cy-NH2的体积比为1:1的CH3CN/CH2Cl2混合溶液，在-5～5℃和氮气保护下，滴加3.5当量的浓盐酸，搅拌均匀，接着，将含有1当量NaNO2的蒸馏水溶液缓慢滴加到上述反应液中，继续搅拌20～30min，然后，加入含有2当量氨基磺酸的水溶液，继续搅拌5～10min，随后，将含有6当量苯酚的乙腈溶液加到上述反应液中并调节pH为6.0～6.5，同样条件下，继续搅拌1.5～2h，反应完成后，加水稀释并用CH2Cl2萃取，收集有机层，用盐水洗涤，无水Na2SO4干燥，过滤并浓缩，粗产品用体积比为20:1的CH2Cl2/CH3OH洗脱剂进行柱层析，得到绿色固体产物Cy-AP，即为所述的荧光探针，其中，Cy-NH2的结构如下：
Figure FDA0003510293710000012
3.根据权利要求1所述的一种基于半花菁染料的缺氧荧光探针的应用，其特征在于，所述荧光探针应用于非疾病诊断与治疗目的的细胞内缺氧程度变化的检测。
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