CN106543226B - 一种定位线粒体的atp荧光探针的制备及应用 - Google Patents
一种定位线粒体的atp荧光探针的制备及应用 Download PDFInfo
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Abstract
本发明公开了一种定位线粒体的ATP荧光探针的制备及应用，该探针的结构式为：
Description
技术领域
本发明属于荧光探针技术领域，具体涉及一种定位线粒体的ATP荧光探针的制备及应用。
背景技术
ATP(三磷酸腺苷)是一种生物阴离子，在细胞呼吸，酶催化，能量和信号传递过程中起关键作用(文献1：Knowles,J.R.Annu.Rev.Biochem.1980,49,877–919.文献2：Dennis,P.B.；Jaeschke,A.；Saitoh,M.；Fowler,B.；Kozma,S.C.；Thomas,G.Science 2001,294,1102–1105.文献3：Khlyntseva,S.V.；Bazel,Y.R.；Vishnikina,A.B.；Andruch,V.；J.Anal.Chem.2009,64,657–673.)。线粒体是细胞进行有氧呼吸的主要场所，以ATP的形式提供细胞代谢能量(文献4：Knowles,J.R.Annu.Rev.Biochem.1980,49,877–919.文献5：Higgins,C.F.；Hiles,I.D.；Salmond,G.P.；Gill,D.R.；Downie,J.A.；Evans,I.J.；Holland,I.B.；Gray,L.；Buckel,S.D.；Bell,A.W.；Hermodson,M.A.Nature 1986,323,448–450.文献6：Lin,M.T.；Beal,M.F.Nature 2006,443,787–795.)。因此，ATP的含量变化必然会影响线粒体的功能(文献7：Carlson,K.；Ehrich,M.Toxicol.Appl.Pharmacol.1999,160,33–42.文献8：Green,D.R.；Kroemer,G.Science 2004,305,626–629.文献9：McBride,H.M.；Neuspiel,M.；Wasiak.S.Curr.Biol.2006,16,R551–R560.)。线粒体功能紊乱与许多疾病有关，比如：心血管病(文献10：Yokoshiki,H.；Sunagawa,M.；Seki,T.；Sperelakis,N.Am.J.Physiol.1998,274,C25–C37.)，恶性肿瘤(文献11：Wallace,D.C.Science 1999,283,1482–1488.)和帕金森症(文献12：Zhou,Z.；Du,Y.；Dong,S.Anal.Chem.2011,83,5122–5127.)。因此，实时监控线粒体中ATP含量的变化，对阐明与线粒体受损有关的细胞功能障碍及疾病的发病机制有着重要的意义。
近年来，发展了许多检测ATP的方法，比如：高效液相色谱法(文献13：Mora,L.；Hernández-Cázares,A.S.；Aristoy,M.C.；Toldrá,F.Food Chem.2010,123,1282–1288.)，质谱法(文献14：Huang,Y.F.；Chang,H.T.Anal.Chem.2007,79,4852–4859.)和电化学法(文献15：Yu,P.；He,X.；Zhang,L.；Mao,L.Anal.Chem.2015,87,1373–1380.)。相比于这些传统的方法，荧光分析方法更简单，灵敏，高效并且能对生物体内外的检测进行实时监控。到目前为止，有许多荧光探针被报道(文献16：Zhou,Y.；Xu,Z.；Yoon,J.Chem.Soc.Rev.2011,40,2222–2235.文献17：R.；Sancenón,F.Chem.Rev.2003,103,4419–4476.文献18：Xu,Z.；Kim,S.K.；Yoon,J.Chem.Soc.Rev.2010,39,1457–1466.文献19：Gale,P.A.Chem.Soc.Rev.2010,39,3746–3771.文献20：Kim,S.K.；Sessler,J.L.Chem.Soc.Rev.2010,39,3784–3809.文献21：Li,A.F.；Wang,J.H.；Wang,F.；Jiang,Y.B.Chem.Soc.Rev.2010,39,3729–3745.文献22：Xu,Z.；Chen,X.；Kim,H.N.；Yoon,J.Chem.Soc.Rev.2010,39,127–137.)。虽然这些探针可以检测ATP，但它们存在两个方面的问题：一方面，大多数探针缺少线粒体的定位基团，不利于检测线粒体中的ATP；另一方面，有些探针只有一个识别位点，因此对生物阴离子的选择性差。
到目前为止，有许多定位线粒体的荧光探针被设计用于检测信号分子，比如一氧化氮(文献23：Sun,Y.Q.；Liu,J.；Zhang,H.；Huo,Y.；Lv,X.；Shi,Y.；Guo,W.J.Am.Chem.Soc.2014,136,12520–12523.文献24：Yu,H.；Zhang,X.；Xiao,Y.；Zou,W.；Wang,L.；Jin,L.Anal.Chem.2013,85,7076–7084.)，硫化氢(文献25：Pak,Y.L.；Li,J.；Ko,K.C.；Kim,G.；Lee,J.Y.；Yoon,J.Anal.Chem.2016,88,5476–5481.文献26：Liu,J.；Guo,X.；Hu,R.；Liu,X.；Wang,S.；Li,S.；Li,Y.；Yang,G.Anal.Chem.2016,88,1052–1057.)，过氧化氢(文献27；Liu,J.；Ren,J.；Bao,X.；Gao,W.；Wu,C.；Zhao,Y.Anal.Chem.2016,88,5865-70.文献28：Yang,L.；Li,N.；Pan,W.；Yu,Z.；Tang,B.Anal.Chem.2015,87,3678–3684.)，次氯酸(文献29：Yuan,L.；Wang,L.；Agrawalla,B.K.；Park,S.J.；Zhu,H.；Sivaraman,B.；Peng,J.；Xu,Q.H.；Chang,Y.T.J.Am.Chem.Soc.2015,137,5930–5938.文献30：Xu,Q.；Heo,C.H.；Kim,J.A.；Lee,H.S.；Hu,Y.；Kim,D.；Swamy,K.M.；Kim,G.；Nam,S.J.；Kim,H.M.；Yoon,J.Anal.Chem.2016,88,6615-6620.)和pH(文献31：Zhu,W.；Chai,X.；Wang,B.；Zou,Y.；Wang,T.；Meng,Q.；Wu,Q.Chem.Commun.2015,51,9608–9611.文献32：Lee,M.H.；Park,N.；Yi,C.；Han,J.H.；Hong,J.H.；Kim,K.P.；Kang,D.H.；Sessler,J.L.；Kang,C.；Kim,J.S.J.Am.Chem.Soc.2014,136,14136–14142.)等等。然而可以用于检测线粒体中ATP的探针很少，仅仅只有两个探针被报道(文献33：Kurishita,Y.；Kohira,T.；Ojida,A.；Hamachi,I.J.Am.Chem.Soc.2012,134,18779–18789.文献34：Srivastava,P.；Razi,S.S.；Ali,R.；Srivastav,S.；Patnaik,S.；Srikrishna,S.；Misra,A.Biosens.Bioelectron.2015,69,179–185.)，但这两个探针存在一些局限性，比如：不能有效的区分ATP和其它有机磷酸阴离子，或者检测范围与线粒体中ATP的浓度(1～5mM)不一致。因此，发展一个合适的探针来监控线粒体中ATP的含量变化仍然具有挑战性。
罗丹明是一个构建关-开型荧光探针的优良荧光基团(文献35：Chen,X.；Pradhan,T.；Wang,F.；Kim,J.S.；Yoon,J.Chem.Rev.2012,112,1910–1956.)。二乙烯三胺经常被用作识别基团，因为它的氨基能与分析物发生作用(文献36：Xiang,Y.；Tong,A.Org.Lett.2006,8,1549-1552.文献37：Roberto,C.；Arnaldo,D.；Gianni G.；Rosangela M.；Anna P.；Giorgio,S.Org.Chem.1997,62,6283–6289.)。三苯基膦是线粒体的定位基团，能定位于线粒体(文献38：Lim,C.S.；Masanta,G.；Kim,H.J.；Han,J.H.；Kim,H.M.；Cho,B.R.J.Am.Chem.Soc.2011,133,11132-11135.文献39：Dodani,S.C.；Leary,S.C.；Cobine,P.A.；Winge,D.R.；Chang,C.J.J.Am.Chem.Soc.2011,133,8606-8616.)。
由此，我们设计了探针Mito-Rh，它以罗丹明为荧光团，以二乙烯三胺为识别基团，以三苯基膦为定位基团，探针本身无荧光，加入ATP后，由于ATP和二乙烯三胺作用，能诱导罗丹明开环，发出很强的红色荧光。该探针对ATP具有高效的选择性，不受其它生物阴离子和无机阴离子的影响。同时，该探针可以定位于线粒体，并实时监控线粒体中ATP的含量变化。
发明内容
为了克服现有技术中的缺点，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，提供了一种高灵敏度，高选择性的定位线粒体的ATP荧光探针。
本发明的技术方案是，一种定位线粒体的ATP荧光探针，其结构式如下：
一种定位线粒体的ATP荧光探针的制备方法。步骤如下：1)在含有50mL乙醇的100mL的圆底烧瓶中加入罗丹明B，完全溶解后溶液呈现紫红色，搅拌下，把二乙烯三胺滴加到上述反应体系中，回流24h。减压蒸馏除去溶剂。粗产品用二氯甲烷/乙醇(体积比为10:1)的洗脱剂柱层析分离得黄色固体(化合物1)。2)将化合物1，5-溴戊酸，1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)和4-二甲氨基吡啶(DMAP)按摩尔比1:1:1:1混合于二氯甲烷溶液中，在室温下，搅拌12小时后，加入三倍当量的三苯基膦反应24小时。减压蒸馏除去溶剂，粗产品用二氯甲烷/乙醇(体积比为50:1)为洗脱剂柱层析分离得黄色固体(探针Mito-Rh)。
一种定位线粒体的ATP荧光探针的性能研究。首先，研究了该探针的荧光光谱性质，加入ATP之前，荧光探针没有罗丹明的荧光发射峰，说明探针分子处于内酰胺闭环结构；随着ATP的加入，在583nm处出现了罗丹明的最大发射峰，并且随着ATP浓度的增大，探针分子的荧光强度不断增强，当加入10mM ATP时，荧光强度增强81倍，说明该探针可以高灵敏的对ATP进行检测。其次，还研究了探针对生物阴离子和无机阴离子的选择性，分别检测了探针与ATP，ADP，AMP，GTP，CTP，UTP，P3O10 5-，P2O7 4-，H2PO4 -，HPO4 2-，PO4 3 -，Cl-，SO4 2 -，NO3-，CH3CO2-和CO3 2 -的紫外响应和荧光响应情况。加入ATP之后，在560nm处出现一个很强的紫外吸收峰，而相同的条件下加入其他的阴离子，除了ADP有较弱的紫外吸收峰外，其它阴离子都没有明显的改变。同时，加入ATP之后，在583nm处出现很强的荧光发射峰，加入ADP有微弱的荧光发射峰，而其他阴离子无响应。由此可见，该探针对ATP有较好的选择性。最后，研究了pH值对该探针测定ATP的影响，当pH值在6.0到8.0之间时，不影响该探针对ATP的测定。
一种定位线粒体的ATP荧光探针的应用。该探针与线粒体定位染料(Mito-TrackerGreen)的Pearson相关系数为0.95，因此可以说明该探针能定位于线粒体；同时，在HeLa细胞中加入探针Mito-Rh培养30分钟,然后加入0.5U/L三磷酸腺苷双磷酸酶(一种把ATP转换为AMP和无机磷酸根的水解酶)，荧光强度随时间不断减弱，15分钟后荧光强度达到最低，可以证明该探针能实时监控线粒体中ATP的含量变化。
附图说明
图1为荧光探针的合成路线。(a)二乙烯三胺,乙醇，回流,24h；(b)5-溴戊酸,三苯基膦，EDC，DMAP，CH2Cl2，12h。
图2为荧光探针与不同浓度的ATP作用后的荧光光谱图。
横坐标为波长，纵坐标为荧光强度。ATP荧光探针的浓度均为10μM，ATP浓度分别为：0,0.1,0.6,1.2,2.0,3.0,4.4,5.6,6.4,8.4,10.0mM。荧光激发波长为520nm。插图为探针对ATP浓度的线性响应图。
图3为荧光探针与ATP的作用机理图。
图4为荧光探针与不同阴离子作用后的荧光光谱图及在紫外灯下的荧光照片。
图5为荧光探针与不同阴离子作用后的紫外吸收光谱图及在可见光下的颜色照片。
图6为pH对荧光探针测定ATP的影响图。
图7为荧光探针的细胞毒性实验图。横坐标为荧光探针的浓度，纵坐标为细胞的存活率。
图8为荧光探针的线粒体定位成像图。
图9为荧光探针实时监控线粒体中的ATP含量变化图。
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：
合成路线如图1所示。
化合物1的合成:在含有50mL乙醇的100mL的圆底烧瓶中加入罗丹明B(2.0g，4.18mmol)，完全溶解后溶液呈现紫红色，搅拌下滴加二乙烯三胺(10.0mL，92.00mmol)到上述反应体系中，回流24h。减压蒸馏除去溶剂。粗产品用二氯甲烷/乙醇为10:1(体积比)的洗脱剂柱层析分离得黄色固体(化合物1)(0.15g，产率:6.40％)。1H NMR(400MHz,CDCl3):δ7.88(d,J＝8.0Hz,1H),7.42(m,2H),7.08(m,1H),6.42(d,J＝8.0Hz,2H),6.36(s,2H),6.26(d,J＝8.0Hz,2H),3.35-3.18(m,10H),2.71(d,J＝8.0Hz,2H),2.55(t,J＝8.0Hz,2H),2.20(t,J＝8.0Hz,2H),1.16(t,J＝8.0Hz,12H).13C NMR(100MHz,CDCl3):δ167.9,153.6,153.3,148.7,132.2,131.3,129.0,128.9,128.0,127.9,123.7,122.7,108.1,105.7,97.9,64.9,51.7,51.3,50.5,39.2,29.7,12.6.MS(TOF)m/z 528.3.
ATP荧光探针Mito-Rh的合成：将化合物1(0.14g,0.25mmol)，5-溴戊酸(0.04g,0.25mmol)，EDC(0.04g,0.25mmol)和DMAP按摩尔比1:1:1:1混合于二氯甲烷(20mL)中，在室温下，搅拌12小时后，加入三倍当量的三苯基膦(0.96g,0.75mmol)反应24小时。减压蒸馏除去溶剂，粗产品用二氯甲烷/乙醇(体积比为50:1)为洗脱剂柱层析分离得黄色固体(探针Mito-Rh)(0.15g,产率：13％)。1H NMR(400MHz,CDCl3):δ7.89(d,J＝8.0Hz,1H),7.45(m,2H),7.36(m,15H),7.09(m,1H),6.43(d,J＝8.0Hz,2H),6.38(s,2H),6.27(d,J＝8.0Hz,2H),3.33(m,10H),3.05(m,8H),2.57(t,J＝8.0Hz,2H),2.23(m,4H),1.38(t,J＝8.0Hz,12H),1.26(s,2H).13C NMR(100MHz,CDCl3):δ171.3,167.7,153.3,153.1,148.5,137.0,136.9,133.5,133.3,131.7,128.6,128.4,128.3,127.9,123.6,122.4,107.8,105.2,105.2,97.5,97.4,64.6,51.2,51.1,46.1,44.2,29.7,22.1,18.9,12.4.Anal.calcd.forC55H63N5O3P+(Mito-Rh):C,75.66；H,7.27；N,8.02.Found:C,75.65；H,7.25；N,7.99.MS(TOF)m/z 872.4.
实施例2：
荧光探针Mito-Rh与ATP作用的溶液配制
将一定量的荧光探针溶解在水中,得到浓度为100μM的探针备用溶液。将一定量的ATP溶解在水中，倒入500mL的容量瓶中，加水稀释至刻度线，得到浓度为1000mM的ATP。将1000mM的ATP水溶液用二次蒸馏水逐渐稀释得到0.1～100mM的ATP水溶液。将1.0mL探针的备用溶液和1.0mL的ATP水溶液加入到10mL的容量瓶中，用缓冲溶液PBS定容，得到浓度为10μM的荧光探针和0.01～100mM的ATP待测溶液。
实施例3：
荧光探针Mito-Rh与ATP作用的荧光光谱性质的测定
以pH值为7.0的PBS缓冲溶液为溶剂用Perkin Elmer LS 55荧光分光光度计测定了荧光探针与ATP作用的荧光光谱，结果如图2。荧光探针的浓度为10μM，ATP的浓度依次为0,0.1,0.6,1.2,2.0,3.0,4.4,5.6,6.4,8.4,10.0mM，激发波长固定为520nm，发射波长范围为530～650nm，狭缝宽度为5nm/5nm。加入ATP之前，荧光探针几乎无荧光，加入ATP后，在583nm处出现了罗丹明的发射峰，这是因为探针分子的结构发生了变化，结构从罗丹明的闭环形式转变为开环形式。并且随着ATP浓度的增大，探针分子的荧光强度不断增强，当加入10.0mM的ATP时，荧光强度增强至未加入ATP时的81倍。如图2的插图所示，荧光强度跟ATP的浓度呈现线性关系，线性范围是0.1～10mM，检测限是0.033mM。这些结果表明探针对ATP的检测具有很好的灵敏度。
实施例4：
荧光探针Mito-Rh与ATP的作用机理研究
图3为荧光探针Mito-Rh与ATP的作用机理图，由于探针Mito-Rh中的罗丹明部分是内酰胺环，加入ATP后,罗丹明的内酰胺环被打开，从而发出红光，据推测这主要是两个因素(氢键和π–π堆积影响)共同导致内酰胺开环。
实施例5：
荧光探针Mito-Rh与不同阴离子荧光光谱性质的测定
图4a为荧光探针与不同阴离子作用后的荧光光谱图，被测阴离子有ATP,ADP,AMP,GTP,CTP,UTP,Mx(P3O10 5-，P2O7 4-，H2PO4 -，HPO4 2-，PO4 3-，Cl-，SO4 2-，NO3 -，CH3CO2 -,和CO3 2-)。ATP的浓度是5mM，其他阴离子的浓度为10mM。从图4a中可以看出，加入ATP之后，在583nm处出现很强的荧光发射峰，加入ADP之后有微弱的荧光发射峰，而其他阴离子无荧光发射峰。如图4b所示，我们也考察了探针Mito-Rh与被测阴离子在紫外灯下的荧光变化情况，只有ATP使溶液有红色荧光，其他阴离子不能使溶液产生荧光。这些结果表明探针对ATP具有很好的选择性。
实施例6：
荧光探针Mito-Rh与不同阴离子紫外吸收光谱性质的测定
图5a为荧光探针与不同阴离子作用后的紫外吸收光谱图，被测阴离子有ATP,ADP,AMP,GTP,CTP,UTP,Mx(P3O10 5-，P2O7 4-，H2PO4 -，HPO4 2-，PO4 3-，Cl-,SO4 2-，NO3 -，CH3CO2 -,和CO3 2-)。ATP的浓度是5mM，其他阴离子的浓度为10mM。从图5a中可以看出，加入ATP之后，在560nm处出现很强的紫外吸收峰，加入ADP之后有微弱的紫外吸收峰，而其他阴离子无紫外吸收峰。如图5b所示，我们也考察了探针Mito-Rh与被测阴离子在可见光下的颜色变化情况，只有ATP使溶液的颜色从无色变成红色，其他阴离子不能使溶液颜色发生改变。这些结果表明探针对ATP具有很好的选择性。
实施例7：
溶液pH值对荧光探针Mito-Rh测定ATP的荧光性质的影响
我们考察了pH值对荧光探针测定ATP的荧光强度的影响，结果如图6。我们研究的pH范围为2.0～11.0，荧光探针的浓度为10μM，ATP的浓度为5mM。实验结果如图6所示，当pH＜6.0，荧光探针随着pH的降低荧光强度增大，这是因为在酸性条件下，探针发生质子化，使得罗丹明结构处于开环状态；当pH＞6.0，由于罗丹明处于闭环结构，随着pH的变化，荧光强度基本不变。然而，加入ATP之后，在pH＜8.0，荧光强度基本不变，这是因为在酸性条件下的质子化或与ATP的结合作用，都能使得罗丹明内酰胺结构处于开环状态。综上所述，当pH值在6.0到8.0之间时，不影响荧光探针对ATP的测定，这有利于该探针实际样品中的应用。
实施例8：
荧光探针Mito-Rh在活细胞中的毒性实验
首先，我们做了细胞毒性试验，结果如图7所示。当加入0～20μM ATP探针，30分钟后，细胞的成活率均在88％以上，因此可以说明，此ATP荧光探针毒性较小可应用于检测活细胞内的ATP。
实施例9：
荧光探针Mito-Rh的线粒体定位实验
接着，我们将HeLa细胞培养于细胞培养基中，然后添加有10％胎牛血清(FBS)和5％CO2，在37℃的环境下放置。将细胞接种于共聚焦培养皿中，培育孵化24小时使细胞贴壁。做细胞成像实验之前，我们先用磷酸盐缓冲溶液(PBS)将HeLa细胞清洗三次。然后分别加入10μM的荧光探针Mito-Rh和Mito-Tracker Green(线粒体定位染料)在37℃下恒温放置1小时，再用PBS缓冲溶液清洗三次，最终在OLYMPUS FV1000激光共聚焦显微镜下进行成像。图8a为探针Mito-Rh在红色通道的成像，图8b为Mito-Tracker Green在绿色通道的成像，图c为图a和图b的合并图，图8d为细胞的明场成像，图8e为红色通道和绿色通道荧光强度的散点图，图8f为图8e中白色线上的荧光强度图。比例尺：5μm。从而得到探针Pearson相关系数为0.95。因此可以说明，此探针Mito-Rh可用于定位线粒体。
实施例10：
荧光探针Mito-Rh实时监控细胞中线粒体ATP含量变化的应用
首先，在HeLa细胞中加入探针Mito-Rh(1μM)在37℃下恒温放置30分钟,然后在HeLa细胞中加入0.5U/L三磷酸腺苷双磷酸酶(一种把ATP转换为AMP和无机磷酸根的水解酶)分别培养0分钟，5分钟，10分钟和15分钟。随时间变化的荧光成像如图9a-9d所示，图9e代表图9a-9d的相对像素强度。由图可知，荧光强度随时间不断减弱，15分钟后荧光强度达到最低。这些可以证明，该探针能实时监控线粒体中的ATP含量变化。
Claims (3)
1.一种定位线粒体的ATP荧光探针，其结构式如下：
。
2.根据权利要求1所述的一种定位线粒体的ATP荧光探针的制备方法，其特征在于它的具体制备步骤为：
1)在含有50mL乙醇的100mL的圆底烧瓶中加入罗丹明B，完全溶解后溶液呈现紫红色，搅拌下，把二乙烯三胺滴加到上述反应体系中，回流24h；减压蒸馏除去溶剂，粗产品用体积比为10:1的二氯甲烷/乙醇洗脱剂柱层析分离得黄色固体，即化合物1，其结构如下：
2)将化合物1，5-溴戊酸，1-(3-二甲氨基丙基)-3-乙基碳二亚胺和4-二甲氨基吡啶按摩尔比1:1:1:1混合于二氯甲烷溶液中，在室温下，搅拌12小时后，加入三倍当量的三苯基膦反应24小时；减压蒸馏除去溶剂，粗产品用体积比为50:1的二氯甲烷/乙醇洗脱剂柱层析分离得黄色固体，即探针Mito-Rh。
3.根据权利要求1所述的一种定位线粒体的ATP荧光探针的应用，其特征在于：该探针与线粒体定位染料Mito-Tracker Green的Pearson相关系数为0.95；同时，在HeLa细胞中加入探针Mito-Rh培养30分钟,然后加入0.5U/L三磷酸腺苷双磷酸酶，荧光强度随时间不断减弱，15分钟后荧光强度达到最低。
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