CN106753341B - 一种近红外碱性磷酸酶荧光探针的制备方法和应用 - Google Patents
一种近红外碱性磷酸酶荧光探针的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种近红外碱性磷酸酶荧光探针的制备方法和应用。该荧光探针的结构式为:。本发明以IR‑780、间苯二酚和三氯氧磷为原料,设计合成了一种基于半花菁生物的近红外荧光探针。该探针可用来检测溶液中ALP浓度,表现好的灵敏度,线性范围为0.01‑2.0 U/mL,检测限为0.003 U/mL。该探针表现出很好的选择性,不受其它酶、生物硫醇、氨基酸以及金属离子的影响。更为重要的是,该探针还可应用于生物成像,检测细胞内和组织中的ALP含量,这对于深入研究ALP在生物体内生理和病理过程具有重要意义。
Description
技术领域
本发明属于荧光探针技术领域,具体涉及一种近红外碱性磷酸酶荧光探针的制备方法和应用。
背景技术
碱性磷酸酶(ALP)广泛分布于人体肝脏、骨骼、肠、肾和胎盘等组织中,是经肝脏向胆外排出的一种酶,它在生理过程中发挥着关键的作用(文献1:J.E.Coleman,Annu.Rev.Biophys.Biomol.Struct.,1992,21,441-483)。ALP在生物体内的浓度的变化与某些生命过程正常与否息息相关。研究表明,ALP的水平异常与许多疾病有关,包括乳腺癌,前列腺癌,糖尿病,骨疾病,以及肝功能异常(文献2:J.A.Lorente,H.Valenzuela,J.Morote,A.Gelabert,Eur.J.Nucl.,1999,26,625-632;文献3:D.L.Robertson,G.F.Joyce,Nature,1990,344,467-468;文献4:P.Colombatto,A.Randone,G.Civitico,J.M.Gorin,L.Dolci,N.Medaina,F.Oliveri,G.Verme,G.Marchiaro,R.Pagni,P.Karayiannis,H.C.Thomas,G.Hess,F.Bonino,M.R.Brunetto,J.Viral Hepat.,1996,3,301-306;文献5:M.M.Couttenye,P.C.D'Haese,V.O.Van Hoof,E.Lemoniatou,W.Goodman,G.A.Verpooten,M.E.De Broe,Nephrol.Dial.Transplant.,1996,11,1065-1072)。因此,设计一种有效检测ALP的方法,以便更好地了解其生理和病理功能,是具有重要意义的。
荧光探针以其高选择性、灵敏度,操作简单,并且可应用于生物成像等优点,受到了广泛的关注。近年来,报道了一些用于检测ALP的荧光探针(文献6:J.J.Deng,P.Yu,Y.X.Wang,L.Q.Mao,Anal.Chem.,2015,87,3080-3086;文献7:Y.Li,Y.N.Li,Z.G.Liu,X.G.Su,RSC Advances.,2014,4,42825-42830;文献8:X.G.Gu,G.X.Zhang,Z.Wang,W.W.Liu,L.Xiao,D.Q.Zhang,Analyst,2013,138,2427-2431;文献9:M.Kawaguchi,K.Hanaoka,T.Komatsu,T.Terai,T.Nagano,Bioorg Med Chem Lett.,2011,21,5088-5091;文献10:X.F.Hou,Q.X.Yu,F.Zeng,J.H.Ye,S.Z.Wu,Journal of Materials Chemistry B.,2015,3,1042-1048;文献11:Z.X.Lu,J.S.Wu,W.M.Liu,G.Y.Zhang,P.F.Wang,RSCAdvances.,2016,6,32046-32051)。我们注意到,尽管这些荧光探针实现了对ALP的检测,但他们仍然存在一个共同的问题,这些探针的发射波长都是在可见光区域,这将会影响探针在生物系统中的应用。因此,设计和合成一个近红外探针检测ALP是很有必要的。
近红外染料的荧光发射波长范围在650-900nm,与可见光区的染料相比,具有一些独特的优势,如组织渗透能力强,对生物样品光损伤小,背景干扰少(文献12:L.Yuan,W.Lin,Y.S.Zhao,W.S.Gao,B.Chen,L.W.He,S.S.Zhu,J.Am.Chem.Soc.,2012,134,13510-13523)。到目前为止,一些近红外荧光探针已被设计合成,并成功用于酶,pH,小分子及金属离子的检测(文献13:H.Yu,M.Sun,K.Zhang,H.Zhu,Z.Liu,Y.Zhang,J.Zhao,L.Wu,Z.Zhang,S.Wang,Sens.Actuators.B.,2015,219,294-300;文献14:K.Xu,F.Wang,X.Pan,R.Liu,J.Ma,F.Kong,B.Tang,Chem.Commun.,2013,49,2554-2556;文献15:J.Zhang,B.Yu,L.Ning,X.Zhu,J.Wang,Z.Chen,X.Liu,X.Yao,X.Zhang,H.Zhang,Eur.J.Org.Chem.,2015,2015,1711-1718;文献16Y.Y.Yang,T.Cheng,W.Zhu,Y.Xu,X.Qian,Org.Lett.,2011,13,264-267)。值得我们注意的是,这些近红外荧光探针基本上都是基于花菁染料的。
与传统花菁染料相比,半花菁染料耐光性好,荧光量子产率高,更有利于成像。到目前为止,半花菁染料已被设计合成,并用于检测硒化氢,肼,半胱氨酸,pH,一氧化氮,β-内酰胺酶,硒醇,超氧根离子,硝基还原酶(文献17:F.Kong,L.Ge,X.Pan,K.Xu,X.Liu,B.Tang,Chem.Sci.,2016,7,1051-1056;文献18:J.Zhang,L.Ning,J.Liu,J.Wang,B.Yu,X.Liu,X.Yao,Z.Zhang,H.Zhang,Anal.Chem.,2015,87,9101-9107;文献19:J.Zhang,J.Wang,J.Liu,L.Ning,X.Zhu,B.Yu,X.Liu,X.Yao,H.Zhang,Anal.Chem.,2015,87,4856-4863;文献20:Y.Li,Y.Wang,S.Yang,Y.Zhao,L.Yuan,J.Zheng,R.Yang,Anal.Chem.,2015,87,2495-2503;文献21:A.T.Wrobel,T.C.Johnstone,A.Deliz Liang,S.J.Lippard,Rivera-Fuentes,P.J.Am.Chem.Soc.,2014,136,4697-4705;文献22:L.Li,Z.Li,W.Shi,X.Li,H.Ma,Anal.Chem.,2014,86,6115-6120;文献23:H.Chen,B.Dong,Y.Tang,W.Lin,Chemistry–AEuropean Journal.,2015,21,11696-11700;文献24:J.Zhang,C.Li,R.Zhang,F.Zhang,W.Liu,X.Liu,X.M.Lee,H.Zhang,Chem.Commun.,2016,52,2679-2682;文献25:Z.Li,X.He,Z.Wang,R.Yang,W.Shi,H.Ma,Biosens.Bioelectron.,2015,63,112-116)。但是用于检测ALP的近红外荧光探针几乎没有。因此,设计一个基于半花菁染料的近红外荧光探针用于生物体内ALP的检测是非常重要的。
本文设计并合成了一种基于半花菁生物的近红外荧光探针,此探针与ALP反应后,产生强烈的红色荧光,表现出很高的灵敏度。该荧光探针与ALP作用迅速,并具有高选择性。并且可成功用于活细胞以及组织中的荧光成像。
发明内容
本发明的目的是提供一种近红外ALP的荧光探针,该荧光探针具有长波长(在近红外区)、高灵敏度以及高选择性的检测ALP,并能够应用于活细胞以及组织中的荧光成像。
本发明的技术方案是,一种基于半花菁衍生物的ALP荧光探针,其结构式如下:
所述的一种基于近红外ALP荧光探针的制备方法,步骤如下:
1)在100mL圆底烧瓶中,依次加入间苯二酚和碳酸钾,其摩尔比为1:1,加入20mL乙腈作为溶剂,室温下,磁力搅拌10min后,将溶解在20mL乙腈中的IR-780溶液逐滴加入到反应液中,其中IR-780和间苯二酚的摩尔比为1:2,N2保护下,加热至50℃,继续搅拌约4h,反应完成,用旋转蒸发仪除去溶剂,粗产品以二氯甲烷/甲醇为20:1(体积比)为淋洗剂,柱层析分离提纯,得蓝绿色固体(CyOH)。
2)将CyOH溶解在无水吡啶中,搅拌下逐滴加入三氯氧磷,其中CyOH和三氯氧磷的摩尔比为1:2,室温下搅拌4h以后,往混合溶液中加入冰,继续在室温条件下搅拌24h,反应完成,减压蒸馏除去溶剂,粗产品用二氯甲烷/甲醇为2:1(体积比)的洗脱剂,柱层析分离提纯,得蓝色固体(CyP)。
反应路线如下:
本发明的有益效果是:本发明的近红外荧光探针在ALP存在下荧光发生显著变化,可以用于高灵敏度的检测ALP,该荧光探针的线性范围为0.01-2.0U/mL,检测限为0.003U/mL。同时,该荧光探针对ALP的响应迅速,响应时间在20min以内。并且该近红外荧光探针对ALP表现出很好的选择性,不受其它酶(Gox,trypsin,AchE,GDH,Gal,thrombin)、生物硫醇(Cys,Hcy,ALP)、氨基酸(Pro,Tyr,Asn,Met,Phe,Nleu,Asp,Orn,Ala,Trp,His,Ser,Gly,Val,Lys,Glu,Arg,Heu,Thr)及其它的金属离子(K+,Ca2+,Zn2+,Na+,Mg2+)的影响。
所述的一种近红外碱性磷酸酶荧光探针的应用:将该荧光探针应用于细胞成像中,细胞内的ALP含量较多,因此直接向细胞内加入探针,能检测到强的红色荧光信号。但是,当在加入探针之前加入一定量的ALP抑制剂Na3VO4时,细胞内没有荧光信号。将该荧光探针应用于组织成像中,组织中的ALP含量也较多,因此直接向组织中加入探针,能检测到强的红色荧光信号。但是,当在加入探针之前加入一定量的ALP抑制剂Na3VO4时,组织中没有荧光信号。这些现象表明该近红外荧光探针不仅能检测溶液中的ALP,还可应用于检测细胞内和组织中的ALP含量,这对于深入研究ALP在生物体内生理和病理过程具有重要意义。
附图说明
图1为荧光探针与不同浓度的ALP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,ALP浓度分别为:0,0.01,0.25,0.5,0.75,1.0,1.25,1.5,1.75,2.0U/mL。荧光激发波长为670nm。插图为探针对ALP浓度的线性响应图。
图2为荧光探针与ALP的作用机理图。
图3为荧光探针与ALP作用后的紫外可见吸收光谱图。
图4为荧光探针在相同ALP浓度下,荧光强度随时间变化的关系曲线图。
图5为荧光探针的选择性图。F0和F表示探针溶液加入ALP前后的荧光强度。
图6为细胞毒性试验。横坐标为荧光探针的浓度,纵坐标为细胞的存活率。
图7为ALP的细胞成像图。
图8为ALP的组织成像图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明,但不限于此。
实施例1:
荧光探针的合成
CyOH的合成:在100mL的圆底烧瓶中,依次加入间苯二酚(1.0mmol,110mg)和碳酸钾(1.0mmol,138mg),再加入20mL乙腈作为溶剂。室温下,磁力搅拌10min后,将溶解在20mL乙腈中的IR-780(0.5mmol,305mg)溶液逐滴加入到反应液中。N2保护下,加热至50℃继续搅拌约4h,反应完成,用旋转蒸发仪除去溶剂,粗产品以二氯甲烷/甲醇为20:1(体积比)为淋洗剂,柱层析分离提纯,得蓝绿色固体169mg,产率为82%。1H NMR(400MHz,CDCl3):δ8.02(d,1H,J=13.2Hz),7.25-7.21(m,2H),7.14(t,1H,J=7.6Hz),7.06-7.03(m,2H),6.97(s,1H),6.90(d,1H,J=8.0Hz),6.81(d,1H,J=8.0Hz),5.56(d,1H,J=13.2Hz),3.76(t,2H,J=7.2Hz),2.67(t,2H,J=6.0Hz),2.59(t,2H,J=6.0Hz),1.91-1.82(m,4H),1.67(s,6H),1.04(t,3H,J=7.6Hz).MS(TOF)m/z 412.2.
CyP探针的合成:在100mL的圆底烧瓶中,将CyOH(0.2mmol,82.4g)溶解在25mL无水吡啶中,在室温下,逐滴加入三氯氧磷(0.4mmol,61.3mg),搅拌4小时以后,向混合溶液中加入冰,继续搅拌24小时,反应完成。减压蒸馏除去溶剂,粗产品用二氯甲烷/甲醇为2:1(体积比)的洗脱剂,柱层析分离提纯,得蓝色固体70.9mg,产率为72%。1H NMR(400MHz,CDCl3):δ8.64(d,1H,J=15.2Hz),7.55-7.51(m,2H),7.44(t,1H,J=7.6Hz),7.36-7.33(m,2H),7.27(s,1H),7.20(d,1H,J=8.0Hz),7.11(d,1H,J=8.0Hz),6.08(d,1H,J=13.2Hz),4.16(t,2H,J=7.6Hz),2.77(t,2H,J=6.0Hz),2.69(t,2H,J=6.0Hz),2.00-1.93(m,4H),1.83(s,6H),1.12(t,3H,J=7.6Hz).13C NMR(100MHz,CDCl3):δ174.4,166.8,163.1,155.5,143.1,142.0,141.2,137.7,132.6,128.8,125.7,123.8,123.7,122.5,118.3,114.7,111.0,103.0,100.1,49.9,46.4,29.7,28.6,24.3,22.7,20.9,11.7.MS(TOF)m/z492.2.Anal.calcd.for C28H31NO5P+(CyP):C,81.52,H,7.33,N,3.40.Found:C,83.56,H,7.45,N,3.47.结果表明,所得产物结构正确。
实施例2:
荧光探针与ALP作用的溶液配制
将一定量的荧光探针溶解在EtOH中,得到浓度为1.0×10-4mol·L-1探针的备用溶液。将1.0mL探针的备用溶液加入到10mL的容量瓶中,用缓冲溶液定容后,得到浓度为1.0×10-5mol·L-1的荧光探针溶液。将ALP分别配制为以下浓度(0,0.01,0.25,0.5,0.75,1.0,1.25,1.5,1.75,2.0U/mL)。
实施例3:
荧光探针与ALP作用的荧光光谱的测定
用pH值为8的缓冲溶液为溶剂测定了荧光探针与ALP作用的荧光光谱,结果如图1。荧光探针的浓度为10μM,ALP的浓度依次为0,0.01,0.25,0.5,0.75,1.0,1.25,1.5,1.75,2.0U/mL,激发波长固定为595nm,发射波长范围为685~850nm,狭缝宽度为10.0nm/10.0nm。从图1可以看出,随着ALP的加入,在703nm处发射峰大幅度的增强,并且随着ALP浓度的增大,探针的荧光强度不断增强。如图1的插图所示,荧光强度跟ALP的浓度呈现线性关系,线性范围是0.01-2.0U/mL,检测限是0.003U/mL。所用的荧光测定仪器为Perkin Elmer LS 55荧光分光光度计。图2为荧光探针与ALP作用的机理图,从图中可以看出,荧光探针与ALP发生反应后,使得磷酸根离去,生成CyOH,从而导致荧光发生显著变化。
实施例4:
荧光探针与ALP作用的紫外可见吸收光谱性质的测定
图3为荧光探针与ALP作用后的紫外可见吸收光谱图。从图3中可以看出,加入ALP之后,686nm处的吸收峰大幅度增强。紫外可见吸收光谱测定用的仪器为Perkin ElmerLambda 25型紫外可见分光光度计。
实施例5:
荧光探针与ALP作用的响应时间的测定
为了研究荧光探针对ALP的响应时间,我们考察了荧光探针在相同ALP浓度下(2.0U/mL)的荧光光谱的变化情况,其结果如图4。从图中可以看出,该探针对ALP的响应时间不到20min,满足在实际样品中进行实时监测时对响应时间的要求。
实施例6:
荧光探针对ALP测定的选择性
在浓度为10μM的荧光探针溶液中加入酶(ALP,Gox,trypsin,AchE,GDH,Gal,thrombin)、生物硫醇(Cys,Hcy,GSH)、氨基酸(Pro,Tyr,Asn,Met,Phe,Nleu,Asp,Orn,Ala,Trp,His,Ser,Gly,Val,Lys,Glu,Arg,Heu,Thr)及金属离子(K+,Ca2+,Zn2+,Na+,Mg2+)。从图5中可以看出,除ALP外,加入其它的酶,生物硫醇和其它氨基酸,荧光强度都没有明显的改变。F0和F表示探针溶液加入ALP前后的荧光强度。这些现象表现探针CyP对ALP的测定表现出良好的选择性。
实施例7:
荧光探针在活细胞中的应用
首先,我们做了细胞毒性试验,如图6所示,当加入0~30μM ALP探针,20min之后,细胞的成活率均在97%以上,因此可以说明,该荧光探针可应用于检测活细胞内的ALP,并且毒性较小。
一般情况下,细胞内的ALP含量非常丰富,因此直接向细胞内加入探针,也能检测到强的红色荧光信号。但是,当在加入探针之前加入一定量的ALP抑制剂Na3VO4时,细胞内没有荧光信号。(图7)。因此可以说明,该探针可高选择性的检测细胞内的ALP。
实施案例8:
荧光探针在组织中的应用
近红外的探针具有背景低,穿透性强的优点。如图8所示,(a)图是在小鼠肝冷冻切片中先加入10μM探针CyP的荧光成像图。(b)图是在小鼠肝冷冻切片中先加入ALP抑制剂Na3VO4,20min后再加入10μM探针CyP的荧光成像图。从图可以看出,直接向组织中加入探针,能检测到强的红色荧光信号。但是,当在加入探针之前加入一定量的ALP抑制剂Na3VO4时,组织中没有荧光信号。探针CyP的穿透深度范围为40-140μm,由此我们能得出探针CyP的组织穿透能力比较强。
Claims (5)
1.一种近红外碱性磷酸酶荧光探针,该探针被命名为CyP,其结构式如下:
。
2.根据权利要求1所述的一种近红外碱性磷酸酶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100 mL圆底烧瓶中,依次加入间苯二酚和碳酸钾,其摩尔比为1:1,加入20 mL乙腈作为溶剂,室温下,磁力搅拌10 min后,将溶解在20 mL乙腈中的IR-780溶液逐滴加入到反应液中,其中IR-780和间苯二酚的摩尔比为1:2,N2保护下,加热至50 ℃,继续搅拌4 h,反应完成,用旋转蒸发仪除去溶剂,粗产品以体积比为20:1的二氯甲烷/甲醇为淋洗剂,柱层析分离提纯,得蓝绿色固体,即CyOH,结构如下图所示:
;
2)将CyOH溶解在无水吡啶中,搅拌下逐滴加入三氯氧磷,其中CyOH和三氯氧磷的摩尔比为1:2,室温下搅拌4 h以后,往混合溶液中加入冰,继续在室温条件下搅拌24 h,反应完成,减压蒸馏除去溶剂,粗产品以体积比为2:1的二氯甲烷/甲醇为洗脱剂,柱层析分离提纯,得蓝色固体,即化合物CyP。
3.根据权利要求1所述的一种近红外碱性磷酸酶荧光探针的应用,其特征在于:用于溶
液中碱性磷酸酶的检测;所述荧光探针本身无荧光,与碱性磷酸酶反应生成CyOH,产生强烈
的红色荧光,线性范围为0.01-2.0 U/mL,检测限为0.003 U/mL,该探针表现出很高的灵敏
度;该荧光探针与碱性磷酸酶作用迅速,响应时间在20分钟以内;该荧光探针对碱性磷酸酶
表现出很好的选择性,不受其它酶、生物硫醇、氨基酸以及金属离子的影响;所述CyOH结构
式为。
4.根据权利要求1所述的一种近红外碱性磷酸酶荧光探针在制备成像试剂中的应用,其特征在于:用于检测细胞内的碱性磷酸酶,细胞内的碱性磷酸酶含量非常丰富,因此向细胞内加入探针后,能检测到强的红色荧光信号,但是,再加入一定量的抑制剂Na3VO4时,细胞内荧光信号消失。
5.根据权利要求1所述的一种近红外碱性磷酸酶荧光探针在制备成像试剂中的应用,其特征在于:用于检测组织中的碱性磷酸酶,组织中的碱性磷酸酶含量也非常丰富,因此向组织中加入探针后,能检测到强的红色荧光信号,但是,再加入一定量的抑制剂Na3VO4时,组织内荧光信号消失,另外,该探针的穿透深度范围为40-140 μm,说明该近红外探针具有良好的组织穿透性。
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EP2910550A2 (en) * | 2006-11-14 | 2015-08-26 | Enzo Therapeutics, Inc. | Compositions and methods for bone formation and remodeling |
CN101784897A (zh) * | 2007-05-23 | 2010-07-21 | 应用生物系统有限公司 | 通过从活化的化学发光的基质将能量转移至能量受体染料来检测生物分子的试剂、试剂盒和方法 |
CN104109176A (zh) * | 2014-06-06 | 2014-10-22 | 浙江工业大学 | 一种化合物及其应用于碱性磷酸酶活性的荧光检测方法 |
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Chunmiao Han.Mitochondria-Targeted Near-Infrared Fluorescent Off−On Probe for Selective Detection of Cysteine in Living Cells andin Vivo.《ACS Appl. Mater. Interfaces》.2015,27968−27975. |
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