CN106753341A - 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0.01‑2.0U/mL,检测限为0.003U/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的制备方法和应用,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 ℃,继续搅拌约4 h,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),结构如下所示:;
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)。
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ALP的检测;所述荧光探针本身无荧光,与ALP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ALP作用迅速,响应时间在20分钟以内;该荧光探针对ALP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。
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的应用,其特征在于:用于检测细胞内的ALP,细胞内的ALP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的应用,其特征在于:用于检测组织中的ALP,组织中的ALP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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