CN105693736A - 一种含有甘氨酸结构的罗丹明光控荧光开关探针及其应用 - Google Patents
一种含有甘氨酸结构的罗丹明光控荧光开关探针及其应用 Download PDFInfo
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 title claims abstract description 50
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 title claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims description 33
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 24
- 239000007850 fluorescent dye Substances 0.000 claims description 18
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 17
- 238000010438 heat treatment Methods 0.000 claims description 16
- 239000007787 solid Substances 0.000 claims description 16
- -1 amino acid compounds Chemical class 0.000 claims description 14
- KQSSATDQUYCRGS-UHFFFAOYSA-N methyl glycinate Chemical compound COC(=O)CN KQSSATDQUYCRGS-UHFFFAOYSA-N 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 238000010992 reflux Methods 0.000 claims description 11
- 239000012295 chemical reaction liquid Substances 0.000 claims description 9
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 238000004440 column chromatography Methods 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 8
- 238000001704 evaporation Methods 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 6
- VYXSBFYARXAAKO-WTKGSRSZSA-N chembl402140 Chemical compound Cl.C1=2C=C(C)C(NCC)=CC=2OC2=C\C(=N/CC)C(C)=CC2=C1C1=CC=CC=C1C(=O)OCC VYXSBFYARXAAKO-WTKGSRSZSA-N 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- MUSLHCJRTRQOSP-UHFFFAOYSA-N rhodamine 101 Chemical compound [O-]C(=O)C1=CC=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MUSLHCJRTRQOSP-UHFFFAOYSA-N 0.000 claims description 5
- 229940043267 rhodamine b Drugs 0.000 claims description 5
- WGTODYJZXSJIAG-UHFFFAOYSA-N tetramethylrhodamine chloride Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C(O)=O WGTODYJZXSJIAG-UHFFFAOYSA-N 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 3
- MYIOYATURDILJN-UHFFFAOYSA-N rhodamine 110 Chemical compound [Cl-].C=12C=CC(N)=CC2=[O+]C2=CC(N)=CC=C2C=1C1=CC=CC=C1C(O)=O MYIOYATURDILJN-UHFFFAOYSA-N 0.000 claims 1
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- JNGRENQDBKMCCR-UHFFFAOYSA-N 2-(3-amino-6-iminoxanthen-9-yl)benzoic acid;hydrochloride Chemical compound [Cl-].C=12C=CC(=[NH2+])C=C2OC2=CC(N)=CC=C2C=1C1=CC=CC=C1C(O)=O JNGRENQDBKMCCR-UHFFFAOYSA-N 0.000 description 4
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- 238000002189 fluorescence spectrum Methods 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 238000013461 design Methods 0.000 description 2
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- 102000034287 fluorescent proteins Human genes 0.000 description 2
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- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/10—Spiro-condensed systems
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- C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
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Abstract
本发明公开一种含有甘氨酸结构的罗丹明光控荧光开关探针,其结构式如下所示。本发明的光控荧光开关探针可用于水溶液中pH 4-8的荧光检测,该探针分子能够快速的进入活细胞内部,其对光响应迅速,具有较高的灵敏度,并且具有良好的光可逆性,能够对线粒体进行光活化定位检测,另外,该探针还具有较好的化学稳定性,较好的溶解性和生物兼容性,不受其他常见的金属离子等物种的干扰。
Description
技术领域
本发明涉及一种含甘氨酸结构的罗丹明光控荧光开关探针及其作为生物体内的光控荧光开关探针的应用。
背景技术
近年来,荧光材料被广泛用于存储材料、化学传感以及生物体成像中,尤其是近期光控荧光开关材料被成功用于光活化定位、受激发射损耗显微技术、随机光学活化重构技术,并形成了新颖的荧光超分辨成像技术。然而目前超分辨成像技术中所使用的荧光材料多是荧光蛋白,基于有机荧光染料的光控开关材料却并不多见,加之荧光蛋白的制备以及生物染色方法较有机荧光染料相比复杂,因此急需研发具有光控荧光开关的荧光材料。S.Hell利用罗丹明的开关环反应即罗丹明隐色体来设计光控罗丹明荧光开关,是目前已知较为成功的设计模式。罗丹明染料具有优异的光稳定性、良好的水溶性、光谱可调节范围宽(500-700nm)、较高的发光效率(Φ=0.9-0.5),最为重要的是,其对pH(4.0-10.0)不敏感。比较典型的罗丹明有罗丹明6G,罗丹明B,四甲基罗丹明TMR,罗丹明101,罗丹明110等。罗丹明的2-羧基与伯胺反应后由于氨基的亲核性最终会形成闭环且结构稳定的罗丹明螺内酰胺结构,该结构在可见区无荧光无吸收,在光的诱导下激活发生开环反应,生成强荧光的罗丹明衍生物。罗丹明优异的光物理、光化学以及生物兼容性,使其成为设计光控荧光开关探针的首选。
发明内容
本发明目的提供一种含甘氨酸结构的罗丹明光控荧光开关探针,该光控荧光开关探针可用于水溶液中pH4-8的荧光检测,该探针分子能够快速的进入活细胞内部,其对光响应迅速,具有较高的灵敏度,并且具有良好的光可逆性,能够对线粒体进行光活化定位检测,另外,该探针还具有较好的化学稳定性,较好的溶解性和生物兼容性,不受其他常见的金属离子等物种的干扰。
本发明采用的技术方案为:
一种含有甘氨酸结构的罗丹明光控荧光开关探针,所述荧光探针为Rh-Gly荧光探针,其结构通式如(Ⅰ)所示:
其中,
R1=R2=R3=R4=H;
或R1=R4=H,R2=-CH2CH3,R3=-CH3;
或R1=R2=-CH3,R3=R4=H;
或R1=R2=-CH2CH3,R3=R4=H;
或R1=R4=-(CH2)3-,R2=R3=-(CH2)3-。
所述的一种含有甘氨酸结构的罗丹明光控荧光开关探针,所述的Rh-Gly荧光探针的制备方法包括如下步骤:
(1)罗丹明甘氨酸甲酯的制备
称取罗丹明类化合物于圆底烧瓶中,加入1,2-二氯乙烷至完全覆盖烧瓶底部,加入三氯氧磷,加热反应3-5小时后,冷却至室温,加入氨基酸类化合物的乙腈溶液,再向圆底烧瓶中逐滴滴入三乙胺,室温搅拌12-24h得反应液A;将反应液A用二氯甲烷萃取,取下层液,加入无水硫酸镁干燥,过柱提纯,得到白色固体;
(2)罗丹明甘氨酸(Rh-Gly)的制备
将白色固体溶于甲醇和过量的氢氧化钠溶液中,加热回流5-8小时后得反应液B,反应液B蒸干后,经柱层析提纯得到Rh-Gly荧光探针。
所述的含有甘氨酸结构的罗丹明光控荧光开关探针,罗丹明类化合物与三氯氧磷的摩尔比为1:(3-6);所用的氨基酸类化合物为甘氨酸甲酯。
所述的含有甘氨酸结构的罗丹明光控荧光开关探针,所述罗丹明类化合物为罗丹明B,罗丹明6G,四甲基罗丹明TMR,罗丹明110或罗丹明101。
所述的含有甘氨酸结构的罗丹明光控荧光开关探针,步骤1)中采用80-100℃的油浴加热反应3-5小时。
所述的含甘氨酸结构的罗丹明光控荧光开关探针,能够准确测定溶液中的pH,其pKa为5.8,pH检测范围为4.0-8.0。
一种含甘氨酸结构的罗丹明光控荧光开关探针可作为光控荧光开关在活细胞内光控荧光开与关的应用。所述的活细胞为Hela细胞株、MCF-7细胞株或RAW264.7细胞株。
本发明中罗丹明光控荧光开关荧光探针的制备反应式如下:
本发明的有益效果是:本发明的光控荧光开关探针可用于水溶液中pH4-8的荧光检测,该探针分子能够快速的进入活细胞内部,其对光响应迅速,具有较高的灵敏度,并且具有良好的光可逆性,能够对线粒体进行光活化定位检测,另外,该探针还具有较好的化学稳定性,较好的溶解性和生物兼容性,不受其他常见的金属离子等物种的干扰。
附图说明
图1是实施例1中RhB-Gly对pH变化的荧光的光谱响应。
图2a是在pH为4.5时不同金属离子对RhB-Gly荧光强度的影响
图2b是在pH为7时不同金属离子对RhB-Gly荧光强度的影响
图3是实施例1中RhB-Gly荧光强度的pH可逆性。
图4是实施例1中RhB-Gly与Rh123的线粒体共定位成像。
图5是实施例1中RhB-Gly光控荧光开与关的细胞成像。
具体实施方式
一种含有甘氨酸结构的罗丹明光控荧光开关探针,所述荧光探针为Rh-Gly荧光探针,其结构通式如(Ⅰ)所示:
其中,
R1=R2=R3=R4=H;
或R1=R4=H,R2=-CH2CH3,R3=-CH3;
或R1=R2=-CH3,R3=R4=H;
或R1=R2=-CH2CH3,R3=R4=H;
或R1=R4=-(CH2)3-,R2=R3=-(CH2)3-。
一种含有甘氨酸结构的罗丹明光控荧光开关探针(Rh-Gly)的制备方法包括如下步骤:
(1)罗丹明甘氨酸甲酯的制备
称取罗丹明类化合物于圆底烧瓶中,加入1,2-二氯乙烷至完全覆盖烧瓶底部,加入三氯氧磷,80-100℃的油浴加热反应3-5小时,冷却至室温,加入氨基酸类化合物的乙腈溶液,再向圆底烧瓶中逐滴滴入三乙胺,室温搅拌12-24h得反应液A;将反应液A用二氯甲烷萃取,取下层液,加入无水硫酸镁干燥,过柱提纯,得到白色固体;其中罗丹明类化合物与三氯氧磷的摩尔比为1:(3-6);所用的氨基酸类化合物为甘氨酸甲酯。罗丹明类化合物为罗丹明B,罗丹明6G,四甲基罗丹明TMR,罗丹明110或罗丹明101。
(2)罗丹明甘氨酸(Rh-Gly)的制备
将白色固体溶于甲醇和过量的氢氧化钠溶液中,加热回流5-8小时后得反应液B,反应液B蒸干后,经柱层析提纯得到Rh-Gly荧光探针。
上述含甘氨酸结构的罗丹明光控荧光开关探针,能够准确测定溶液中的pH,其pKa为5.8,pH检测范围为4.0-8.0。
上述含甘氨酸结构的罗丹明光控荧光开关探针可作为光控荧光开关在活细胞内光控荧光开与关的应用。所述的活细胞为Hela细胞株、MCF-7细胞株或RAW264.7细胞株。
实施例1一种含有甘氨酸结构的罗丹明光控荧光开关探针(RhB-Gly荧光探针)
将1摩尔量的罗丹明B与5摩尔量的POCl3加入到干燥的1,2-二氯乙烷中,90℃油浴反应4h,冷却至室温后,加入1摩尔量的甘氨酸甲酯的乙腈溶液(称取甘氨酸甲酯0.35g用乙腈溶解),再逐滴滴入3摩尔量的三乙胺,室温下搅拌24小时得反应液A,反应液A用二氯甲烷萃取,取下层液,再用无水硫酸镁干燥,经硅胶柱层析提纯,得到白色固体。将该白色固体溶于甲醇中,再加入3摩尔量氢氧化钠饱和水溶液,70-90℃加热回流7小时后得反应液B,反应液B蒸干后,经柱层析提纯得到目标产物RhB-Gly荧光探针。HRMS:500.2540
实施例2Rh6G-Gly荧光探针
将1摩尔量罗丹明6G与3-6摩尔量POCl3加入到干燥的1,2-二氯乙烷中,90℃油浴反应4h,冷却至室温后,加入1摩尔量的甘氨酸甲酯的乙腈溶液,再逐滴滴入3摩尔量的三乙胺,室温下搅拌24小时得反应液A,反应液A用二氯甲烷萃取,取下层液,再用无水硫酸镁干燥,经硅胶柱层析提纯,得到白色固体。将该白色固体溶于甲醇中,再加入3摩尔量氢氧化钠饱和水溶液,70-90℃加热回流7小时后得反应液B,反应液B蒸干后,经柱层析提纯得到目标产物Rh6G-Gly荧光探针。HRMS:471.2143
实施例3RhT-Gly荧光探针
将1摩尔量四甲基罗丹明TMR与3-6摩尔量POCl3加入到干燥的1,2-二氯乙烷中,加热回流反应3-5小时,冷却至室温后,加入1摩尔量的甘氨酸甲酯的乙腈溶液,再逐滴滴入3摩尔量的三乙胺,室温下搅拌24小时得反应液A,反应液A用二氯甲烷萃取,取下层液,再用无水硫酸镁干燥,经硅胶柱层析提纯,得到白色固体。将该白色固体溶于甲醇中,再加入3摩尔量氢氧化钠饱和水溶液,70-90℃加热回流7小时后得反应液B,反应液B蒸干后,经柱层析提纯得到目标产物RhT-Gly荧光探针。HRMS:444.1898
实施例4Rh110-Gly荧光探针
将1摩尔量罗丹明110与3-6摩尔量的POCl3加入到干燥的1,2-二氯乙烷中,加热回流反应3-5小时,冷却至室温后,加入1摩尔量的甘氨酸甲酯的乙腈溶液,再逐滴滴入3摩尔量的三乙胺,室温下搅拌24小时得反应液A,反应液A用二氯甲烷萃取,取下层液,再用无水硫酸镁干燥,经硅胶柱层析提纯,得到白色固体。将该白色固体溶于甲醇中,再加入3摩尔量氢氧化钠饱和水溶液,70-90℃加热回流7小时后得反应液B,反应液B蒸干后,经柱层析提纯得到目标产物Rh110-Gly荧光探针。HRMS:388,1321
实施例5Rh101-Gly荧光探针
将1摩尔量的罗丹明101与3-6摩尔量的POCl3加入到干燥的1,2-二氯乙烷中,加热回流反应3-5小时,冷却至室温后,加入1摩尔量的甘氨酸甲酯的乙腈溶液,再逐滴滴入3摩尔量的三乙胺,室温下搅拌24小时得反应液A,反应液A用二氯甲烷萃取,取下层液,再用无水硫酸镁干燥,经硅胶柱层析提纯,得到白色固体。将该白色固体溶于甲醇中,再加入3摩尔量氢氧化钠饱和水溶液,70-90℃加热回流7小时后得反应液B,反应液B蒸干后,经柱层析得到目标产物Rh101-Gly荧光探针。HRMS:548,2540
实施例6应用试验
本应用试验采用实施例1制备的RhB-Gly荧光探针进行。
[1]荧光光谱的测定。配制浓度为5×10-5mol/L的含有30%乙醇的RhB-Gly探针水溶液,该探针溶液分别用氢氧化钠和盐酸调节pH,测试荧光光谱,如图1所示。结果表明,随着pH值的降低,荧光强度不断增强。
[2]金属离子干扰性测定,分别配制浓度为5×10-5mol/L的含有30%乙醇的探针RhB-Gly溶液,其pH分别为4.5和7。pH为4.5的探针溶液中分别加入金属盐的量为探针摩尔量的100倍,进行荧光光谱测定。pH为7的探针溶液中分别加入金属盐的量为探针摩尔量的100倍,进行吸收光谱测定。结果如图2a和图2b所示:Ni2+,Sn2+,k+,Fe2+,Zn2+,Cu+,Cu2+,Na+,Cd2+等金属离子均对探针没有影响,说明探针稳定性较好,适用于复杂环境下的光致变色应用。
[3]可逆性测试。光谱测定探针RhB-Gly在pH在~4和~7.5荧光变化的可逆性。用NaOH,HCl调节pH在4和7.5左右,进行荧光光谱测定。如图3所示结果表明,该探针响应迅速,荧光强度变化可逆。探针RhB-Gly在pH为4和7区间内,荧光变化迅速且稳定。
[4]线粒体共定位。向上述呈现明显红色荧光的细胞中加入线粒体荧光染色剂Rh123(终浓度2μM)并在37℃下抚育5min,之后用磷酸盐缓冲液冲洗三次。将清洗后的MCF-7细胞置于共聚焦荧光显微镜下观测,先经370-400激光激活后,分别采用488nm和559nm的激发光源,进行双通道荧光信号的采集,如4图所示。Ch1信号采集范围500-530nm,观测到线粒体中呈现出强烈的绿色荧光,Ch2信号采集范围560-690nm,可以观测到较强的红色荧光。由于Rh123具有线粒体专一性定位功能,这为细胞内线粒体位置的确定提供了参比。将Ch1和Ch2通道进行合并叠加,合并后的细胞线粒体中呈现出明显的黄色荧光,说明通道Ch1和Ch2能够较好的重叠。以上实验结果均表明探针RhB-Gly能够定位于线粒体中并有良好的光控荧光开关性能。
[5]细胞成像:将探针RhB-Gly(5mM)的二甲基亚砜溶液2μL加入到含有2mL培养基(RPMI-1640,含10%小牛血清)的MCF-7细胞中37℃下抚育5分钟,然后将抚育后的MCF-7细胞用PBS磷酸盐缓冲液冲洗(pH=7.2,1mL×3次),将清洗后的细胞置于共聚焦荧光显微镜下观察,细胞内没有荧光。经370-400激光激活后,采用559nm的激发光源,进行单通道荧光信号的采集,间隔不同的时间(0,1,3,5min)采集荧光图片,结果如5所示,发现探针RhB-Gly可以实现线粒体中光控荧光的开与关。
Claims (8)
1.一种含有甘氨酸结构的罗丹明光控荧光开关探针,其特征在于,所述荧光探针为Rh-Gly荧光探针,其结构通式如(Ⅰ)所示:
其中,
R1=R2=R3=R4=H;
或R1=R4=H,R2=-CH2CH3,R3=-CH3;
或R1=R2=-CH3,R3=R4=H;
或R1=R2=-CH2CH3,R3=R4=H;
或R1=R4=-(CH2)3-,R2=R3=-(CH2)3-。
2.如权利要求1所述的一种含有甘氨酸结构的罗丹明光控荧光开关探针,其特征在于,所述的Rh-Gly荧光探针的制备方法包括如下步骤:
(1)罗丹明甘氨酸甲酯的制备
称取罗丹明类化合物于圆底烧瓶中,加入1,2-二氯乙烷至完全覆盖烧瓶底部,加入三氯氧磷,加热反应3-5小时后,冷却至室温,加入氨基酸类化合物的乙腈溶液,再向圆底烧瓶中逐滴滴入三乙胺,室温搅拌12-24h得反应液A;将反应液A用二氯甲烷萃取,取下层液,加入无水硫酸镁干燥,过柱提纯,得到白色固体;
(2)罗丹明甘氨酸(Rh-Gly)的制备
将白色固体溶于甲醇和过量的氢氧化钠溶液中,加热回流5-8小时后得反应液B,反应液B蒸干后,经柱层析提纯得到Rh-Gly荧光探针。
3.如权利要求2所述的含有甘氨酸结构的罗丹明光控荧光开关探针,其特征在于,罗丹明类化合物与三氯氧磷的摩尔比为1:(3-6);所用的氨基酸类化合物为甘氨酸甲酯。
4.如权利要求2所述的含有甘氨酸结构的罗丹明光控荧光开关探针,其特征在于,所述罗丹明类化合物为罗丹明B,罗丹明6G,四甲基罗丹明TMR,罗丹明110或罗丹明101。
5.如权利要求2所述的含有甘氨酸结构的罗丹明光控荧光开关探针,其特征在于,步骤1)中采用80-100℃的油浴加热反应3-5小时。
6.一种如权利要求1所述的含甘氨酸结构的罗丹明光控荧光开关探针,能够准确测定溶液中的pH,其pKa为5.8,pH检测范围为4.0-8.0。
7.一种如权利要求1所述的含甘氨酸结构的罗丹明光控荧光开关探针可作为光控荧光开关在活细胞内光控荧光开与关的应用。
8.如权利要求7所述的应用,其特征在于,所述的活细胞为Hela细胞株、MCF-7细胞株或RAW264.7细胞株。
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