CN104530102A - 一种检测生物体内硫离子的荧光铜配合物及其应用 - Google Patents
一种检测生物体内硫离子的荧光铜配合物及其应用 Download PDFInfo
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Abstract
本发明公开了一种检测生物体内硫离子的荧光铜配合物及其应用,能够实现对生物体内硫离子的定量检测。荧光铜配合物的通式如下:检测生物体内硫离子的荧光铜配合物的应用包括:荧光铜配合物的体外细胞毒性测试;荧光铜配合物检测活体细胞内硫离子。检测活体细胞内硫离子包括:荧光铜配合物共聚焦荧光显微成像法检测活体细胞内硫离子;荧光铜配合物流式细胞术定量检测活体细胞内硫离子。本发明提供的铜荧光配合物具有较好的光稳定性,较高的荧光量子产率,较强的硫离子选择性,可以有效避免水体系以及复杂的生物体系内竞争物种的干扰。此外,该类铜配合物还具有较好的细胞通透性和较低的毒副作用。
Description
技术领域
本发明涉及检测生物体内硫离子的试剂及其应用,特别是一种检测生物体内硫离子的荧光铜配合物及其应用。
背景技术
硫化物是主要的环境污染物之一,它损害人的中枢神经、呼吸、消化和内分泌等系统。同时硫化氢是仅次于一氧化氮和一氧化碳的气体信号分子,在调节血压、调解神经递质、控制炎症、抗氧化以及清除活性氧等生理活动中发挥着重要的作用。细胞内硫化氢水平的变化与唐氏综合症、阿尔茨海默氏病、糖尿病等疾病的发生密切相关。因此,开发用于生物体内硫离子定性和定量检测的试剂,具有重要的生物医学应用价值。
荧光成像技术是一种能在亚细胞层次观察的可视化方法,可以实现对细胞内微细结构和分子的动态变化进行定性、定量、定时和定位分析。流式细胞术(Flowcytometry,FCM),是20世纪70年代发展起来的一项自动分析的技术。FCM通过激光光源激发细胞上所标记的荧光物质的强度和颜色以及散射光的强度可以得到细胞内部各种各样的生物信息。流式细胞术具有测量速度快、测量参数多、采集数据量大、分析全面、方法灵活以及对所需细胞进行分选等优点。荧光成像技术与流式细胞术技术在应用中都需要荧光标记物对细胞进行预先染色,而荧光标记物的靶向性是决定应用效果的一个关键因素。荧光探针是荧光成像所用到的一种主要的成像试剂,它是一种特殊的分子,具有灵敏度高、专一性强的优点。用硫离子荧光探针试剂对活体细胞进行特异性标记,进而通过荧光成像技术和流式细胞术研究活体细胞内硫离子的分布状态以及动态含量,在生物医学领域将具有广泛的应用前景。
发明内容
本发明提供了一种检测生物体内硫离子的荧光铜配合物及其应用,能够实现对生物体内硫离子的定量检测。
本发明提供的一种检测生物体内硫离子的荧光铜配合物的通式如下:
所述通式中的取代基R=H,或R=CH3,或R=NO2,或R=OCH3,或R=NH(C2H5)2,或R=OH,或R=C(CH3)3;所述通式中的荧光团F=荧光素,或F=香豆素,或F=1,8-萘酰亚胺,或F=4-氯-7-硝基-2,1,3-苯并氧杂恶二唑,或F=咔唑,或F=氟硼吡咯,或F=罗丹明。
一种检测生物体内硫离子的荧光铜配合物的应用,该应用的步骤包括:
一.荧光铜配合物的体外细胞毒性测试
采用噻唑蓝试验法测试荧光铜配合物对人乳腺癌细胞MDA-MB-231的毒性,取人乳腺癌细胞MDA-MB-231制成细胞悬液,接种于96孔的培养板上,每个剂量设置5个复孔,然后向每个孔中加入含定量荧光铜配合物的新鲜培养液,设立6个浓度组,置于含5%二氧化碳的培养箱内37℃下孵化,PBS洗涤后再加入MTT培养液,继续孵化后漂洗掉过量的MTT培养液,然后加入二甲基亚砜,摇床震荡,使用酶标仪测定各孔吸光度,以细胞活性百分比作为荧光铜配合物对细胞活性影响的评价指标,其计算公式为:
细胞活性百分比(%)=荧光铜配合物组各孔OD均值/对照组OD均值×100%;
二.荧光铜配合物共聚焦荧光显微成像法检测活体细胞内硫离子
在37℃下,将定量荧光铜配合物加入培养好的人乳腺癌细胞MDA-MB-231中,在培养基中培养20分钟,再向上述经荧光铜配合物染色的细胞培养液中加入定量硫离子,继续孵化30分钟,用PBS漂洗残留物,选择适当激发波长,通过共聚焦荧光显微镜成像。
所述一种检测生物体内硫离子的荧光铜配合物的应用,步骤二为荧光铜配合物流式细胞术定量检测活体细胞内硫离子,检测方法为:
将人乳腺癌细胞MDA-MB-231加入到6孔培养板中孵化,然后加入定量的荧光铜配合物并在湿润的37℃培养箱中孵化,继续再用适量的硫离子继续孵化,采用RPMI-1640作为分析对照,通过trypsin-EDTA溶液使细胞脱离培养板,用PBS洗涤3次后移入流式管中,进行流式细胞仪分析。
本发明与现有同类技术相比,其显著的有益效果体现在:
本发明选择的香豆素和1,8-萘酰亚胺等量子产率高、性能稳定、价格低廉。选择易制备的染料为荧光信号输出单元,制备了一系列水杨醛席夫碱衍生物,进而与铜离子络合制备了一种荧光铜配合物,应用于生物体内硫离子的定量检测。其识别动力来至于硫离子夺取了配合物中的铜离子并生成了稳定的硫化铜(CuS),从而恢复了因铜离子而淬灭的探针荧光。以荧光“关”到“开”这一模式识别硫离子,可以有效避免水体系以及更加复杂的生物体系内各种竞争物种对探针荧光的干扰,增强了铜配合物对硫离子识别的专一性和灵敏度。通过激光共聚焦成像和流体细胞术等技术的支持,该铜荧光配合物可应用于活体单细胞内硫离子的定性和定量分析。
本发明提供的铜荧光配合物具有较好的光稳定性,较高的荧光量子产率,较强的硫离子选择性,可以有效避免水体系以及复杂的生物体系内竞争物种的干扰。此外,该类铜配合物还具有较好的细胞通透性和较低的毒副作用。
附图说明
图1是人乳腺癌细胞MDA-MB-231的活性百分比与铜配合物浓度的关系图。
图2是依次用荧光铜配合物NL-Cu2+和硫离子孵化的活体人乳腺癌细胞MDA-MB-231内的共聚焦荧光成像图。
图3是流式细胞仪分析荧光铜配合物NL-Cu2+孵化的人乳腺癌细胞MDA-MB-231内的荧光强度。
图4是铜离子与硫离子调节细胞内荧光强度的“开-关-开”变化图。
具体实施方式
为了更清楚地说明本发明内容,用具体实施例说明,具体实施例不限定本发明内容范围。
实施例1
一种检测生物体内硫离子的荧光铜配合物NL-Cu2+的结构式如下:
荧光铜配合物NL-Cu2+的制备方法:
取4-肼基-7-硝基-2,1,3-苯并氧杂恶二唑(0.195g,1mmol)溶于15mL无水甲醇中,然后滴加含有1倍量水合肼的10mL甲醇溶液。加热回流6小时得到红黑色沉淀。冷却至室温,过滤,滤饼用冷的甲醇洗涤3次,得到目标荧光探针配体NL,产率80%。在HEPES缓冲体系(THF∶H2O=3∶7,20mM,pH=7.4)中,NL的浓度为10μM,然后加入20μM的Cu2+,制备的配合物NL-Cu2+经电喷雾电离质谱(ESI-MS)进行表征。m/z=468.0处的分子离子峰可归属于[NL-H+Cu+Cl+H]+,理论值:m/z=468.3。荧光铜配合物NL-Cu2+加入2倍量的硫离子,出现新的峰,m/z=369.1,可归属于[NL-H]-,证明硫离子夺去了配合物中的铜离子,生成了难溶性的硫化铜(CuS)产物,并释放出荧光探针配体NL,从而恢复了由于铜离子的顺磁性作用淬灭的荧光。
一种检测生物体内硫离子的荧光铜配合物的应用:
一.荧光铜配合物NL-Cu2+的细胞毒性测试
选用人乳腺癌细胞MDA-MB-231,采用噻唑蓝试验法(MTT)。取人乳腺癌细胞MDA-MB-231制成5×104个/毫升的细胞悬液,以每孔100μL接种于96孔的培养板上,每个剂量设置5个复孔。然后向每个孔中加入含荧光铜配合物NL-Cu2+的新鲜培养液,使NL-Cu2+的浓度分别为0(对照组)、2、4、6、10、20μM。在含5%二氧化碳的培养箱内37℃下孵化24小时,弃培养液,PBS洗涤。然后每孔加入PBS培养液,再加入100μL,0.5mg/mL的MTT培养液,继续孵化4小时后轻弃过量的MTT培养液,然后加入100μL的二甲基亚砜,摇床震荡。使用酶标仪于590nm波长测定各孔吸光度(OD值)。以细胞活性百分比作为荧光铜配合物NL-Cu2+对细胞活性影响的评价指标。实验结果如附图2所示,在37℃下,依次用2-20μM的NL孵化人乳腺癌细胞(MDA-MB-231)24小时后,没有发现显著的细胞增殖现象,即使用20μM的NL-Cu2+孵化24小时,细胞活性依然大于90%,证明荧光铜配合物NL-Cu2+对活体细胞具有较低的毒性,适合生物应用;
二.检测活体人乳腺癌细胞MDA-MB-231内的硫离子
(一)共聚焦荧光成像法检测活体人乳腺癌细胞MDA-MB-231内的硫离子
在37℃下,用5μM的荧光铜配合物NL-Cu2+孵化乳腺癌细胞MDA-MB-23120分钟,用PBS缓冲溶液冲洗过量的探针,共聚焦荧光显微成像,测量细胞的荧光强度。最后再用10μM的硫离子孵化细胞30分钟,共聚焦荧光显微成像。结果如附图3所示,孵化了NL-Cu2+的MDA-MB-231细胞内几乎没有荧光;然而继续用硫离子孵化后,细胞内呈现强的绿色荧光强度,实验结果证实荧光铜配合物NL-Cu2+具有较好的细胞通透性,可用于活体细胞内硫离子的检测;
(二)流式细胞法定量检测人乳腺癌细胞MDA-MB-231内的硫离子
活体人乳腺癌细胞MDA-MB-231(1×105个/毫升)加入6孔培养板中孵化24小时。然后加入荧光铜配合物NL-Cu2+(5μM)并在湿润的37℃培养箱中孵化半小时。去除上清液,并用PBS洗涤3次。然后再用10μM的硫离子继续孵化10分钟。采用RPMI-1640作为分析对照。结果如附图4所示,NL-Cu2+染色了99.3%的MDA-MB-231细胞。加入10μM的硫离子后大约98.6%的MDA-MB-231细胞内荧光铜配合物NL-Cu2+的荧光得到回复。另外,通过改变细胞内铜离子与硫离子浓度,实现对单细胞内荧光铜配合物NL-Cu2+荧光强度的“开-关-开”调控。
实施例2
一种检测生物体内硫离子的荧光铜配合物CL-Cu2+的结构式如下:
荧光铜配合物CL-Cu2+的制备:
同实施例1.
一种检测生物体内硫离子的荧光铜配合物的应用:
同实施例1。
实施例3
一种检测生物体内硫离子的荧光铜配合物GL-Cu2+的结构式如下:
荧光铜配合物GL-Cu2+的制备:
同实施例1。
一种检测生物体内硫离子的荧光铜配合物的应用:
同实施例1。
实施例4
一种检测生物体内硫离子的荧光铜配合物KL-Cu2+的结构式如下:
荧光铜配合物KL-Cu2+的制备:
同实施例1。
一种检测生物体内硫离子的荧光铜配合物的应用:
同实施例1。
实施例5
一种检测生物体内硫离子的荧光铜配合物BL-Cu2+的结构式如下:
荧光铜配合物BL-Cu2+的制备:
同实施例1。
一种检测生物体内硫离子的荧光铜配合物的应用:
同实施例1。
Claims (3)
1.一种检测生物体内硫离子的荧光铜配合物,其特征是该荧光铜配合物的通式如下:
所述通式中的取代基R=H,或R=CH3,或R=NO2,或R=OCH3,或R=NH(C2H5)2,或R=OH,或R=C(CH3)3;所述通式中的荧光团F=荧光素,或F=香豆素,或F=1,8-萘酰亚胺,或F=4-氯-7-硝基-2,1,3-苯并氧杂恶二唑,或F=咔唑,或F=氟硼吡咯,或F=罗丹明。
2.一种检测生物体内硫离子的荧光铜配合物的应用,其特征是该应用的步骤包括:
一.荧光铜配合物的体外细胞毒性测试
采用噻唑蓝试验法测试荧光铜配合物对人乳腺癌细胞MDA-MB-231的毒性,取人乳腺癌细胞MDA-MB-231制成细胞悬液,接种于96孔的培养板上,每个剂量设置5个复孔,然后向每个孔中加入含定量荧光铜配合物的新鲜培养液,设立6个浓度组,置于含5%二氧化碳的培养箱内37℃下孵化,PBS洗涤后再加入MTT培养液,继续孵化后漂洗掉过量的MTT培养液,然后加入二甲基亚砜,摇床震荡,使用酶标仪测定各孔吸光度,以细胞活性百分比作为荧光铜配合物对细胞活性影响的评价指标,其计算公式为:
细胞活性百分比(%)=荧光铜配合物组各孔OD均值/对照组OD均值×100%;
二.荧光铜配合物共聚焦荧光显微成像法检测活体细胞内硫离子
在37℃下,将定量荧光铜配合物加入培养好的人乳腺癌细胞MDA-MB-231中,在培养基中培养20分钟,再向上述经荧光铜配合物染色的细胞培养液中加入定量硫离子,继续孵化30分钟,用PBS漂洗残留物,选择适当激发波长,通过共聚焦荧光显微镜成像。
3.根据权利要求2所述的一种检测生物体内硫离子的荧光铜配合物的应用,其特征是步骤二为荧光铜配合物流式细胞术定量检测活体细胞内硫离子,检测方法为:
将人乳腺癌细胞MDA-MB-231加入到6孔培养板中孵化,然后加入定量的荧光铜配合物并在湿润的37℃培养箱中孵化,继续再用适量的硫离子继续孵化,采用RPMI-1640作为分析对照,通过trypsin-EDTA溶液使细胞脱离培养板,用PBS洗涤3次后移入流式管中,进行流式细胞仪分析。
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