CN108896522A - 一种基于纳米荧光染料对水生生物肠道pH测定的方法 - Google Patents
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Abstract
本发明涉及一种基于纳米荧光染料对水生生物肠道pH测定的方法,其步骤如下:(1)将磷脂酰胆碱(DOPC)溶解于三氯甲烷的溶液中,制备成储备液并加入到玻璃瓶中;将大型溞培养液加入到玻璃瓶中;将10µM的荧光纳米染料加入到大型溞培养液中;利用荧光仪对不同pH条件下TPE‑Cy的荧光特性进行测定;利用激光扫描共聚焦显微镜对大型溞肠道进行扫描,对大型溞肠道整体荧光进行分析;本发明利用荧光纳米染料实现对大型溞肠道pH整体分布的测定方法,且操作简单便捷、灵敏度高,大幅提高了测定准确度。
Description
技术领域
本发明属于环境分析化学领域,涉及一种基于纳米荧光染料对水生生物肠道pH测定的方法,用于对大型溞肠道内pH的测定。
背景技术
大型溞作为一种常见的水生生物,广泛分布于湖泊、河流中。由于其容易养殖,生命周期较短以及半透明的特殊身体结构,大型溞已经成为一种广泛应用于毒理学,基因组学等研究中的模式生物。近年来,大型溞的肠道被广受关注。肠道作为大型溞主要的排泄器官,大量的食物经过摄取后,会长时间停留在肠道中,经过酶解以及代谢过程,将蛋白质以及糖类物质转化为生命活动所需要的营养物质,因此肠道对大型溞的生命活动起着重要作用。而肠道内的pH对消化酶的活性有着直接影响,最终影响食物慢慢的被迁移转化,吸收利用过程,因此,有必要对大型溞肠道内pH进行检测以更好的理解和调节肠道代谢过程。
微电极技(Brune, A., Emerson, D. and Breznak, J.A. Applied andEnvironmental Microbiology,1995,61,2681-2687)、荧光成像技术(Pond, D.W.,Harris, R.P. and Brownlee, C. Marine Biology, 1995, 123, 75-79.)因其具有操作简单,准备度高等优点,是目前测定水生生物肠道pH测定最为常用的两种技术。但是这两种技术都存在一定的局限性。微电极技术并不能实现对活体无损检测,在操作过程中,会对生物体造成致命性的损伤,同时这种技术只能实现对肠道内某些部位的pH的测定,并不能实现对整个肠道pH分布的测定。相反,荧光技术可以实现对浮游动物整个肠道pH分布的测定,但是传统的荧光材料的生物相容性较差,因此会对生物体产生一定程度的毒性。另外传统的荧光染料灵敏度较差,精确度相对较低,长时间激发可能导致荧光淬灭。近年来,聚集诱导发光技术在生物成像,重金属检测等领域取得重要突破,因此引起多领域学者的广发关注。因此,将聚集诱导发光纳米荧光染料用于浮游动物肠道pH的分布可实现对大型溞肠道内pH分布进行简单便捷以及准确的测定,对于研究大型溞肠道内代谢过程以及一些污染物在肠道内迁移转化、吸收利用有着重要意义。
发明内容
本发明的目的在于首次提供一种利用荧光纳米染料实现对大型溞肠道pH整体分布的测定方法,且操作简单便捷、灵敏度高,大幅提高了测定准确度。
本发明的技术方案如下:
一种基于纳米荧光染料对水生生物肠道pH测定的方法,其步骤如下:
(1)将磷脂酰胆碱(DOPC)溶解于三氯甲烷的溶液中,制备成储备液;
(2)将储备液加入到玻璃瓶中,利用氮气吹扫仪,去除三氯甲烷的溶液,使玻璃瓶瓶底形成一层DOPC薄膜;将大型溞培养液加入到玻璃瓶中;在超声震荡条件下,使得磷脂酰胆碱(DOPC)均匀分散在溶液中;
(3)将10µM的荧光纳米染料(tetraphenylethene-cyanine, TPE-Cy)加入到大型溞培养液中;利用荧光仪对不同pH条件下TPE-Cy的荧光特性进行测定;
(4)加入体积为10µL,浓度为10mM的TPE-Cy,然后加入大型溞;3小时后将大型溞取出,置于干净的培养液中清洗约2分钟;
(5)利用激光扫描共聚焦显微镜对大型溞肠道进行扫描,选择两个荧光通道,对大型溞肠道整体荧光进行分析;
(6)利用matlab对每个像素中两个荧光通道中I489/I615进行分析,将大型溞肠道内各个部位的I489/I615转换为相应的pH值。
优选地,所述步骤(1)中的储备液的浓度为1mg/mL。
优选地,所述步骤(2)中培养液由0.04 mM NaHCO3、0.35 mM CaSO4、0.50 mMMgSO4、和0.05 mM KNO3组成,并加入1 M的NaOH以及3-(N-吗啉)丙磺酸钠盐来调节溶液pH。
优选地,所述步骤(2)中磷脂酰胆碱(DOPC)在加入培养液后,其浓度为0.1mg/mL。
优选地,所述步骤(5)中两个荧光通道的激发光均为405nm,发射波长分别为450−520nm和580−650nm。
本发明的有益效果是:
与现有的可视化检测方法相比,本发明具有以下优点:
1、此方法可实现对大型溞进行活体成像,实现对肠道内pH的测定,对大型溞本身的伤害较小;
2、此方法操作简便,只需要将纳米荧光染料暴露于大型溞培养液中,一定时间后,对大型溞肠道内荧光特性进行分析,即可实现对肠道pH分布的测定;
3、灵敏度较高,分辨率达到0.25;
4、纳米荧光染料的生物相容性较强,对大型溞的毒性较低,基本可以忽略不计;
5、纳米荧光染料的稳定性较强,可实现在显微镜下,对大型溞长时间观测。
附图说明
图1为TPE-Cy在不同pH的溶液中荧光发射光谱;
图2为在DOPC存在条件下,TPE-Cy在不同pH的溶液中荧光发射光谱以及对应地溶液pH与I489/I615之间相关关系图;TPE-Cy浓度为10−5 M; DOPC浓度为0.1 mg/mL;I489与I615分别代表在激发波长为380 nm时,发射峰在489 nm以及615nm处的荧光强度;
图3为将大型溞暴露在pH=6.8的培养液后相应的荧光成像图;A代表激波长为405 nm,发射波段为450-520 nm通道所得到的的荧光图;B代表激波长为405 nm,发射波段为580−650 nm通道所得到的的荧光图;C代表A和B通道合并图;D代表将C图根据肠道内各个部位I489/I615不同,转化为相应的pH值,并且只保留了肠道;
图4为大型溞在不同pH的培养液中肠道pH的分布。A代表培养液pH=6.0, B代表 pH=6.8, C代表pH=7.8。
具体实施方式
为了便于本领域技术人员的理解,下面结合实施例对本发明技术方案做进一步的说明,实施方式提及的内容并非对本发明的限定。
一种基于纳米荧光染料对水生生物肠道pH测定的方法如下:
培养液的制备
大型溞培养液主要由0.04 mM NaHCO3, 0.35 mM CaSO4, 0.50 mM MgSO4, 0.05 mMKNO3组成,通过1 M的NaOH以及3-(N-吗啉)丙磺酸钠盐调节溶液的pH。
储备液的制备
将磷脂酰胆碱(DOPC)溶解于三氯甲烷的溶液中,制备成浓度为1mg/mL的储备液;
对照组设置
取八个相同的玻璃瓶并加入等体积的储备液,利用氮气吹扫仪,去除三氯甲烷的溶液,使玻璃瓶瓶底形成一层DOPC薄膜;取八份不同PH的大型溞培养液分别加入到八个玻璃瓶中,并使得磷脂酰胆碱(DOPC)的浓度为0.1 mg/mL;在超声震荡条件下,使得磷脂酰胆碱(DOPC)均匀分散在溶液中;
建立pH与TPE-Cy荧光特性相关关系
将10µM的荧光纳米染料(tetraphenylethene-cyanine, TPE-Cy)加入到不同pH的大型溞培养液;利用荧光仪对不同pH条件下TPE-Cy的荧光特性(激发波长,发射波长,峰高等性质)进行测定。当溶液pH<6.3时,TPE-Cy在615nm处有很强的发射峰,而当溶液pH>6.3时,TPE-Cy在488nm处有较强的发射峰(激光波长均为380nm);纳米荧光染料TPE-Cy在pH范围为4.5-8.0时,其在488nm与615nm处发射峰的峰高之间的比例(I489/I615)与pH之间有较好的的线性相关关系(y=0.481-2.865x,R2=0.976)。
大型溞肠道暴露实验
在每份大型溞培养液中均加入体积为10µL,浓度为10mM的TPE-Cy,然后在每份大型溞培养液中均加入一只大型溞;3小时后,溶液中的纳米荧光染料TPE-Cy会被大型溞摄入,大部分存在于肠道中,少部分会被吸附到身体表面;将大型溞取出,置于干净的培养液中清洗约2分钟,洗去表面吸附的荧光染料,减少干扰;
大型溞肠道荧光成像
利用激光扫描共聚焦显微镜对大型溞肠道进行扫描,选择激发光为405nm,发射波长分别为450−520 nm以及580−650 nm的两个荧光通道,对大型溞肠道整体荧光进行分析,其结果如图3(A),3(B)以及3(C)所示;
大型溞肠道pH的测定
利用matlab对每个像素中两个荧光通道中I489/I615进行分析,利用上述所建立的线性相关关系,将大型溞肠道内各个部位的I489/I615转换为相应的pH值。利用伪色对肠道内各个部位pH进行描述,其结果如图3D所示。
聚集诱导发光纳米荧光染料TPE-Cy已经被成功应用于水体环境中pH的测定(Chen, S.J., Liu, J.Z., Liu, Y., et al. Chemical Science, 2012, 3, 1804-1809)以及细胞内不同部位的pH的测定(Chen, S.J., Hong, Y.N., Liu, Y., Liu, J.Z., et al, Journal of the American Chemical Society, 2014, 136, 11196-11196)。与传统的荧光染料相比,TPE-Cy具有更好的水溶性以及生物相容性,对生物体的损坏极小,同时可提供较为精确的测定结果。
大型溞最为一种模式生物,其肠道内主要的脂类物质是甘油磷脂,对细胞膜的结构和功能起着重要作用。磷脂酰胆碱(1,2-dioleoyl-glycero-3-phosphocholine,DOPC)作为一种模式磷脂,因此常常被用作研究磷脂与其他物质之间的相互作用。甘油磷脂与磷脂酰胆碱都是甘油的衍生物,甘油磷脂严格意义上属于一种磷脂。因此通过研究DOPC与聚集诱导发光纳米荧光染料之间相互作用,在不同pH条件下,建立纳米荧光染料的荧光特性与pH之间的相关关系。将大型溞加入到含有浓度为10 µM的TPE-Cy的大型溞培养液中,一段时间后,TPE-Cy会被摄入到大型溞体内,大部分存在于肠道内。大型溞肠道内各个部位pH不同,肠道内荧光不均匀分布。基于pH与TPE-Cy荧光特性相关关系,可实现对大型溞肠道内pH分布进行简单便捷以及准确的测定。
本发明提供一种可视化的分析方法,可实现对大型溞的肠道内pH分布进行简单便捷以及准确的测定;与现有的可视化检测方法相比,本发明具有以下优点:
1、此方法可实现对大型溞进行活体成像,实现对肠道内pH的测定,对大型溞本身的伤害较小;
2、此方法操作简便,只需要将纳米荧光染料暴露于大型溞培养液中,一定时间后,对大型溞肠道内荧光特性进行分析,即可实现对肠道pH分布的测定;
3、灵敏度较高,分辨率达到0.25;
4、纳米荧光染料的生物相容性较强,对大型溞的毒性较低,基本可以忽略不计;
5、纳米荧光染料的稳定性较强,可实现在显微镜下,对大型溞长时间观测。
以上为本发明的较佳实施方式,但实施方式并不是用来限定本发明,在不脱离本发明之精神和范围内,所做的任何等效变化或润饰,同样属于本发明之保护范围,因此本发明的保护范围应当以本申请的权利要求所界定的内容为标准。
Claims (5)
1.一种基于纳米荧光染料对水生生物肠道pH测定的方法,其步骤如下:
(1)将磷脂酰胆碱(DOPC)溶解于三氯甲烷的溶液中,制备成储备液;
(2)将储备液加入到玻璃瓶中,利用氮气吹扫仪,去除三氯甲烷的溶液,使玻璃瓶瓶底形成一层DOPC薄膜;将大型溞培养液加入到玻璃瓶中;在超声震荡条件下,使得磷脂酰胆碱(DOPC)均匀分散在溶液中;
(3)将10µM的荧光纳米染料(tetraphenylethene-cyanine, TPE-Cy)加入到大型溞培养液中;利用荧光仪对不同pH条件下TPE-Cy的荧光特性进行测定;
(4)加入体积为10µL,浓度为10mM的TPE-Cy,然后加入大型溞;3小时后将大型溞取出,置于干净的培养液中清洗约2分钟;
(5)利用激光扫描共聚焦显微镜对大型溞肠道进行扫描,选择两个荧光通道,对大型溞肠道整体荧光进行分析;
(6)利用matlab对每个像素中两个荧光通道中I489/I615进行分析,将大型溞肠道内各个部位的I489/I615转换为相应的pH值。
2.根据权利要求1所述的一种基于纳米荧光染料对水生生物肠道pH测定的方法,其特征在于:所述步骤(1)中的储备液的浓度为1mg/mL。
3. 根据权利要求1所述的一种基于纳米荧光染料对水生生物肠道pH测定的方法,其特征在于:所述步骤(2)中培养液由0.04 mM NaHCO3、0.35 mM CaSO4、0.50 mM MgSO4、和0.05mM KNO3组成,并加入1 M的NaOH以及3-(N-吗啉)丙磺酸钠盐来调节溶液pH。
4.根据权利要求1所述的一种基于纳米荧光染料对水生生物肠道pH测定的方法,其特征在于:所述步骤(2)中磷脂酰胆碱(DOPC)在加入培养液后,其浓度为0.1mg/mL。
5.根据权利要求1所述的一种基于纳米荧光染料对水生生物肠道pH测定的方法,其特征在于:所述步骤(5)中两个荧光通道的激发光均为405nm,发射波长分别为450−520nm和580−650nm。
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