CN117740743A - 基于荧光共轭聚电解质的多巴胺传感体系、制备及检测方法 - Google Patents
基于荧光共轭聚电解质的多巴胺传感体系、制备及检测方法 Download PDFInfo
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Abstract
本发明公开了一种基于荧光共轭聚电解质的多巴胺传感体系、制备及检测方法。分别合成含二甲氨基侧基的二乙炔基单体、二溴芳烃单体,再通过Sonogashira偶联反应合成含二甲氨基侧基的共轭聚电解质前驱体,经季铵化反应并脱除保护基团,得到含苯硼酸侧基的荧光共轭聚电解质,溶解于甲醇与水的混合溶剂中,加入甘氨酸‑氢氧化钠缓冲液,得到多巴胺传感体系。检测液能随体系中多巴胺浓度的增加表现出迅速高效的荧光淬灭响应。本发明提供的传感体系及检测方法实现了对多巴胺快速、灵敏和有选择性的检测,可应用于复杂样品的分析、检验。
Description
技术领域
本发明涉及一种多巴胺传感材料,特别涉及一种含高密度苯硼酸侧基的荧光共轭聚电解质,及用荧光分析法对多巴胺浓度进行测定的方法,属于高分子荧光材料应用领域。
背景技术
多巴胺(DA)是儿茶酚胺神经递质之一,可调节多种生物过程。多巴胺的异常释放可导致体内多种疾病,包括亨廷顿氏病、精神疾病和帕金森氏病。此外,体内多巴胺的水平还与对某些药物(如大麻和可卡因)的成瘾有关。因此,灵敏地检测多巴胺对于临床诊断和生物医学研究至关重要。
高效液相色谱法(HPLC)和电化学法是目前在复杂生物环境中检测多巴胺的常用方法,可实现多巴胺的高效分离和精确检测。然而,它们都需要大型仪器作为技术支撑。基于荧光的传感技术因其灵敏度高、反应迅速和操作简单而引起了极大的研究兴趣基于不同类型荧光材料(如碳点、纳米粒子和荧光有机小分子)的多巴胺传感方法已有很多文献报道(参见文献:Sens. Actuators B Chem. 2015, 220, 146-153;J. Photochem. Photobiol.A. 2020, 392, 112438)。这些方法为不同应用领域的多巴胺检测提供了多种选择,但每种方法也都存在或多或少的缺点,如表面修饰复杂、孵育时间相对较长等。
共轭聚电解质是具有离子性侧基的共轭聚合物,因其良好的亲水性经常被用于检测水介质中的生物分子。这类材料不仅可以利用“分子导线效应”增强检测信号,同时还可以通过引入特殊官能团,实现特异性响应。(参见文献:Angew Chem Int Edit. 2009, 48,4300-4316)苯硼酸可与路易斯碱和多元醇进行可逆共价结合,因此已被用作检测碳水化合物的受体以及相关的细胞和组织成像。多巴胺是一种可溶于水相的小分子神经递质,其儿茶酚结构也可与苯硼酸结合。(参见文献:Sens. Actuators B Chem. 2014, 191, 227-232),因此,将共轭聚合物与苯硼酸结合起来有望构建一种新型多巴胺荧光传感体系。相关文献曾报道过一种含苯硼酸侧基的聚合物(参见文献:ACS Appl Mater Interfaces.2015, 7, 18581-18589),首先采用Suzuki偶联法制备一种侧基末端含氨基的聚合物前驱体,接着通过酰胺化反应在聚合物侧链修饰聚乙二醇和苯硼酸两种基团,并将其制备成纳米粒子用于多巴胺检测及体内成像。这种方法的检测机理为光诱导电子转移,具有响应迅速、灵敏度高等优势;然而,该聚合物需制备成纳米粒子才能够溶于水中;且在报道中聚合物的荧光未被完全淬灭,可能是聚合物侧基引入的苯硼酸基团密度不高。因此,设计合成一种水溶性好、含高密度苯硼酸侧基的共轭聚电解质有望实现更高效的多巴胺检测。
发明内容
本发明针对现有技术存在的不足,提供一种荧光强度稳定、可选择性与儿茶酚基团可逆结合,并通过光诱导电子转移(PET)过程迅速表现出高效淬灭响应的含高密度苯硼酸侧基的荧光共轭聚电解质检测液的制备方法及其应用。
实现本发明目的的技术方案是提供一种基于荧光共轭聚电解质的多巴胺传感体系,它包括浓度为0.02~0.1 mol/L的甘氨酸-氢氧化钠缓冲液,甲醇和水按体积比为1:1~9:1的混合溶剂,按重复单元计浓度为10-6~10-4mol/L的含苯硼酸侧基的荧光共轭聚电解质;所述传感体系的pH为8~10;所述的含苯硼酸侧基的荧光共轭聚电解质的结构式为:
,
其中,n为重复单元数,n=8~21。
本发明技术方案包括一种基于荧光共轭聚电解质的多巴胺传感体系的制备方法,如下步骤:
(1)按物质的量计,将1份2,5-双(3'-(N,N-二甲基氨基)丙基)-1,4-二(三甲基硅基乙炔)苯,2~2.5 份n-Bu4NF与1100~1200份四氢呋喃的混合物,搅拌至固体溶解,再在室温下搅拌0.5~1小时后,经萃取、旋蒸、中性氧化铝柱层析分离提纯和干燥处理,得到一种二乙炔基单体;
(2)在无水无氧条件下,按物质的量计,将1份二溴芳烃单体,1份步骤(1)得到的二乙炔基单体,0.015~0.05份四(三苯基膦)二氯化钯,0.015~0.05份碘化亚铜,100~200份二异丙胺,200~300份甲苯加入到反应器中混合搅拌,在氩气保护、温度为70~75℃的条件下反应30~50小时,再经过滤、重结晶、离心、干燥处理,得到一种共轭聚电解质前驱体,其结构式为;
,
其中,n为重复单元数,n=8~21;
(3)按物质的量计,将1份步骤(2)制备的共轭聚电解质前驱体与3500~3600份四氢呋喃混合,在温度为60~66℃的油浴条件下加入35~55份4-溴甲基苯硼酸频哪醇酯进行反应,反应时间为48~72小时,反应期间分2~3次共加入400~900份甲醇; 反应结束后,冷却至室温,加入35~55份4-溴甲基苯硼酸和80~100份盐酸,搅拌20~40分钟后,置于丙酮/乙醚体积比为1:9~1:7的混合溶剂中沉淀,再经离心、干燥处理,得到一种含苯硼酸侧基的荧光共轭聚电解质;
(4)将步骤(3)制备得到的含苯硼酸侧基的荧光共轭聚电解质溶解于体积比为1:1~9:1的甲醇与水的混合溶剂中,加入pH为8~10的甘氨酸-氢氧化钠缓冲液充分混合,得到按重复单元计含苯硼酸侧基的荧光共轭聚电解质浓度为10-6~10-4 mol/L,甘氨酸-氢氧化钠浓度为0.02~0.1 mol/L的多巴胺传感体系。
本发明技术方案还提供一种基于荧光共轭聚电解质的多巴胺传感体系的检测方法,按10~30 mL多巴胺传感体系中加入5~20 μL的多巴胺待测溶液为待测样,以多巴胺传感体系为空白样,分别将空白样和待测样用滴管吸入石英比色皿中,采用395 nm波长的激发光,分别记录空白样、待测样最大发射波长处的荧光强度I 0 和I,依据公式 (1 -I /I 0 ) ×100%计算得到淬灭效率。
由于多巴胺是一种小分子神经递质,在水环境中,可通过邻二酚结构与苯硼酸侧基可逆共价结合,并将电子转移至聚合物激发态下的空电子轨道内,从而使聚合物的激发态电子无法返回基态轨道,表现为聚合物的蓝色荧光显著淬灭。本发明利用这一特性,提供一种新型的荧光传感器,在多巴胺存在下产生荧光淬灭,实现快速、高效、便捷地检测多巴胺。
与现有技术相比,本发明的有益效果在于:
1.本发明制备的含高密度苯硼酸侧基的荧光共轭聚电解质的高密度苯硼酸侧基与多巴胺间为可逆共价相互作用,相比于氢键、静电等相互作用稳定性更高。因此在结合过程中精确度更好。该传感体系的机理为光诱导电子转移,与血清、尿液等样品中可能存在的干扰物如氨基酸和蛋白质(丝氨酸、半胱氨酸、谷胱甘肽、γ-球蛋白、人血清白蛋白)、多元醇(葡萄糖、果糖、半乳糖、抗坏血酸)、阳离子(Na+、K+、Ca2+)和代谢产物(尿素、尿酸)等相比,本传感体系对多巴胺具有高选择性。
2. 本发明制备的含高密度苯硼酸侧基的荧光共轭聚电解质在极性溶剂中溶解良好,且具有优异的光稳定性。
3.与基于HPLC/比色法/电化学的方法和基于量子点/有机小分子/金属复合物的荧光传感相比,本发明提供的含高密度苯硼酸侧基的荧光共轭聚电解质表现出快速响应和相对低的检测限的综合优势。与荧光共轭聚合物、荧光有机小分子这类化学传感体系相比,本发明提供的检测体系其灵敏度(以计算所得的Stern-Volmer常数(KSV)表示)也具有一定优势。
4.与现有的多巴胺传感材料相比,本发明提供的含高密度苯硼酸侧基的荧光共轭聚电解质溶液浓度很低,可以减少聚合物的用量,降低检测成本,减少浪费。
5.本发明提供的检测方法操作过程方便简单,无需任何前处理或后处理,响应过程迅速,能够在几分钟之内得出检测结果。
6.与现有的含苯硼酸基团的聚合物传感方法相比,本发明提供的含高密度苯硼酸侧基的荧光共轭聚电解质不仅合成高效简捷,在极性溶剂中溶解良好,并且重复单元中含有更高密度的苯硼酸基团,提供了更多与多巴胺的反应位点,因此具有更高的荧光淬灭响应范围。
附图说明
图1和2分别是本发明实施例1中含高密度苯硼酸侧基的荧光共轭聚电解质(PFPE-PBA)的核磁谱图和红外谱图;
图3是本发明实施例1中含高密度苯硼酸侧基的荧光共轭聚电解质(PFPE-PBA)的光物理性能数据图;
图4是本发明实施例2中聚电解质(PFPE-PBA)对多巴胺响应的荧光发射谱图;
图5是452 nm处荧光强度I0/I与多巴胺浓度之间的Stern-Volmer图;
图6是本发明实施例3中向聚电解质(PFPE-PBA)溶液中加入15 μM DA后,荧光淬灭效率随时间变化图。
图7是聚电解质(PFPE-PBA)溶液的452 nm处的荧光强度在干扰物质存在的情况下,加入多巴胺前后的柱状图;
图8是本发明实施例3中聚电解质(PFPE-PBA)在不同pH值情况下添加多巴胺之前和之后452 nm处荧光强度的柱状图。
实施方式
下面结合附图和实施例对本发明技术方案作进一步的阐述。
实施例1
本实施例提供一种含高密度苯硼酸侧基的荧光共轭聚电解质多巴胺传感体系及检测方法。
多巴胺传感体系的制备方法具体操作步骤如下:
(1)将2,5-双(3'-(N,N-二甲基氨基)丙基)-1,4-二(三甲基硅基乙炔)苯(1.42 g,3 mmol)置于100 mL的烧瓶中,加入 20 mL的四氢呋喃搅拌至固体溶解,然后加入6 mL溶于四氢呋喃的n-Bu4NF (1 M)。将该混合物在室温下搅拌0.5小时。结束反应后,向反应混合物中加入30 mL去离子水,并用3 ´ 100 mL的二氯甲烷萃取。合并有机相,加入无水硫酸钠以除去水分。在减压下旋转蒸发除去多余的溶剂,得到粗品,在中性氧化铝柱上以二氯甲烷/石油醚(2∶1,v/v)为淋洗剂纯化,得到一种二乙炔基单体记作M1,其结构式为:
。
(2)在氩气下,向烧瓶中加入2,7-二溴芴(3.05 g,9.4 mmol)和60 mL DMSO并搅拌至溶解,接着加入四丁基溴化铵(60 mg)和8 mL质量分数为50%的氢氧化钠水溶液。同样在氩气氛围下向该混合物中滴加20 mL的3-二甲基氨基氯丙烷盐酸盐(3.51 g,22 mmol)的DMSO溶液。反应混合物在室温下搅拌6小时。停止反应,然后用50 mL水稀释以溶解所有的盐。用乙醚(3 ´ 100 mL)萃取产物,并用去离子水(3 ´ 100 mL)和饱和氯化钠(100 mL)洗涤。合并有机层,经MgSO4干燥,过滤,减压除去溶剂,得到的粗产物为绿色固体。将粗产物在MeOH/H2O中重结晶,得到一种二溴芴单体记作M2,其结构式为:
;
(3)在氩气的保护下,将M1(0.082 g,0.25 mmol)、M2(0.12 g,0.25 mmol)、(PPh3)2PdCl2(0.0142 g,0.02 mmol)和CuI(0.0042 g,0.02 mmol)加入烧瓶中,在氩气氛围下向烧瓶中加入干DIPA(3 mL)和甲苯(8 mL),得到的混合物在氩气下回流48小时后,冷却至室温,用孔径为0.22 μm的滤头过滤该混合物。滤液经减压浓缩并滴入100 mL的正己烷中,置于冰箱上层过夜。离心处理(3分钟,3000 rpm)后,收集沉淀物,在真空条件下干燥处理24小时,得到暗红色固体为共轭聚电解质前驱体,记作PFPE-NMe2,其结构式为:
,
其中,n为重复单元数,n约为10。
(4)将步骤(3)制备的前驱体PFPE-NMe2(0.075 g,0.1 mmol)和四氢呋喃(10 mL)放入100 mL的烧瓶中,搅拌至聚合物溶解,并在60 ℃的油浴条件下向烧瓶中加入4-溴甲基苯硼酸频哪醇酯(1.20 g,4 mmol)。在此期间,一旦烧瓶中出现固体,就加入约3 mL的甲醇,共消耗9 mL的甲醇。反应48小时后,停止加热并冷却至室温,加入4-溴甲基苯硼酸(0.85 g,4 mmol)和盐酸(0.5 mL),在室温下搅拌30分钟后停止反应;得到的混合液通过减压蒸馏除去大部分溶剂,并在100 mL的混合溶剂(丙酮/乙醚,1∶9,v/v)中沉淀,经离心、干燥处理,得到一种含苯硼酸侧基的荧光共轭聚电解质,记作PFPE-PBA,其结构式为:
,
其中,n为重复单元数,n约为10。
(5)将步骤(4)制备得到的含苯硼酸侧基的荧光共轭聚电解质PFPE-PBA固体溶于甲醇与水体积比为9:1的混合溶液中,再与pH为10的甘氨酸-氢氧化钠混合,得到检测液,聚合物浓度为10-5 mol/L(按重复单元计),检测液中缓冲剂的最终浓度浓度为0.02 mol/L。
参见附图1和2,分别是本实施例提供的聚电解质PFPE-PBA的核磁谱图和红外谱图。
参见附图3,它是本实施例提供的聚电解质PFPE-PBA的光物理性能的表征图。由图3可知,PFPE-PBA的最大吸收波长在400 nm左右,最大发射波长在460 nm附近。
实施例2
本实施例利用实施例1提供的聚电解质PFPE-PBA检测液,用于检测多巴胺。
待测样制备:取20 mL检测液置于50 mL锥型瓶中,每次滴加极小体积的分析物溶液(10 μL),摇匀后用移液枪吸取2.5 mL液体转移至比色皿中进行荧光测试;多巴胺溶液用去离子水配制,浓度已知。每次滴加之后20 mL检测液中多巴胺的浓度可由计算得出。
荧光测试:将待测样溶液用一次性滴管吸入石英比色皿中,然后进行荧光测试。测试时,选择395 nm作为激发波长(狭缝宽度为3 nm/3 nm)。将不加入待测物的检测液作为空白样,记录其最大发射波长处荧光强度为I0;记录添加待测样30s之后的最大发射波长处的荧光强度为I。所有的荧光测试都至少重复三次。
参见附图4,它是本实施例按照上述方法测得的荧光共轭聚电解质PFPE-PBA在甲醇/水=9:1混合溶剂中与不同浓度多巴胺响应后的荧光发射光谱。由图4可以看出聚合物溶液的荧光强度随多巴胺浓度增加有明显的淬灭,。
参见附图5,它是本实施例检测过程中452 nm处荧光强度I0/I与多巴胺浓度之间的Stern-Volmer图,由图5 中的曲线在低浓度下的线性部分计算,检测液的Stern-Volmer常数(KSV,代表灵敏度)为5.9 × 105 M-1。
参见附图6,它是本实施例检测过程中荧光淬灭效率随时间变化图,由图6可以看出聚合物对多巴胺的响应十分迅速,加入多巴胺后荧光强度迅速下降,30秒后保持在一个稳定的数值。
实施例3
实施例2中只对仅含多巴胺的待测样品进行测定,在实施例2的基础上,本实施例对聚电解质PFPE-PBA在检测过程中的抗干扰性能进行检测。实验方法:按实施例1提供的方法配制总体积为300 mL的检测液;选择可能的干扰物质:氨基酸和蛋白质类(丝氨酸、半胱氨酸、谷胱甘肽、γ-球蛋白、人血清白蛋白)、多元醇类(葡萄糖、果糖、半乳糖、抗坏血酸)、阳离子(Na+、K+、Ca2+)以及代谢物(尿素、尿酸),并将上述物质配制成水溶液待测。
荧光测试采用实施例2提供的方法,具体步骤如下:对于每种干扰物质,取20 mL检测液于锥形瓶中,分别测试检测液初始的最大发射波长处荧光强度I0、添加10 μL干扰物溶液30s后的荧光强度I1以及继续添加10 μL多巴胺溶液30s后的荧光强度I2,并分别计算添加多巴胺之前和之后最大发射波长处的荧光淬灭效率。检测液中蛋白质类干扰物质的浓度为0.2 g/L,其余干扰物浓度与多巴胺相同,均为15 μM。
参见附图7,它是本实施例中,在含有不同干扰物的检测液在加入多巴胺(15 μM)前后,检测液在452 nm处的淬灭效率。荧光淬灭效率计算方法为 (1 - Ix/I 0 ) ×100%,其中I 0 是检测液在452 nm处的初始荧光强度,I是加入相应物质后在452 nm处的荧光强度。
如附图7所示,在多巴胺不存在的情况下,干扰物的淬灭效率都低于10%;在存在多巴胺的情况下,所有的淬灭效率都非常相似,约为90%。由此可见,聚电解质PFPE-PBA对于多巴胺的检测具有很好的的抗干扰性。
参见附图8,它是本实施例中检测液在不同pH值条件下添加15 μM多巴胺前后452nm处的荧光强度。由图8可以看出,在8≤pH≤10范围内,聚电解质PFPE-PBA可以实现对多巴胺的检测。
Claims (3)
1. 一种基于荧光共轭聚电解质的多巴胺传感体系,其特征在于:包括浓度为0.02~0.1mol/L的甘氨酸-氢氧化钠缓冲液,甲醇和水按体积比为1:1~9:1的混合溶剂,按重复单元计浓度为10-6~10-4mol/L的含苯硼酸侧基的荧光共轭聚电解质;所述传感体系的pH为8~10;所述的含苯硼酸侧基的荧光共轭聚电解质的结构式为:
,
其中,n为重复单元数,n=8~21。
2.一种基于荧光共轭聚电解质的多巴胺传感体系的制备方法,其特征在于包括如下步骤:
(1)按物质的量计,将1份2,5-双(3'-(N,N-二甲基氨基)丙基)-1,4-二(三甲基硅基乙炔)苯,2~2.5 份n-Bu4NF与1100~1200份四氢呋喃的混合物,搅拌至固体溶解,再在室温下搅拌0.5~1小时后,经萃取、旋蒸、中性氧化铝柱层析分离提纯和干燥处理,得到一种二乙炔基单体;
(2)在无水无氧条件下,按物质的量计,将1份二溴芳烃单体,1份步骤(1)得到的二乙炔基单体,0.015~0.05份四(三苯基膦)二氯化钯,0.015~0.05份碘化亚铜,100~200份二异丙胺,200~300份甲苯加入到反应器中混合搅拌,在氩气保护、温度为70~75℃的条件下反应30~50小时,再经过滤、重结晶、离心、干燥处理,得到一种共轭聚电解质前驱体,其结构式为;
,
其中,n为重复单元数,n=8~21;
(3)按物质的量计,将1份步骤(2)制备的共轭聚电解质前驱体与3500~3600份四氢呋喃混合,在温度为60~66℃的油浴条件下加入35~55份4-溴甲基苯硼酸频哪醇酯进行反应,反应时间为48~72小时,反应期间分2~3次共加入400~900份甲醇; 反应结束后,冷却至室温,加入35~55份4-溴甲基苯硼酸和80~100份盐酸,搅拌20~40分钟后,置于丙酮/乙醚体积比为1:9~1:7的混合溶剂中沉淀,再经离心、干燥处理,得到一种含苯硼酸侧基的荧光共轭聚电解质;
(4)将步骤(3)制备得到的含苯硼酸侧基的荧光共轭聚电解质溶解于体积比为1:1~9:1的甲醇与水的混合溶剂中,加入pH为8~10的甘氨酸-氢氧化钠缓冲液充分混合,得到按重复单元计含苯硼酸侧基的荧光共轭聚电解质浓度为10-6~10-4 mol/L,甘氨酸-氢氧化钠浓度为0.02~0.1 mol/L的多巴胺传感体系。
3. 一种基于荧光共轭聚电解质的多巴胺传感体系的检测方法,其特征在于:按10~30mL多巴胺传感体系中加入5~20 μL的多巴胺待测溶液为待测样,以多巴胺传感体系为空白样,分别将空白样和待测样用滴管吸入石英比色皿中,采用395 nm波长的激发光,分别记录空白样、待测样最大发射波长处的荧光强度I 0 和I,依据公式 (1 -I /I 0 ) ×100%计算得到淬灭效率。
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