CN115015347B - 基于微管的离子液体胶体/水界面的搭建及其应用 - Google Patents
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Abstract
本发明属于液/液界面电化学及分析化学技术领域,具体公开一种基于微管的离子液体胶体/水界面的构建及其应用。本发明介绍的高稳定性、高选择性离子液体胶体/水界面的方法包括:首先将聚离子液体加入离子液体中形成离子液体胶体,来增强有机相的界面稳定性;其次向有机相中加入钾离子载体,形成选择性离子液体胶体/水界面,构建高稳定性、高选择性离子液体胶体/水界面。最终将该界面应用于大脑皮层的K+检测,这对于研究K+在生物体内的行为以及与疾病的关系具有重要意义。
Description
技术领域
本发明属于离子选择性传感检测技术领域,具体涉及一种基于微管的离子液体胶体/水界面的构建以及使用差分脉冲法(DPV)对钾离子检测的电化学分析方法。
背景技术
目前,脑科学研究是最富挑战性的重大科学问题之一,它有助于理解大脑复杂生理过程的本质、脑疾病的病理机制。脑神经信号的传递与神经递质、自由基、离子等神经化学物质的参与密切相关。这些神经化学物质的失衡会导致精神疾病(如抑郁症)、神经退行性疾病(如老年痴呆症等)等多种脑疾病的发生。因此,研究神经生理和病理的分子机制对脑疾病的预防、诊断和治疗具有重要意义。常见的脑科学研究方法有核磁共振成像法、电生理法、高分辨荧光成像法等。其中,电化学分析法具有灵敏度高、时空分辨率高、电极易于微型化等优点,在脑化学物质的植入式活体检测上有很大的优势。在体植入式活检测中,基于伏安法的液/固界面活体电化学分析应用十分广泛,例如碳纤维、金、铂等微米修饰电极。但是由于非电活性物质存在较大的过电势以及氧化还原电位超出水的分解电位,使得在水溶液基于伏安法的非电活性物质检测仍然是一个巨大的挑战。
液/液界面作为一种新型的界面,在非电活性物质的检测上有很大的优势。因为它不依赖于氧化还原反应产生的电信号,而依赖于离子在界面迁移产生的电信号。传统的液/液界面中有机相由支持电解质和有机溶剂组成,如1,2-二氯乙烷、硝基苯,但鼠脑存在颅内压,使界面的稳定性变差。为了解决液/液界面稳定性的问题,通常加入PVC来固化有机相,这使有机相导电性变差、离子传输速率变慢,从而导致检测的灵敏度下降。因此利用液/液界面对鼠脑非电活性物质的植入式检测仍然是一个很大的挑战。
发明内容
针对上述现有技术存在的问题,本发明提供了一种离子液体胶体/水界面,具有界面稳定性高、选择性高、抗蛋白污染能力强等优点。本发明为了获得具有界面稳定性高、选择性高、抗蛋白污染能力强等优点的离子液体胶体/水界面,具体采用以下技术手段:首先,为了增强界面的稳定性,将聚离子液体加入离子液体中进行固化,形成离子液体胶体;其次,将钾离子载体引入离子液体胶体中,选择性地协助钾离子从水相迁移至有机相。此外,蛋白质具有亲水性和大分子量,从水相迁移至有机相需要很高的能量,因此液/液界面自身具有优异的抗蛋白污染能力。最终,本发明将该液/液界面应用于选择性和准确地植入式检测鼠脑中钾离子的水平。
本发明提供的离子液体胶体/水界面的构建方法,具体步骤如下:
(1)合成并筛选宽界面电位窗的疏水性离子液体;
(2)合成聚离子液体;
(3)合成钾离子载体;
(4)将步骤(2)的聚离子液体和步骤(3)的钾离子载体加入步骤(1)的离子液体中,制得离子液体胶体;
(5)将步骤(4)的离子液体胶装填于微米管中,构建离子液体胶体/水界面。
步骤(1)中,所述离子液体为C2M、C4M、C10M,结构分别如下式(a)所示:
步骤(2)中,所述聚离子液体为聚(1-丁基-3-乙烯基咪唑双(三氟甲基磺酰亚胺)),其结构如式(b)所示:
其中,所述的聚(1-丁基-3-乙烯基咪唑双(三氟甲基磺酰亚胺))通过溴化1-丁基-3-乙烯基咪唑单体自聚、与LiTFSI阴离子交换两步法制得。
步骤(3)中,所述钾离子载体结构如式(c)所示:
步骤(4)中,所述离子液体胶体的制备方法是,将聚(1-丁基-3-乙烯基咪唑双(三氟甲基磺酰亚胺))加入离子液体中,所述聚(1-丁基-3-乙烯基咪唑双(三氟甲基磺酰亚胺))与离子液体的固化比例为0.5-3:1(优选地,为1:1),再加入钾离子载体,使钾离子载体浓度为30mM,制得离子液体胶体。
步骤(5)中,所述装填离子液体胶体于微米管的方法是,利用注射器将离子液体胶体装填于激光拉制的微米管中。
步骤(6)中,所述离子液体胶体/水界面的构建方法是,将装有离子液体胶体的微米管置于水溶液中,构建了离子液体胶体/水界面。
本发明还提供了如上所述方法制备得到的离子液体胶体。
本发明还提供了如上所述方法制备得到的离子液体胶体/水界面。
本发明还提供了利用如上所述构建的离子液体胶体/水界面在体外检测K+浓度的应用。
本发明还提供了利用如上所述构建的离子液体胶体/水界面检测鼠脑皮层K+浓度的应用。
本发明利用钾离子载体能与K+高选择性地结合,同时在电场的协同作用下,K+从水相迁移至有机相,从而得到相应的DPV曲线及其线性范围,进而可以根据DPV检测K+浓度的变化。
本发明还提供了一种基于微管的离子液体胶体/水界面体外检测K+的方法,所述方法包括:向所述的离子液体胶体/水界面的水相中加入含一定浓度K+的溶液,将两根镀有氯化银的银丝分别置于离子液体胶体相和水相中,然后施加电压,K+在电场和离子载体的协同作用下,从水相迁移至有机相;通过差分脉冲伏安法DPV记录K+在界面迁移的电流大小,实现体外对K+的定量检测。
本发明还提供了一种通过离子液体胶体/水界面在体外检测K+浓度的方法,具体步骤如下:
(1)将装填离子液体胶体的微米管置于水溶液中;
(2)将两根镀有氯化银的银丝分别置于离子液体胶体和水溶液中
(3)向水溶液中加入含不同K+浓度的溶液;
(4)DPV进行检测,通过DPV曲线的峰电流,来测定K+的浓度。
其中,所述方法适用检测K+的线性范围为0.8mM-60mM。
其中,所述方法适用检测K+的最低检测限为20μM。
在本发明的一个具体实施方式中,所述通过离子液体胶体/水界面体外检测K+浓度的方法,包括:
(1)制作标准曲线:
在构成离子液体胶体/水界面的水相中加入不同K+浓度的溶液,使得水相中K+浓度分别为0.8,1,2,5,10,15,20.0,25,30,40,50,60mM,记录不同浓度下的DPV曲线,然后做出各组峰电流和K+浓度的关系曲线,得出线性范围为0.8mM-60mM。
(2)测定样品溶液中K+浓度
将装有离子液体胶体的微米管置于样品溶液中,形成离子液体胶体/水界面,测定DPV曲线,并根据K+浓度和峰电流的关系,计算样品溶液中K+浓度。
本发明根据上述方法得到的DPV曲线及其线性范围,可以用于体外检测K+浓度的变化。
本发明还提供了一种基于微管的离子液体胶体/水界面鼠脑皮层K+浓度检测的方法,所述方法包括:利用立体定位仪,将所述制备的装填离子液体胶体的微米管植入鼠脑皮层,在鼠脑内形成离子液体胶体/水界面;将镀有氯化银的银丝和参比电极分别置于离子液体胶体相和鼠脑硬脑膜中,然后施加电压,K+在电场和离子载体的协同作用下,从水相迁移至有机相;通过DPV测定鼠脑皮层K+在该界面迁移的电流大小,实现活体层次上对K+的定量检测。
本发明还提供了一种通过离子液体胶体/水界面检测鼠脑皮层K+浓度的方法,具体步骤如下:
(1)将装有离子液体胶体的微米管植入鼠脑皮层;
(2)将Ag/AgCl参比电极置于硬脑膜中;
(3)DPV测定,通过DPV曲线的峰电流大小,测定皮层中K+浓度。
其中,所述实验鼠为wistar雄鼠,体重200-250g。
其中,所述植入的皮层分别为运动皮层、视觉皮层和感觉皮层的L2/3区。
在本发明的一个具体实施方式中,所述通过离子液体胶体/水界面检测鼠脑皮层中K+浓度的方法,包括:
(1)利用立体定位仪,将装填离子液体胶体的微米管植入鼠脑皮层,在鼠脑内形成离子液体胶体/水界面,记录K+在界面上迁移的DPV曲线;
(2)根据DPV曲线峰电流大小,对照标准曲线得出皮层中K+的浓度。
本发明中,将装有离子液体胶体的微米管分别植入鼠脑的感觉皮层、运动皮层和视觉皮层中,在鼠脑中形成离子液体胶体/水界面,然后进行DPV检测。利用DPV曲线的峰电流大小,测定鼠脑不同皮层区域的K+含量。
本发明还提供了一种研究离子液体胶体电极/水界面抗蛋白污能力的方法。
在本发明的一个具体实施方式中,所述研究离子液体胶体/水界面抗蛋白污能力的方法,包括:
向离子液体胶体/水界面的水相中加入FITC-BSA溶液,FITC-BSA浓度为5mg mL-1,每隔一段时间记录K+在界面处迁移的DPV曲线,同时利用共聚焦观察界面处受荧光蛋白污染的程度。
本发明根据上述方法得到DPV曲线及荧光成像图,表明离子液体胶体/水界面具有良好的抗蛋白污染能力,可长时间植入鼠脑而界面不受蛋白污染。
本发明还提供了一种研究离子液体胶体电极/水界面稳定性的方法。
在本发明的一个具体实施方式中,所述研究离子液体胶体/水界面稳定性的方法,包括:
(1)观察装有离子液体胶体的微米管被植入鼠脑前后液面高度的变化;
(2)将荧光素加入离子液体胶体中,观察在鼠脑内形成离子液体胶体/水界面后脑切片的荧光成像。
本发明根据上述方法得到微管内液面变化图和脑切片图,表明离子液体胶体/水界面具有良好的稳定性,鼠脑颅内压的存在对界面稳定性的影响较小。
本发明的有益效果在于,通过钾离子载体构建具有选择性的离子液体胶体/水界面,线性范围为0.8-60mM,最低检测限为20μM(S/N=3)。所述离子液体胶体/水界面具有优异的抗蛋白污染能力,在BSA溶液中浸泡60d后对钾离子仍有83%以上的电流响应。另外,该电极可以选择性检测K+,神经递质、氨基酸、金属离子和其他生物活性物质对K+检测的干扰很小(<10%),且植入鼠脑检测的稳定性高。因此,本发明的高选择性、高抗污能力、高稳定性的离子液体胶体/水界面能够满足体外和体内对K+浓度的检测。
附图说明
图1:不同疏水性离子液体的界面电位窗。扫速:10mV/s。
图2:(A)K+在液/液界面迁移的示意图。(B)界面无钾离子载体时,水溶液中含有不同钾离子浓度的DPV曲线。(C)界面存在钾离子载体时,水溶液中含有不同钾离子浓度的DPV曲线,其中黑色虚线为背景电流。(D)离子液体胶体电极检测钾离子的校准曲线。
图3:离子液体胶体电极对金属离子、氨基酸、阴离子和其他生物分子的选择性。
图4:(A)离子液体胶体电极放置于5mg mL-1FITC-BSA溶液中0d、5d、10d、25d、50d、60d的荧光图像;(B)离子液体胶体电极放置于5mg mL-1BSA溶液0-60d期间的DPV曲线;(C)离子液体胶体电极浸入BSA溶液前后DPVs的峰电流比值图。
图5:脑损伤切片图。
图6:运动皮层、感觉皮层、视觉皮层的钾离子浓度图。
具体实施方式
结合以下具体实施例和附图,对本发明作进一步的详细说明。实施本发明的过程、条件、实验方法等,除以下专门提及的内容之外,均为本领域的普遍知识和公知常识,本发明没有特别限制内容。
实施例1:聚(1-丁基-3-乙烯基咪唑双(三氟甲基磺酰亚胺))的合成
将1-丁基-3-乙烯基咪唑溴化盐(2.31g,10.00mmol)和2,2-偶氮双(2-甲基丙腈)(0.016g,0.10mmol)和乙醇(5mL)加入到25mL圆底烧瓶中,搅拌24小时。然后,旋蒸除去乙醇,向混合物加入过量的丙酮,产生白色沉淀,抽滤。沉淀干燥后溶于水中,逐滴加入5mLLiTFSI(3.00g)溶液至沉淀完全,抽滤,洗涤,干燥。
实施例2:不同离子液体电位窗的测定
将C2M、C4M、C10M三种离子液体分别装于疏水化处理的微米玻璃管中,将离子液体电极置于LiCl溶液中,采用循环伏安法(CV)测定不同离子液体/水界面的电位窗大小。由图1可知,C10M具有最宽的电位窗,达0.8V,满足后续离子检测的需求。因此将C10M作为有机相。
实施例3:离子液体胶体/水界面用于体外K+的检测
将含不同K+浓度的溶液加入水相中,记录无钾离子载体存在的离子液体胶体/水界面上检测不同K+浓度的DPV曲线。由图2B可知,该界面无钾离子载体辅助时,K+不会从水相迁移至有机相,从而不会产生迁移电流。将含不同K+浓度的溶液加入水相中,记录钾离子载体存在的离子液体胶体/水界面上检测不同K+浓度的DPV曲线,然后做出峰电流和K+浓度的关系曲线,得出线性范围为0.8mM-60mM(图2C-D),满足后续生物应用的检测需求。
实施例4:选择性实验
离子液体胶体/水界面在存在不同浓度的金属离子、阴离子、氨基酸、生物活性物质时,进行选择性实验。
向水溶液中加入金属离子神经递质(10mMNa+,1mM的Ca2+和Mg2+,10μM的Cu2+、Fe3+、Zn2+、Co2+、Ni2+)(图3A),阴离子(10μM NO3-、HCO3-、OH-、CO3 2-、SO4 2-、SO3 2-、Cl-)(图3B),氨基酸(10μM Phe、Met、Gly、Glu、Cys、Arg、Lys、Leu、Ser、Thr、Val)(图3C)以及其他生物活性物质(10mM glucose,10μMAA、DA、UA、5-HT、DOPAC、lact)(图3D)。通过DPV测得对应的峰电流,再向每组溶液中加入5mM K+,再得到对应的峰电流,根据各组曲线绘制I(i)/IK+图。
实施例5:界面的抗蛋白污染能力
将装有离子液体胶体的微米管插入富含5mg mL-1FITC-BSA的水溶液中,形成离子液体胶体/水界面,观察离子液体胶体/水界面在FITC-BSA存在0d、5d、10d、25d、50d、60d后受蛋白污染的情况。第60天,离子液体胶体/水界面界面开始受到一定程度的污染(图4A)。同时用DPV测定FITC-BSA存在0d-60d后,K+在离子液体胶体/水界面处迁移的峰电流大小。由图4B-C可知,即使该界面在BSA溶液中存在60d,峰电流大小仍然保持原来的83%以上。以上数据表明,离子液体胶体/水界面具有优异的抗蛋白污染能力。
实施例6:脑损伤切片
将装有离子液体胶体的微米管植入鼠脑分别30min、60min、120min后,取出微米管,取脑,制成脑切片。然后将脑切片浸于TTC溶液中染色5-10min,观察脑切片的受损情况。图5表明,微米管植入鼠脑30-120min期间,鼠脑皮层没有明显的损伤。
实施例7:检测鼠脑皮层的K+含量
将装有离子液体胶体的微米管分别植入鼠脑的运动皮层、感觉皮层和视觉皮层,在鼠脑内构建离子液体胶体/水界面,用DPV测定不同皮层的钾离子浓度。根据DPV曲线峰电流的大小计算得K+浓度。运动皮层、感觉皮层和视觉皮层钾离子浓度分别为3.3±0.37mM、3.1±0.25mM、3.4±0.31mM(图6)。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下,本领域技术人员能够想到的变化和优点都被包括在本发明中,并且以所附的权利要求书为保护范围。
Claims (8)
1.一种基于微管的离子液体胶体/水界面的构建方法,其特征在于,具体步骤如下:
(1)合成并筛选离子液体;所述离子液体的结构如式(a)所示:
(2)合成聚离子液体;所述聚离子液体为聚(1-丁基-3-乙烯基咪唑双(三氟甲基磺酰亚胺)),其结构如式(b)所示:
(3)合成钾离子载体;所述钾离子载体的结构如式(c)所示:
(4)将所述步骤(2)的聚离子液体和所述步骤(3)的钾离子载体加入所述步骤(1)的离子液体中,制得离子液体胶体;
(5)在步骤(4)的基础上,构建离子液体胶体/水界面;所述离子液体胶体/水界面的构建方法是,利用注射器将所述离子液体胶体装填于微米管中,然后将其置于水溶液中,构建所述离子液体胶体/水界面。
2.根据权利要求1所述的方法,其特征在于,所述聚(1-丁基-3-乙烯基咪唑双(三氟甲基磺酰亚胺))通过溴化1-丁基-3-乙烯基咪唑单体自聚、与LiTFSI阴离子交换两步法制得。
3.根据权利要求1所述的方法,其特征在于,步骤(4)中,所述离子液体胶体的制备方法是,将聚(1-丁基-3-乙烯基咪唑双(三氟甲基磺酰亚胺))加入离子液体中,所述聚(1-丁基-3-乙烯基咪唑双(三氟甲基磺酰亚胺))与离子液体的固化比例为0.5-3:1,再加入钾离子载体,得到所述离子液体胶体。
4.根据权利要求1-3之任一项所述的方法制备得到的离子液体胶体。
5.根据权利要求1-4之任一项所述的方法构建得到的离子液体胶体/水界面。
6.根据权利要求5所述的离子液体胶体/水界面在以非疾病的诊断和治疗为目的体外检测K+中的应用。
7.一种基于微管的离子液体胶体/水界面体外检测K+的方法,其特征在于,所述方法以非疾病的诊断和治疗为目的,所述方法包括:向根据权利要求5所述的离子液体胶体/水界面的水相中加入含一定浓度K+的溶液,将两根镀有氯化银的银丝分别置于离子液体胶体相和水相中,然后施加电压,K+在电场和离子载体的协同作用下,从水相迁移至有机相;通过差分脉冲伏安法DPV记录K+在界面迁移的电流大小,实现体外对K+的定量检测。
8.一种基于微管的离子液体胶体/水界面鼠脑皮层K+浓度检测的方法,其特征在于,所述方法以非疾病的诊断和治疗为目的,所述方法包括:利用立体定位仪,将根据权利要求1中所述制备的装填离子液体胶体的微米管植入鼠脑皮层,在鼠脑内形成离子液体胶体/水界面;将镀有氯化银的银丝和参比电极分别置于离子液体胶体相和鼠脑硬脑膜中,然后施加电压,K+在电场和离子载体的协同作用下,从水相迁移至有机相;通过DPV测定鼠脑皮层K+在该界面迁移的电流大小,实现活体层次上对K+的定量检测。
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