CN106928477B - 电化学发光聚合物纳米粒的制备方法及应用 - Google Patents
电化学发光聚合物纳米粒的制备方法及应用 Download PDFInfo
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Abstract
本发明涉及一种电化学发光聚合物纳米粒的制备方法,包括以下步骤:将电化学发光聚合物与非发光聚合物在溶剂中混合,发生交联反应后得到电化学发光聚合物纳米粒,非发光聚合物为导电性聚合物。本发明还提供了采用上述方法所制备的电化学发光聚合物纳米粒在生物标记中的应用。采用本发明的方法,可制备出信号增强且结构稳定的电化学发光纳米粒,其具有良好的水溶性,粒径均一,粒径约为20nm,由于表面存在大量的活泼基团,有利于生物标记,在实现高灵敏度的生物医药检测方面将具有广泛的应用。
Description
技术领域
本发明涉及电化学发光材料领域,尤其涉及一种电化学发光聚合物纳米粒的制备方法及应用。
背景技术
在小分子电化学发光(ECL)物质中,以三联吡啶钌Ru(bpy)3 2+为发光组分,衍生化上活泼基因进而标记生物分子,以实现ECL生物分析,这是当前临床医学的主要检测手段。理论上,对于每一个被标记的生物大分子(如抗体),标记上的ECL小分子越多,检测信号就越强,检测灵敏度也越高。但事实上,标记的信号分子过多,生物大分子的反应活性会明显降低,检测灵敏度反而变差,即“过标记”。
为了实现高灵敏的生物医学检测,人们常常采用间接标记,即ECL小分子不直接标记在生物大分子上,而采用纳米粒作为载体,固载大量的ECL小分子,再用纳米粒标记生物分子。由于Ru(bpy)3 2+没有活泼基团,目前主要采用掺杂、包合以及静电吸附等方式固载于纳米粒表面或内部,它们大多数是非化学键方式固载ECL小分子。例如,(1)以阴离子配合物或阴离子聚电解质与阳离子组分Ru(bpy)3 2+之间的静电吸附而固载于纳米粒内部;(2)以阴离子聚电解质与阳离子组分Ru(bpy)3 2+之间的静电吸附而掺杂于硅纳米粒内部;(3)以反相微乳法掺杂壳聚糖和联吡啶钌制备ECL二氧化硅复合纳米粒;(4)以纳米粒的微孔吸附和(或)静电吸附固载ECL小分子;(5)以纳米粒作为膜(如Nafion膜)载体,在成膜后利用静电相互作用固载ECL小分子。
上述固载方法依靠弱作用力,所制备的纳米粒具有结构不稳定、ECL小分子容易泄漏、发光信号随保存时间较快衰减等缺点,且会明显影响ECL信号的强度。通常由于ECL小分子外的包裹壳的影响,固载于纳米粒内部的ECL小分子组分的发光信号明显减弱,然而若采用表面固载ECL小分子的方法,其固载量又较小。以上这些问题对于生物标记特别是检测试剂是极其不利的。
发明内容
为解决上述技术问题,本发明的目的是提供一种电化学发光聚合物纳米粒的制备方法及应用,采用本发明的方法,可制备出信号增强且结构稳定的电化学发光纳米粒,其具有良好的水溶性,粒径均一,粒径约为20nm,由于表面存在大量的活泼基团,有利于生物标记,在实现高灵敏度的生物医药检测方面将具有广泛的应用。
本发明提供了一种电化学发光聚合物纳米粒的制备方法,包括以下步骤:
将电化学发光聚合物与非发光聚合物在溶剂中混合,在搅拌下,发生交联反应后得到电化学发光聚合物纳米粒,非发光聚合物为导电性聚合物;溶剂为水、N,N-二甲基甲酰胺和二甲亚砜中的一种或几种。
进一步地,导电性聚合物为离子型导电聚合物和/或非离子型导电聚合物。
进一步地,离子型导电聚合物为离子型有机聚合物或离子型无机聚合物,离子型有机聚合物为聚丙烯酸、聚甲基丙烯酸、聚苯乙烯磺酸、聚乙烯磺酸、聚乙烯磷酸、聚谷氨酸、聚(吡咯-羧基吡咯)、聚乙烯亚胺、聚乙烯胺、聚乙烯吡啶、聚二烯丙基二甲基季铵盐、氨基化的聚对苯撑乙炔、聚赖氨酸、壳聚糖或核酸,离子型无机聚合物为聚磷酸盐或聚硅酸盐;非离子型导电聚合物为聚吡咯、聚苯胺、聚(3,4-乙烯二氧噻吩)或聚对苯撑乙炔。
进一步地,电化学发光聚合物由小分子电化学发光化合物与聚合物偶联得到。
进一步地,小分子电化学发光化合物为钌络合物或锇络合物。
进一步地,钌络合物为二(2,2'-联吡啶)(2,2'-联吡啶-羧酸)钌、二(2,2'-联吡啶)(氨基-2,2'-联吡啶)钌、二(2,2'-联吡啶)(氨基-1,10-菲咯啉)钌、二(2,2'-联吡啶)(羧基-1,10-菲咯啉)钌或二(2,2'-联吡啶)(5,6-环氧-5,6-二氢-[1,10]菲咯啉)钌;锇络合物为二(2,2'-联吡啶)(2,2'-联吡啶-羧酸)锇、二(2,2'-联吡啶)(氨基-2,2'-联吡啶)锇、二(2,2'-联吡啶)(氨基-1,10-菲咯啉)锇、二(2,2'-联吡啶)(羧基-1,10-菲咯啉)锇、二(2,2'-联吡啶)(5,6-环氧-5,6-二氢-[1,10]菲咯啉)锇、(羧基-1,10-菲咯啉)(1,10-菲咯啉)(1,2-双(二苯基膦)乙烯)锇、(氨基-1,10-菲咯啉)(1,10-菲咯啉)(1,2-双(二苯基膦)乙烯)锇、(氨基-1,10-菲咯啉)(碳酰氯)(1,2-双(二苯基膦)乙烯)锇或(羧基-1,10-菲咯啉)(碳酰氯)(1,2-双(二苯基膦)乙烯)锇。
进一步地,聚合物为聚丙烯酸、聚甲基丙烯酸、聚谷氨酸、聚(吡咯-羧基吡咯)、聚乙烯亚胺、聚乙烯胺、聚赖氨酸或壳聚糖。
进一步地,交联反应是在交联剂或活化剂的作用下进行;也可以直接进行交联反应,既没有交联剂也没有活化剂。
进一步地,交联剂为三羟甲基丙烷-三(3-氮丙啶基)丙酸酯、戊二醛、丙二酸、丁二酸、乙二胺、丙二胺或丁二胺;活化剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)和N-羟基丁二酰亚胺(NHS)的组合物、N,N′-二环己基碳二亚胺和N-羟基丁二酰亚胺的组合物、1-(3-二甲氨基丙基)-3-乙基碳二亚胺或N,N′-二环己基碳二亚胺。
进一步的,导电性聚合物的分子量为1800-300000g/mol。
进一步地,电化学发光聚合物与非发光聚合物的质量比为20:1-1:1。优选地,电化学发光聚合物与非发光聚合物的质量比为4:3。
进一步地,交联反应可以发生在发光聚合物之间、发光聚合物与非发光聚合物之间,或者非发光聚合物之间。交联反应时间为0.5-48h。
进一步地,交联反应的温度为10-140℃。优选地,交联反应的温度为20-60℃。
进一步地,电化学发光聚合物的制备方法包括以下步骤:
将小分子电化学发光化合物和聚合物分别溶于碱的水溶液,混匀后,在保护气氛下,于20-140℃下避光反应0.5-48h,透析6-48h后得到电化学发光聚合物。
进一步地,碱为碳酸氢钠、碳酸氢钾、碳酸钠、碳酸钾、氢氧化钠或氢氧化钾。
进一步地,保护气氛为氮气或氦气。
本发明还提供了一种采用上述方法所制备的电化学发光聚合物纳米粒在生物标记中的应用。
本发明的原理如下:
本发明首先将小分子的ECL络合物偶联到聚合物而合成ECL聚合物。若仅仅由ECL聚合物交联制备ECL纳米粒时,固载于纳米粒中的ECL络合物将存在两种情况,即内部固载和表面固载,且发生前者的概率较大。纳米粒的发光需要电激发才能进行,固载于内部的ECL络合物由于有机材料纳米粒的导电性极弱甚至不导电,从而导致了这些ECL络合物不能或不能有效地被外部的电极激发而发光。而固载于表面的ECL络合物是能够有效发光的。因此,仅仅由ECL聚合物交联制备的纳米粒的发光效率是极其低下的。当ECL聚合物与导电性聚合物混合后,再交联制备ECL纳米粒时,导电性聚合物将交联和/或掺杂于纳米粒中,此时,上述的固载于内部的ECL络合物的电激发问题即获得了解决,使固载于内部的大量的ECL络合物也就能够有效地产生ECL信号,从而实现了ECL信号的增强。
借由上述方案,本发明至少具有以下优点:
所制备的电化学发光聚合物纳米粒电化学发光信号明显增强,结构稳定,能够在水溶液中良好分散,粒径为20nm左右;相对于现有技术中不存在非发光聚合物的纳米粒,电化学发光信号明显增强;将本发明制备的电化学发光聚合物纳米粒在溶液中保存至少一个月时,粒子大小和分布、电化学发光强度、溶液悬浮均一性均能够稳定保留。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是本发明所制备的电化学发光聚合物纳米粒的SEM图;
图2是相关各物质的电化学发光强度对比结果;
图3是本发明制备的电化学发光聚合物纳米粒在放置3天后的粒径分布测试结果;
图4是本发明制备的电化学发光聚合物纳米粒在放置19天后的粒径分布测试结果;
图5是本发明制备的电化学发光聚合物纳米粒在放置31天后的粒径分布测试结果。
具体实施方式
下面结合附图和实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1
将二(2,2'-联吡啶)(5,6-环氧-5,6-二氢-[1,10]菲咯啉)钌与聚丙烯酸(PAA)的羧基化学连接,获得钌络合物接枝聚丙烯酸,即电化学发光聚合物(ECL聚合物)。具体方案如下:
称取108mg二(2,2'-联吡啶)(5,6-环氧-5,6-二氢-[1,10]菲咯啉)钌溶解于20mL的NaHCO3水溶液(1mmol/L)中,得到ECL络合物储备液。称取2g PAA(分子量为30000g/mol)溶解于10mL的NaHCO3水溶液(1mmol/L)中,得PAA储备液。取3mL上述ECL络合物储备液置于10mL单颈圆底烧瓶中,并加入1mL的PAA储备液。抽真空,充氮气,反复三次。在110℃油浴中避光、氮气保护下,磁力搅拌反应24h。反应结束后,冷却至室温,用去离子水透析48h,每12h换一次液,最后将透析液定容到20mL,得电化学发光聚合物溶液。
以PAA(分子量为30000g/mol)为非发光聚合物,利用上述获得的电化学发光聚合物溶液,以三羟甲基丙烷-三(3-氮丙啶基)丙酸酯为交联剂,制备化学交联的电化学发光聚合物纳米粒。具体方案为:
将1.6mL上述制备的电化学发光聚合物溶液和0.4mL的PAA水溶液(1.2mg/mL)分别加入单颈圆底烧瓶中,再加入交联剂三羟甲基丙烷-三(3-氮丙啶基)丙酸酯20mg,置于60℃水浴中,搅拌(400转/min)反应10h。最后,将反应液在水中透析24h以除去多余的交联剂,即得电化学发光聚合物纳米粒。
实施例2
取实施例1制备的电化学发光聚合物溶液1.6mL置于圆底烧瓶中,向其中加入0.2mL PAA(分子量为30000g/mol)水溶液(0.4mg/mL),混匀,加入0.1mLPEI(分子量为1800g/mol)水溶液(0.01mg/mL),再加入交联剂三羟甲基丙烷-三(3-氮丙啶基)丙酸酯20mg,置于60℃水浴中,搅拌(400转/min)反应10h。最后,将反应液在水中透析24h以除去多余的交联剂,即得电化学发光聚合物纳米粒。在此实施例中,电化学发光聚合物与PAA发生交联反应,而PEI被静电组装在ECL聚合物或(和)PAA上,之后,再交联固定于纳米粒的结构中,此时PEI是被静电相互作用掺杂于纳米粒内部。
实施例3
取实施例1制备的电化学发光聚合物溶液1.6mL置于圆底烧瓶中,再加入0.2mLPAA(分子量为30000g/mol)水溶液(0.4mg/mL)。在0.1mL PEI(分子量为1800g/mol)水溶液(0.01mg/mL)中加入1mg EDC,摇匀,再加入上述圆底烧瓶中,EDC活化电化学发光聚合物和PAA中的羧基。室温下搅拌(400转/min)反应1h,使活化后的羧基与PEI上的氨基发生交联反应。最后,将反应液在水中透析24h以除去多余的活化剂,即得电化学发光聚合物纳米粒。在此实施例中,电化学发光聚合物、PAA和PEI均发生了交联反应,而固载于纳米粒中。
实施例4
取实施例1制备的电化学发光聚合物溶液1.6mL置于圆底烧瓶中。然后取0.2mLPAA(分子量为30000g/mol)水溶液(0.4mg/mL),用EDC和NHS活化PAA的羧基,将活化后的PAA水溶液加入上述圆底烧瓶中,再加入0.1mL PEI(分子量为1800g/mol)水溶液(0.01mg/mL),室温下搅拌(400转/min)反应3h。最后,将反应液在水中透析24h以除去多余的活化剂,即得电化学发光聚合物纳米粒。在此实施例中,ECL聚合物被静电组装在PEI上,之后,组装的PEI再与活化的PAA发生交联反应,获得交联纳米粒,此时ECL聚合物被静电相互作用掺杂于纳米粒内部。
实施例5
取实施例1制备的电化学发光聚合物溶液1.6mL置于圆底烧瓶中,用EDC和NHS活化发光聚合物的羧基。然后加入0.2mLPAA(分子量为2000g/mol)水溶液(0.4mg/mL),混匀,再加入0.1mL PEI(分子量为60000g/mol)水溶液(0.01mg/mL),室温下搅拌(400转/min)反应3h。最后,将反应液在水中透析24h以除去多余的活化剂,即得电化学发光聚合物纳米粒。在此实施例中,PAA被静电组装在PEI上,组装的PEI再与活化的发光聚合物发生交联反应,获得交联纳米粒,此时PAA被静电相互作用掺杂于纳米粒内部。
实施例6
将(羧基-1,10-菲咯啉)(1,10-菲咯啉)(1,2-双(二苯基膦)乙烯)锇与PEI(分子量为60000g/mol)的氨基化学连接,获得锇络合物接枝PEI,即电化学发光聚合物(ECL聚合物)。具体方案如下:
称取80mg(羧基-1,10-菲咯啉)(1,10-菲咯啉)(1,2-双(二苯基膦)乙烯)锇溶解于20mL的N,N-二甲基甲酰胺(DMF)中,得到ECL络合物储备液。称取2.5g PEI溶解于10mL的DMF中,得PEI储备液。取3mL上述ECL络合物储备液置于10mL单颈圆底烧瓶中,加入1mL的PEI储备液,然后加入1mg EDC活化锇络合物的羧基。抽真空,充氮气,反复三次。在50℃水浴中避光、磁力搅拌反应5h。反应结束后,冷却至室温,用DMF透析24h,每6h换一次液,最后将透析液用DMF定容到20mL,得ECL聚合物溶液。
以非离子型导电聚合物聚吡咯和离子型导电聚合物聚谷氨酸为非发光聚合物,利用上述获得的ECL聚合物溶液,在加热下发生化学交联反应,制备电化学发光聚合物纳米粒。具体方案为:
将1mg聚吡咯(分子量为2100g/mol)和2mg聚谷氨酸(分子量为50000g/mol)置于烧杯中,加入30mLDMF,振摇,使聚合物溶解,过滤后将滤液置于单颈圆底烧瓶中,加入4mL上述制备的ECL聚合物溶液,振摇均匀。抽真空,充氮气,反复三次。置于140℃的油浴中,搅拌(400转/min),加热反应48h。冷却至室温,高速离心(12000rpm),除去多余的各种聚合物,沉淀复溶于DMF中,即得电化学发光聚合物纳米粒。带有大量氨基的ECL聚合物和带有大量羧基的离子型导电聚合物聚谷氨酸在高温下形成酰胺键而发生交联反应,而制备电化学发光纳米粒。非离子型导电聚合物聚吡咯在聚谷氨酸的羧基作用下,部分离子化而静电吸附于聚谷氨酸上,而在聚谷氨酸交联时被包埋于纳米粒内;同时,在发生ECL聚合物与聚谷氨酸两种大分子交联过程中,也能够通过掺杂的方式而将聚吡咯包埋于交联纳米粒之中。
图1是本发明实施例3所制备的电化学发光聚合物纳米粒的SEM图,从图中可看出,其粒径在20nm左右,且粒径较均一。
图2是本发明实施例3相关各物质的电化学发光强度对比结果。从图中可看出,在加入具有导电性的非发光聚合物交联后,其纳米粒的电化学发光强度达到1466a.U.左右,明显高于小分子钌络合物(1196a.U.)和未加入具有导电性的非发光聚合物的ECL纳米粒(即发光聚合物纳米粒的1000a.U.)。说明本发明制备的ECL纳米粒具有明显的信号增强作用。
图3-5是本发明实施例3制备的电化学发光聚合物纳米粒在分别放置3天、19天和31天后的粒径分布测试结果。从图中可看出,纳米粒的各物理参数没有发生明显的变化,说明其粒径大小以及粒径分布具有良好的纳米粒结构稳定性。
以上所述仅是本发明的优选实施方式,并不用于限制本发明,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变型,这些改进和变型也应视为本发明的保护范围。
Claims (5)
1.一种电化学发光聚合物纳米粒的制备方法,其特征在于,包括以下步骤:
将电化学发光聚合物与非发光聚合物在溶剂中混合,发生交联反应后得到所述电化学发光聚合物纳米粒,所述非发光聚合物为导电性聚合物;所述导电性聚合物为离子型导电聚合物和/或非离子型导电聚合物;所述离子型导电聚合物为离子型有机聚合物或离子型无机聚合物,所述离子型有机聚合物为聚丙烯酸、聚甲基丙烯酸、聚苯乙烯磺酸、聚乙烯磺酸、聚乙烯磷酸、聚谷氨酸、聚(吡咯-羧基吡咯)、聚乙烯亚胺、聚乙烯胺、聚乙烯吡啶、聚二烯丙基二甲基季铵盐、氨基化的聚对苯撑乙炔、聚赖氨酸、壳聚糖或核酸,所述离子型无机聚合物为聚磷酸盐或聚硅酸盐;所述非离子型导电聚合物为聚吡咯、聚苯胺、聚(3,4-乙烯二氧噻吩)或聚对苯撑乙炔;所述电化学发光聚合物由小分子电化学发光化合物与聚合物偶联得到;所述小分子电化学发光化合物为钌络合物或锇络合物;所述聚合物为聚丙烯酸、聚甲基丙烯酸、聚谷氨酸、聚(吡咯-羧基吡咯)、聚乙烯亚胺、聚乙烯胺、聚赖氨酸或壳聚糖。
2.根据权利要求1所述的电化学发光聚合物纳米粒的制备方法,其特征在于:所述钌络合物为二(2,2'-联吡啶)(2,2'-联吡啶-羧酸)钌、二(2,2'-联吡啶)(氨基-2,2'-联吡啶)钌、二(2,2'-联吡啶)(氨基-1,10-菲咯啉)钌、二(2,2'-联吡啶)(羧基-1,10-菲咯啉)钌或二(2,2'-联吡啶)(5,6-环氧-5,6-二氢-[1,10]菲咯啉)钌;所述锇络合物为二(2,2'-联吡啶)(2,2'-联吡啶-羧酸)锇、二(2,2'-联吡啶)(氨基-2,2'-联吡啶)锇、二(2,2'-联吡啶)(氨基-1,10-菲咯啉)锇、二(2,2'-联吡啶)(羧基-1,10-菲咯啉)锇、二(2,2'-联吡啶)(5,6-环氧-5,6-二氢-[1,10]菲咯啉)锇、(羧基-1,10-菲咯啉)(1,10-菲咯啉)(1,2-双(二苯基膦)乙烯)锇、(氨基-1,10-菲咯啉)(1,10-菲咯啉)(1,2-双(二苯基膦)乙烯)锇、(氨基-1,10-菲咯啉)(碳酰氯)(1,2-双(二苯基膦)乙烯)锇或(羧基-1,10-菲咯啉)(碳酰氯)(1,2-双(二苯基膦)乙烯)锇。
3.根据权利要求1所述的电化学发光聚合物纳米粒的制备方法,其特征在于:所述交联反应是在交联剂或活化剂的作用下进行。
4.根据权利要求3所述的电化学发光聚合物纳米粒的制备方法,其特征在于:所述交联剂为三羟甲基丙烷-三(3-氮丙啶基)丙酸酯、戊二醛、丙二酸、丁二酸、乙二胺、丙二胺或丁二胺;所述活化剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N-羟基丁二酰亚胺的组合物、N,N′-二环己基碳二亚胺和N-羟基丁二酰亚胺的组合物、1-(3-二甲氨基丙基)-3-乙基碳二亚胺或N,N′-二环己基碳二亚胺。
5.根据权利要求1所述的方法所制备的电化学发光聚合物纳米粒在生物标记中的应用。
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