CN115505146A - 一种基于荧光共振能量转移的聚集诱导发光荧光微球及其制备方法和应用 - Google Patents
一种基于荧光共振能量转移的聚集诱导发光荧光微球及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于纳米材料合成领域，具体涉及一种基于荧光共振能量转移的聚集诱导发光荧光微球及其制备方法和应用。该聚集诱导发光荧光微球的制备方法包括以下步骤：将TCBPEME荧光染料、BAPF荧光染料和聚马来酸酐十八醇酯溶于溶剂后分散于十二烷基硫酸钠水溶液中，制成微乳液后除去溶剂，离心清洗复溶即制得。本发明首次使用并发现两种光谱重叠的AIE染料制备基于荧光共振能量转移的聚集诱导发光荧光微球，制得的聚集诱导发光荧光微球具有荧光信号强，表面的基团具有可以直接用于生物分子偶联的优点；解决了现有AIE分子史托克斯位移小、在应用上常被背景干扰等情况，实现了在试纸条上高灵敏检测样本中的待检物。
Description
技术领域
本发明属于纳米材料合成领域，具体涉及一种基于荧光共振能量转移的聚集诱导发光荧光微球及其制备方法和应用。
背景技术
荧光是一种光致发光现象，当一束波长在物质激发波长范围内的光照射该物质时，会以发射光的形式释放所吸收的能量，这一物质即是荧光物质。荧光信号的输出与入射光(激发光)的强度成正相关关系，相比于传统胶体金的灰度信号，其可以提高入射光的强度以提高发射光的强度，从而达到提高灵敏度的目的。检测基质中常含有有色杂质，荧光探针相比于胶体金探针，抗背景干扰能力也更强。
然而，作为探针的荧光小分子通常在聚集体或固态出现荧光信号大幅下降，这也是常规荧光分子的弊端，即聚集诱导淬灭效应(ACQ)。然而，聚集诱导发光分子(AIE)描述了一种荧光分子在稀溶液中发射微弱或无发射，而在聚集体或固态中发射显著增强的现象，AIE分子发光机制普遍认为是分子内运动限制(RIM)所致。
如何进一步增强AIE分子的发射成为了本领域当中新的挑战和亟待解决的问题。
发明内容
荧光共振能量转移(FRET)是处于激发态的供体将能量转移给邻近处于基态的受体，即是一种表征和探测相邻供体和受体之间相互作用的技术。通常情况下，发生FRET需要满足以下3个重要的条件：(1)供体的发射光谱和受体的吸收光谱需要有很好的重叠；(2)供体和受体之间的距离要足够近(1-10nm范围内)；(3)供体和受体之间具有一定的偶极相互作用的相对取向性。目前，尚未有将荧光共振能量转移为原理制备聚集诱导发光荧光微球及其试纸条应用的相关报道。
基于上述情况，本发明旨在提供一种基于荧光共振能量转移的聚集诱导发光荧光微球，具体技术方案如下：
一种基于荧光共振能量转移的聚集诱导发光荧光微球的制备方法，包括以下步骤：将TCBPEME荧光染料、BAPF荧光染料和聚马来酸酐十八醇酯溶于溶剂后分散于十二烷基硫酸钠水溶液中，在冰浴条件下超声制成微乳液，除去溶剂，离心得到沉淀后清洗，再复溶于缓冲液中，即制得所述聚集诱导发光荧光微球。
该技术方案是发明人经过研究后发现可以满足上述发生FRET条件的特定方案，其制备得到的聚集诱导发光荧光微球(直径为10nm-800nm)的表面有可以直接与蛋白质偶联的活性基团，该活性基团包括但不仅限于羧基、羟基。所述微球荧光信号强，具有较大的史托克斯位移，应用于免疫层析试纸条上定量检测样本中待检物的浓度，具有检测灵敏度高的特点。同时，制备方法也具有高效和易于制备的优点。
在上述制备方法中，TCBPEME荧光染料、BAPF荧光染料和聚马来酸酐十八醇酯溶于所述溶剂后的终浓度分别为5mg/mL-50mg/mL、5mg/mL-50mg/mL和5mg/mL-100mg/mL；十二烷基硫酸钠水溶液的质量浓度为0.1％-10％；离心的条件包括：转速为4000rpm-15000rpm，时间为10min-30min。溶剂优选为三氯甲烷；清洗剂优选为质量浓度0.01-1％的氢氧化钠溶液。
基于上述聚集诱导发光荧光微球，本发明还提供了一种免疫层析试纸条，所述免疫层析试纸条包括玻璃纤维垫，其上喷涂由上述聚集诱导发光荧光微球与待标记抗体制得的探针。
优选地，所述待标记抗体为单克隆抗体、多克隆抗体、纳米抗体或噬菌体表达抗体。
优选地，所述的免疫层析试纸条，包括底板，以及在底板上依次搭接粘贴的样本垫、所述玻璃纤维垫、硝酸纤维素膜和吸水纸。
其中，硝酸纤维素膜上包被有待测物人工偶联抗原或待测物抗体作为检测线，包被有抗鼠抗体或抗兔抗体(二抗)作为质控线；制备方法包括如下步骤：
(1)使用0.01–0.5M pH 6.0–8.0的PBS(磷酸盐缓冲溶液)溶液分别调节包被物待测物人工偶联抗原或待测物抗体、抗鼠源抗体或抗兔源抗体至浓度为0.01-10.0mg/mL；
(2)将调整浓度后的待测物人工偶联抗原或待测物抗体喷涂于硝酸纤维素膜上部，作为检测线，将抗鼠抗体或抗兔抗体喷涂于硝酸纤维素膜下部，作为质控线；其中，检测线和质控线之间间隔一定距离，二者的喷量均为0.25-0.74μL/cm；
(3)将喷涂有检测线和质控线的硝酸纤维素膜于37℃过夜烘干处理后，在室温干燥的环境下保存备用。
本发明的有益效果为：本发明首次使用并发现两种光谱重叠的AIE染料制备基于荧光共振能量转移的聚集诱导发光荧光微球，制得的聚集诱导发光荧光微球具有荧光信号强，表面的基团具有可以直接用于生物分子偶联的优点；解决了聚集诱导发光荧光微球史托克斯位移小、在应用上常被背景干扰等情况，实现了在试纸条上高灵敏检测样本中的待检物。
附图说明
图1所示为TCBPEME染料和BAPF染料的分子式；
图2所示为TCBPEME染料和BAPF染料的激发和发射光谱；
图3是掺杂与不掺杂TCBPEME染料荧光微球的激发光谱；
图4所示为本发明荧光共振能量转移的聚集诱导发光荧光微球的透射电镜图；
图5所示为本发明荧光微球的试纸条应用于检测多效唑的实物图；
图6所示为本发明荧光微球的试纸条应用于检测大肠杆菌O157:H7的实物图。
具体实施方式
以下将结合实施例和附图对本发明的构思及产生的技术效果进行清楚、完整的描述，以充分地理解本发明的目的、方案和效果。
实施例1：
一、100nm粒径荧光共振能量转移的聚集诱导发光荧光微球的制备：
(1)制备疏水相：称取TCBPEME染料，BAPF染料(分子式如图1所示，激发和发射光谱如图2所示)和聚马来酸酐十八醇酯溶于三氯甲烷，终浓度分别为20、20、60mg/mL；
(2)微乳液：吸取1.2mL上述预制液，分散于6mL 0.25％ SDS溶液中，冰浴下，用超声破碎仪超声乳化5min，乳化功率为120W；
(3)荧光微球：在旋转蒸发容器中，将上述乳液旋蒸除去三氯甲烷，浓缩液用12000rpm/min 4℃离心20min，弃上清，沉淀用0.01M NaOH洗涤，水洗数次至离心后上清液澄清，清洗后的沉淀物复溶于5mL缓冲溶液中，4℃保存。
图4所示为制得的荧光共振能量转移的聚集诱导发光荧光微球的透射电镜图；其展现了单个微球的球形形貌和良好的分散性。
二、单独采用BAPF染料制备聚集诱导发光荧光微球，图3所示为掺杂与不掺杂TCBPEME染料荧光微球的激发光谱。从图3可观察到BAPF染料中掺杂了TCBPEME染料合成的荧光微球展现了更宽的激发波长和更高的激发强度，有利于被波长短于发射光的光激发或同一波长的光激发而实现多色标记应用
实施例2：
一种基于荧光共振能量转移的聚集诱导发光荧光微球为免疫标记物竞争法检测农药多效唑的试纸条应用：
免疫层析试纸条包括底板，以及在底板上依次搭接粘贴的样本垫、玻璃纤维垫、硝酸纤维素膜和吸水纸。
(1)硝酸纤维素膜的制备：
多效唑全抗原和抗鼠源抗体包被到硝酸纤维素膜上：用0.01M pH 7.5的PBS(磷酸盐缓冲液)稀释多效唑全抗原的浓度至1mg/mL，所得的溶液在膜上喷涂作为检测线；稀释抗鼠抗体的浓度为0.5mg/mL，所得的溶液在膜上喷涂作为质控线，两线的喷量均为0.74μL/cm，检测线与膜顶边间隔10mm，两线中间间隔5mm，37℃烘干12h，放置于干燥柜中保存备用。
(2)荧光共振能量转移的聚集诱导发光荧光微球探针玻璃纤维垫的制备：
荧光共振能量转移的聚集诱导发光荧光微球探针标记多效唑单克隆抗体的制备：向制得的荧光共振能量转移的聚集诱导发光荧光微球中加入5μg多效唑单克隆抗体，混匀后，孵育1h，之后，加入10％ BSA封闭剂，室温孵育0.5h，离心取沉淀，得到的沉淀用0.01MpH 7.0的PBS复溶为起始体积的1/10，制成荧光共振能量转移的聚集诱导发光荧光微球探针，按照3μL/cm体积喷涂到玻璃纤维垫上，真空干燥2h，放置于干燥柜中保存备用。
(3)基于荧光共振能量转移的聚集诱导发光荧光微球多效唑免疫层析试纸条标准曲线的建立：
制作试纸条标准曲线：在阴性基质中加标多效唑，使其在阴性基质中的浓度分别为：0、0.1、0.25、0.5、1、2.5、5、10、25、50和100ng/mL。
图5所示为本发明荧光微球的试纸条应用于检测多效唑的实物图；由图5展现了本发明荧光微球的试纸条检测不同浓度的多效唑，试纸条的检测线呈现不同荧光信号，随着多效唑的浓度的增加，荧光信号逐渐减弱，说明基于荧光共振能量转移的聚集诱导发光荧光微球应用在试纸条上检测多效唑农药上，性能良好。
实施例3：
一种基于荧光共振能量转移的聚集诱导发光荧光微球为免疫标记物夹心法检测大肠杆菌O157:H7的试纸条应用：
免疫层析试纸条包括底板，以及在底板上依次搭接粘贴的样本垫、玻璃纤维垫、硝酸纤维素膜和吸水纸。
(1)硝酸纤维素膜的制备
大肠杆菌O157:H7多克隆抗体和抗鼠源抗体包被到硝酸纤维素膜上：用0.01M pH7.5的PBS(磷酸盐缓冲液)稀释大肠杆菌O157:H7多克隆抗体的浓度至1mg/mL，所得的溶液在膜上喷涂作为检测线；稀释抗鼠抗体的浓度为0.5mg/mL，所得的溶液在膜上喷涂作为质控线，两线的喷量均为0.74μL/cm，检测线与膜顶边间隔10mm，两线中间间隔5mm，37℃烘干12h，放置于干燥柜中保存备用。
(2)荧光共振能量转移的聚集诱导发光荧光微球探针玻璃纤维垫的制备
荧光共振能量转移的聚集诱导发光荧光微球探针标记大肠杆菌O157:H7单克隆抗体的制备：向制得的荧光共振能量转移的聚集诱导发光荧光微球中加入10μg大肠杆菌O157:H7单克隆抗体，混匀后，孵育1h，之后，加入10％ BSA封闭剂，室温孵育0.5h，离心取沉淀，得到的沉淀用0.01M pH 7.0的PBS复溶为起始体积的1/10，制成荧光共振能量转移的聚集诱导发光荧光微球探针，按照3μL/cm体积喷涂到玻璃纤维垫上，真空干燥2h，放置于干燥柜中保存备用。
(3)基于荧光共振能量转移的聚集诱导发光荧光微球大肠杆菌O157:H7免疫层析试纸条标准曲线的建立：
制作试纸条标准曲线：在阴性基质中加标大肠杆菌O157:H7，使其在阴性基质中的浓度分别为：0、102、2.5×102、5×102、103、2.5×103、5×103、104、2.5×104、105、2.5×105、5×105、106和2.5×106CFU/mL。
图6所示为本发明荧光微球的试纸条应用于检测大肠杆菌O157:H7的实物图；由图6展现了本发明荧光微球的试纸条检测不同浓度的大肠杆菌O157:H7，试纸条的检测线呈现不同荧光信号，随着大肠杆菌O157:H7的浓度的增加，荧光信号逐渐增强，说明基于荧光共振能量转移的聚集诱导发光荧光微球应用在试纸条上检测大肠杆菌O157:H7上，性能良好。
以上所述，只是本发明的较佳实施例而已，本发明并不局限于上述实施方式，只要其以相同的手段达到本发明的技术效果，都应属于本发明的保护范围。在本发明的保护范围内其技术方案和/或实施方式可以有各种不同的修改和变化。
Claims (10)
1.一种基于荧光共振能量转移的聚集诱导发光荧光微球的制备方法，其特征在于，包括以下步骤：将TCBPEME荧光染料、BAPF荧光染料和聚马来酸酐十八醇酯溶于溶剂后分散于十二烷基硫酸钠水溶液中，在冰浴条件下超声制成微乳液，除去溶剂，离心得到沉淀后清洗，再复溶于缓冲液中，即制得所述聚集诱导发光荧光微球。
2.根据权利要求1所述的制备方法，其特征在于，TCBPEME荧光染料、BAPF荧光染料和聚马来酸酐十八醇酯溶于所述溶剂后的终浓度分别为5mg/mL-50mg/mL、5mg/mL-50mg/mL和5mg/mL-100mg/mL。
3.根据权利要求1所述的制备方法，其特征在于，十二烷基硫酸钠水溶液的质量浓度为0.1％-10％。
4.根据权利要求1所述的制备方法，其特征在于，离心的条件包括：转速为4000rpm-15000rpm，时间为10min-30min。
5.一种基于荧光共振能量转移的聚集诱导发光荧光微球，其特征在于，由权利要求1至4任一项所述的制备方法制得。
6.根据权利要求5所述的聚集诱导发光荧光微球，其特征在于，所述聚集诱导发光荧光微球的直径为10nm-800nm。
7.权利要求5或6所述的聚集诱导发光荧光微球在制备免疫层析试纸条中的应用。
8.一种免疫层析试纸条，其特征在于，所述免疫层析试纸条包括玻璃纤维垫，其上喷涂由权利要求6或7所述的聚集诱导发光荧光微球与待标记抗体制得的探针。
9.根据权利要求8所述的免疫层析试纸条，其特征在于，所述待标记抗体为单克隆抗体、多克隆抗体、纳米抗体或噬菌体表达抗体。
10.根据权利要求8所述的免疫层析试纸条，其特征在于，包括底板，以及在底板上依次搭接粘贴的样本垫、所述玻璃纤维垫、硝酸纤维素膜和吸水纸。
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