CN117074664A - 基于SEBS/CoFe2O4的生物柔性传感器及其制备方法及应用 - Google Patents
基于SEBS/CoFe2O4的生物柔性传感器及其制备方法及应用 Download PDFInfo
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Abstract
本发明提出基于SEBS/CoFe2O4的生物柔性传感器及其制备方法及应用，属于生物传感器技术领域；生物柔性传感器包括SEBS/CoFe2O4复合薄膜和经过抗原包覆的金纳米簇；经过抗原包覆的金纳米簇是将HAuCl4水溶液加入至抗原溶液中加热制备得到；将经过抗原包覆的金纳米簇滴加在SEBS/CoFe2O4复合薄膜上进行生物材料检测，可以应用于对5‑羟色胺的检测；本发明基于5‑HT与转铁蛋白结合以及Tf‑AuNCs团聚，导致薄膜荧光强度以及表面应力的变化，通过检测薄膜的磁信号、电信号、以及荧光强可成功检测5‑HT，排除其他干扰，有效提高了5‑HT检测的准确性，可广泛应用于生物检测领域。
Description
技术领域
本发明属于生物传感器技术领域，具体为一种基于SEBS/CoFe2O4的生物柔性传感器及其制备方法及应用。
背景技术
5-羟色胺（5-HT）是中枢神经系统中一种重要的单胺类神经递质，参与调节认知过程，单胺信号失调是许多精神和神经疾病的共同特征，在中枢神经系统之外，胃肠道系统中的5-HT占机体5-HT的95%，密切调节肠道功能和微生物群。目前，已报道的5-HT检测方法包括液相色谱法和电化学技术，虽然这些方法大多可以准确检测5-HT，但它们需要复杂的高成本设备并且耗时（尤其是液相色谱法）。同时由于分子的复杂性和其他具有类似化学结构的神经递质（如多巴胺和肾上腺素）的常见干扰，设计简单的程序来精准检测5-HT仍然是一个挑战。
发明内容
本发明克服了现有技术的不足，提出基于SEBS/CoFe2O4的生物柔性传感器及其制备方法及应用，解决目前对于检测5-HT存在测定复杂和测定不准确的问题。
为了达到上述目的，本发明是通过如下技术方案实现的。
基于SEBS/CoFe2O4的生物柔性传感器，包括SEBS/CoFe2O4复合薄膜和经过抗原包覆的金纳米簇；所述SEBS/CoFe2O4复合薄膜的一侧覆盖有金层，另一侧覆盖有Ag层，且SEBS/CoFe2O4复合薄膜上修饰有抗原；所述的经过抗原包覆的金纳米簇是将HAuCl4水溶液加入至抗原溶液中加热制备得到；将经过抗原包覆的金纳米簇滴加在SEBS/CoFe2O4复合薄膜上进行生物材料检测。
优选的，所述的抗原为转铁蛋白。
基于SEBS/CoFe2O4的生物柔性传感器的制备方法，包括以下步骤：
1）SEBS/CoFe2O4复合薄膜的制备：将SEBS加入到CoFe2O4流体中混合均匀并加热融化之后冷却成膜；
2）SEBS/CoFe2O4复合薄膜表面功能化：将SEBS/CoFe2O4复合薄膜在添加有氯金酸的葡萄糖还原液中还原，使得SEBS/CoFe2O4复合薄膜的一侧含有金层，再在SEBS/CoFe2O4复合薄膜的另一侧滴加AgNWs形成Ag层；之后对SEBS/CoFe2O4复合薄膜进行胺化，再将胺化后的SEBS/CoFe2O4复合薄膜浸入抗原溶液中孵育，将抗原修饰到SEBS/CoFe2O4复合薄膜上；
3）制备抗原包覆的金纳米簇：将HAuCl4水溶液滴加到含有抗原的水溶液中，在碱性环境下加热处理；
4）将含有相应抗体的待测物孵育在经过步骤2）处理得到的SEBS/CoFe2O4复合薄膜上；
5）检测时，将制备得到的抗原包覆的金纳米簇滴加至经过步骤4）处理得到的SEBS/CoFe2O4复合薄膜上。
优选的，步骤1）中SEBS与CoFe2O4的质量比为1:3.5-4.5。
优选的，所述葡萄糖还原液的制备方法为：将0.1g/mL的氯金酸水溶液、0.2 g/mL的葡萄糖水溶液和0.2 g/mL 的碳酸氢钾水溶液，按体积比为1：1：2的比例进行混合，配置成葡萄糖还原液。
更优的，所述的还原是将步骤1）制备得到的SEBS/CoFe2O4复合薄膜浸泡于所述的葡萄糖还原液中，进行14 h的避光还原反应。
优选的，所述的胺化是将SEBS/CoFe2O4复合薄膜浸入巯基乙胺溶液中处理后，再浸入含有1-乙基-3-（3-二甲基氨基丙基）碳二亚胺盐酸盐和N-羟基磺基琥珀酰亚胺的混合溶液中30-40 min。
优选的，步骤3）所述的在碱性环境下加热处理是将滴加了HAuCl4的抗原水溶液搅拌后加入到NaOH溶液中混匀，之后在120oC~140oC下加热5-8min。
优选的，使用PBS溶液将制备的抗原包覆的金纳米簇稀释后避光保存以供后续使用。
本发明相对于现有技术所产生的有益效果为：
1、本发明基于SEBS良好的柔性、CoFe2O4优异的电磁特性和可掺杂性、5-HT与转铁蛋白唾液酸残基的高亲和性以及纳米金粒子的生物相容性等诸多优势，提出一种基于SEBS/CoFe2O4的柔性磁弹性生物传感器，它同时具有灵敏的传感性能和优良的磁弹性能以及成本低、制备简易等等优点。
2、与传统的基于金属和刚性材料的传感器相比，通过将性能优异的软磁材料与柔性材料混合的方式制备磁弹性生物检测试纸，解决了传统传感器成本高、制备工艺复杂昂贵、不可再生等问题，本发明中的生物检测试纸具有优异的柔性、磁弹性、可拉伸性和连续监测性。
3、金纳米粒子（AuNPs）具有良好的生物相容性，通过在SEBS/CoFe2O4复合薄膜表面还原AuNPs实现基于金硫键的表面功能化。由于 CoFe2O4的磁弹性效应和AuNPs的荧光特性，可以将表面应力信号转换为磁电信号、荧光信号的多级信号实现微弱生物信号的捕捉和提取。为生物敏感探针提供修饰界面，并拓展了生物检测试纸的功能和应用范围。
4、蛋白稳定性金纳米簇因其制备简单、生物活性和生物相容性好等优点在生物传感领域备受关注，转铁蛋白包封的金纳米簇（Tf-AuNCs）基于两个双天线络合型聚糖，其末端有四个转铁蛋白SA残基，由于5-HT与转铁蛋白的唾液酸残基之间的高亲和力，不仅使得5-HT与薄膜上修饰的转铁蛋白连接，增强电阻变化，还可以触发Tf-AuNCs的聚集使得薄膜荧光强度增强，因此基于SEBS/CoFe2O4的柔性磁弹性生物检测试纸对5-HT具有良好的识别能力。
5、本发明基于5-HT与转铁蛋白结合以及Tf-AuNCs团聚，导致薄膜荧光强度以及表面应力的变化，通过检测薄膜的磁信号、电信号、以及荧光强可成功检测5-HT，排除其他干扰，有效提高了5-HT检测的准确性。
6、本发明的制备方法简单易行，可批量化生产，可广泛应用于生物检测领域。
附图说明
图1为本实施例所述的基于SEBS/CoFe2O4的柔性磁弹性生物传感器（即检测试纸）的制备方法的流程示意图。
具体实施方式
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚明白，结合实施例和附图，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。下面结合实施例和附图详细说明本发明的技术方案，但保护范围不被此限制。
实施例1
本实施例提出一种基于SEBS/CoFe2O4的柔性磁弹性生物传感器（即检测试纸）及其制备方法，以及在检测5-HT的应用。
制备该生物传感器的基本材料包括：铁酸钴（CoFe2O4）、SEBS、氯金酸（HAuCl4）、银纳米线（AgNWs）、5-HT、转铁蛋白、抗坏血酸；其中，SEBS是以聚苯乙烯为末端段，以聚丁二烯加氢得到的乙烯-丁烯共聚物为中间弹性嵌段的线性三嵌共聚物。
步骤1、SEBS/CoFe2O4复合薄膜的制备
1.1 将称取的CoFe2O4纳米粒子，加入至质量百分数为5%的石蜡溶液中。然后，使用超声波粉碎仪工作20min从而将CoFe2O4纳米粒子充分分散到石蜡溶液中，得到均匀的CoFe2O4流体。
1.2 SEBS/CoFe2O4的制备：（1）按1：4的质量比将SEBS加入到CoFe2O4流体中，使用玻璃棒搅拌使其均匀混合，形成SEBS/CoFe2O4固体。（2）将其在140℃电热板上加热至融化，将融化后的液体滴加到玻璃片上，冷却后可成功制备SEBS/ CoFe2O4薄膜，将固化后薄膜裁剪至5mm*5mm大小。（3）干燥后的SEBS/CoFe2O4薄膜存放在恒温恒湿的环境中备用。
步骤2、SEBS/CoFe2O4复合薄膜表面功能化
2.1 分别配制0.1g/mL的氯金酸水溶液、0.2 g/mL的葡萄糖水溶液和0.2 g/mL 的碳酸氢钾水溶液，将氯金酸水溶液、葡萄糖水溶液、碳酸氢钾水溶液按体积比为1：1：2的比例进行混合，配置成葡萄糖还原液。将固化后的SEBS/ CoFe2O4薄膜浸泡于配置好的还原溶液中，进行14 h的避光还原反应。还原结束后，用去离子水清洗干净，得到一侧含有金层的SEBS/CoFe2O4薄膜。
2.2 将AgNWs乙醇溶液滴在SEBS/CoFe2O4薄膜的另一侧，当乙醇挥发后得到Ag层。
2.3 将每个SEBS/CoFe2O4薄膜在室温下浸入40mmol/L巯基乙胺溶液中12h进行胺化。随后将其取出，用去离子水冲洗并在氮气中干燥以供后续使用。
2.4 将胺化的SEBS/CoFe2O4薄膜浸入含有1-乙基-3-（3-二甲基氨基丙基）碳二亚胺盐酸盐（EDC）和N-羟基磺基琥珀酰亚胺（NHS）的混合溶液中30 min。
2.5 使用PBS溶液稀释转铁蛋白，制备25μg/ml的抗原溶液，将SEBS/CoFe2O4薄膜浸入抗原溶液中，在37℃下孵育1h，使得转铁蛋白成功修饰到SEBS/CoFe2O4薄膜上。
步骤3、转铁蛋白包覆的金纳米簇（Tf-AuNCs）的制备
3.1 在37oC剧烈搅拌下，将1ml（5mM）HAuCl4水溶液滴加到2ml转铁蛋白水溶液中（7.5mg/ml）。
3.2 搅拌5min后，加入200μl的NaOH（1M）到混合溶液中。
3.3 继续搅拌2min到溶液混合均匀，将烧杯放置在130℃加热台上加热5min。
3.4 使用PBS溶液将所制备的Tf-AuNCs稀释2.5倍，避光保存以供后续使用。
步骤4、5-HT检测
4.1 使用PBS溶液稀释得到不同浓度的5 -HT，将不同浓度的5-HT滴加到步骤2制备得到的SEBS/CoFe2O4薄膜上，在37℃摇床中孵育10min。
4.2 取20μl步骤3制备得到的Tf-AuNCs滴加至孵育完成的SEBS/CoFe2O4薄膜上，根据生物柔性传感器，即该薄膜检测试纸的荧光强度，或磁信号变化以及电阻变化检测5-HT。
由于5-HT与转铁蛋白中的唾液酸具有高亲和性，因此5-HT会使得Tf-AuNCs的金纳米簇聚集，使得荧光增强。因此可以通过检测薄膜的荧光强度去检测5-HT；另外，由于转铁蛋白与5-HT在薄膜表面的结合产生表面应力，使得薄膜发生形变导致Ag层的电阻发生相应变化，以及基于磁弹效应薄膜的磁性能同样发生变化，因此可以通过检测薄膜的电阻变化以及磁信号的变化去检测5-HT。
实施例2
本实施例提出一种基于SEBS/CoFe2O4的柔性磁弹性生物传感器（即检测试纸）及其制备方法，以及在检测5-HT的应用。
制备该生物传感器的基本材料包括：铁酸钴（CoFe2O4）、SEBS、氯金酸（HAuCl4）、银纳米线（AgNWs）、5-HT、转铁蛋白、抗坏血酸；其中，SEBS是以聚苯乙烯为末端段，以聚丁二烯加氢得到的乙烯-丁烯共聚物为中间弹性嵌段的线性三嵌共聚物。
步骤1、SEBS/CoFe2O4复合薄膜的制备
1.1 将称取的CoFe2O4纳米粒子，加入至质量百分数为8%的石蜡溶液中。然后，使用超声波粉碎仪工作30min从而将CoFe2O4纳米粒子充分分散到石蜡溶液中，得到均匀的CoFe2O4流体。
1.2 SEBS/CoFe2O4的制备：（1）按1：4.5的质量比将SEBS加入到CoFe2O4流体中，使用玻璃棒搅拌使其均匀混合，形成SEBS/CoFe2O4固体。（2）将其在140℃电热板上加热至融化，将融化后的液体滴加到玻璃片上，冷却后可成功制备SEBS/ CoFe2O4薄膜，将固化后薄膜裁剪至6mm*6mm大小。（3）干燥后的SEBS/CoFe2O4薄膜存放在恒温恒湿的环境中备用。
步骤2、SEBS/CoFe2O4复合薄膜表面功能化
2.1 分别配制0.2g/mL的氯金酸水溶液、0.4 g/mL的葡萄糖水溶液和0.4 g/mL 的碳酸氢钾水溶液，将氯金酸水溶液、葡萄糖水溶液、碳酸氢钾水溶液按体积比为1：1：2的比例进行混合，配置成葡萄糖还原液。将固化后的SEBS/ CoFe2O4薄膜浸泡于配置好的还原溶液中，进行16h的避光还原反应。还原结束后，用去离子水清洗干净，得到一侧含有金层的SEBS/CoFe2O4薄膜。
2.2 将AgNWs乙醇溶液滴在SEBS/CoFe2O4薄膜的另一侧，当乙醇挥发后得到Ag层。
2.3 将每个SEBS/CoFe2O4薄膜在室温下浸入42mmol/L巯基乙胺溶液中14h进行胺化。随后将其取出，用去离子水冲洗并在氮气中干燥以供后续使用。
2.4 将胺化的SEBS/CoFe2O4薄膜浸入含有1-乙基-3-（3-二甲基氨基丙基）碳二亚胺盐酸盐（EDC）和N-羟基磺基琥珀酰亚胺（NHS）的混合溶液中40 min。
2.5 使用PBS溶液稀释转铁蛋白，制备25μg/ml的抗原溶液，将SEBS/CoFe2O4薄膜浸入抗原溶液中，在37℃下孵育1.5h，使得转铁蛋白成功修饰到SEBS/CoFe2O4薄膜上。
步骤3、转铁蛋白包覆的金纳米簇（Tf-AuNCs）的制备
3.1 在37℃剧烈搅拌下，将1ml（5mM）HAuCl4水溶液滴加到2ml转铁蛋白水溶液中（7.5mg/ml）。
3.2 搅拌5min后，加入200μl的NaOH（1M）到混合溶液中。
3.3 继续搅拌2min到溶液混合均匀，将烧杯放置在140℃加热台上加热5min。
3.4 使用PBS溶液将所制备的Tf-AuNCs稀释3倍，避光保存以供后续使用。
步骤4、5-HT检测
4.1 使用PBS溶液稀释得到不同浓度的5 -HT，将不同浓度的5-HT滴加到步骤2制备得到的SEBS/CoFe2O4薄膜上，在37℃摇床中孵育20min。
4.2 取20μl步骤3制备得到的Tf-AuNCs滴加至孵育完成的SEBS/CoFe2O4薄膜上，根据生物柔性传感器，即该薄膜检测试纸的荧光强度，或磁信号变化以及电阻变化检测5-HT。
由于5-HT与转铁蛋白中的唾液酸具有高亲和性，因此5-HT会使得Tf-AuNCs的金纳米簇聚集，使得荧光增强。因此可以通过检测薄膜的荧光强度去检测5-HT；另外，由于转铁蛋白与5-HT在薄膜表面的结合产生表面应力，使得薄膜发生形变导致Ag层的电阻发生相应变化，以及基于磁弹效应薄膜的磁性能同样发生变化，因此可以通过检测薄膜的电阻变化以及磁信号的变化去检测5-HT。
以上内容是结合具体的优选实施方式对本发明所做的进一步详细说明，不能认定本发明的具体实施方式仅限于此，对于本发明所属技术领域的普通技术人员来说，在不脱离本发明的前提下，还可以做出若干简单的推演或替换，都应当视为属于本发明由所提交的权利要求书确定专利保护范围。
Claims (10)
1.基于SEBS/CoFe2O4的生物柔性传感器，其特征在于，包括SEBS/CoFe2O4复合薄膜和经过抗原包覆的金纳米簇；所述SEBS/CoFe2O4复合薄膜的一侧覆盖有金层，另一侧覆盖有Ag层，且SEBS/CoFe2O4复合薄膜上修饰有抗原；所述的经过抗原包覆的金纳米簇是将HAuCl4水溶液加入至抗原溶液中加热制备得到；将经过抗原包覆的金纳米簇滴加在SEBS/CoFe2O4复合薄膜上进行生物材料检测。
2.根据权利要求1所述的基于SEBS/CoFe2O4的生物柔性传感器，其特征在于，所述的抗原为转铁蛋白。
3.如权利要求1或2所述的基于SEBS/CoFe2O4的生物柔性传感器的制备方法，其特征在于，包括以下步骤：
1）SEBS/CoFe2O4复合薄膜的制备：将SEBS加入到CoFe2O4流体中混合均匀并加热融化之后冷却成膜；
2）SEBS/CoFe2O4复合薄膜表面功能化：将SEBS/CoFe2O4复合薄膜在添加有氯金酸的葡萄糖还原液中还原，使得SEBS/CoFe2O4复合薄膜的一侧含有金层，再在SEBS/CoFe2O4复合薄膜的另一侧滴加AgNWs形成Ag层；之后对SEBS/CoFe2O4复合薄膜进行胺化，再将胺化后的SEBS/CoFe2O4复合薄膜浸入抗原溶液中孵育，将抗原修饰到SEBS/CoFe2O4复合薄膜上；
3）制备抗原包覆的金纳米簇：将HAuCl4水溶液滴加到含有抗原的水溶液中，在碱性环境下加热处理；
4）将含有相应抗体的待测物孵育在经过步骤2）处理得到的SEBS/CoFe2O4复合薄膜上；
5）检测时，将制备得到的抗原包覆的金纳米簇滴加至经过步骤4）处理得到的SEBS/CoFe2O4复合薄膜上。
4.根据权利要求3所述的基于SEBS/CoFe2O4的生物柔性传感器的制备方法，其特征在于，步骤1）中SEBS与CoFe2O4的质量比为1:3.5-4.5。
5.根据权利要求3所述的基于SEBS/CoFe2O4的生物柔性传感器的制备方法，其特征在于，所述葡萄糖还原液的制备方法为：将0.1g/mL的氯金酸水溶液、0.2 g/mL的葡萄糖水溶液和0.2 g/mL 的碳酸氢钾水溶液，按体积比为1：1：2的比例进行混合，配置成葡萄糖还原液。
6.根据权利要求5所述的基于SEBS/CoFe2O4的生物柔性传感器的制备方法，其特征在于，所述的还原是将步骤1）制备得到的SEBS/CoFe2O4复合薄膜浸泡于所述的葡萄糖还原液中，进行14 h的避光还原反应。
7.根据权利要求3所述的基于SEBS/CoFe2O4的生物柔性传感器的制备方法，其特征在于，所述的胺化是将SEBS/CoFe2O4复合薄膜浸入巯基乙胺溶液中处理后，再浸入含有1-乙基-3-（3-二甲基氨基丙基）碳二亚胺盐酸盐和N-羟基磺基琥珀酰亚胺的混合溶液中30-40min。
8.根据权利要求3所述的基于SEBS/CoFe2O4的生物柔性传感器的制备方法，其特征在于，步骤3）所述的在碱性环境下加热处理是将滴加了HAuCl4的抗原水溶液搅拌后加入到NaOH溶液中混匀，之后在120℃~140℃下加热5-8min。
9.根据权利要求3所述的基于SEBS/CoFe2O4的生物柔性传感器的制备方法，其特征在于，使用PBS溶液将制备的抗原包覆的金纳米簇稀释后避光保存以供后续使用。
10.如权利要求2所述的基于SEBS/CoFe2O4的生物柔性传感器在检测5-羟色胺中的应用。
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