CN110283275B - 碳量子点分子印迹纳米凝胶荧光传感器的合成及其应用 - Google Patents
碳量子点分子印迹纳米凝胶荧光传感器的合成及其应用 Download PDFInfo
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Abstract
本发明公开碳量子点分子印迹纳米凝胶荧光传感器的合成及其应用,包括(1)碳量子点的制备;(2)分子印迹纳米凝胶荧光传感器的制备。本发明以CQDs为荧光信号,LYZ为模板分子,NIPAAm为温敏单体,AAm、HEMA、MAA为辅助单体,MBA为交联剂,APS为引发剂,以及TEMED为催化剂,合成了对模板分子LYZ具有选择性识别作用的分子印迹纳米凝胶荧光传感器。
Description
技术领域
本发明涉及荧光传感器技术领域。具体地说是碳量子点分子印迹纳米凝胶荧光传感器的合成及其应用。
背景技术
水凝胶是一种亲水性三维网络交联聚合物,可容纳本身重量的数十、数百倍的水,具有高保湿、保水的特性。亲水性单体通过一定的交联可以制备水凝胶,常用的交联方法分为化学交联和物理交联。化学交联是指在光、热、超声波、高能辐射等媒介以及交联剂等的作用下,通过化学键结合起来,从而形成三维网状聚合物的过程,现有的化学交联合成水凝胶的方法有单体交联聚合、聚合物聚合、载体接枝共聚;物理交联是通过静电作用、氢键作用、离子相互作用等物理作用力交联,这种交联是可逆的、非永久性的,因此制备的水凝胶称为可逆水凝胶。相比于物理交联,化学交联水凝胶是稳定、永久性的水凝胶,只会溶胀不会溶解,只有在共价交联点断裂的时候才会被破坏。根据对外界环境刺激的响应情况不同,可将水凝胶分为传统型的水凝胶和环境敏感型水凝胶,环境敏感型水凝胶其容纳水的量随环境因素的改变而变化,可分为(a)温敏水凝胶:容纳水的量随环境温度的改变而变化;(b)盐敏水凝胶:容纳水的量随溶液盐浓度的改变而变化;(c)光敏水凝胶:容纳水的量随光强度的改变而变化;(d)pH敏水凝胶:容纳水的量随溶液酸碱度的改变而变化,除此之外还有红外激光、电场响应、形状记忆水凝胶等。目前最为常用的环境响应型水凝胶有温敏水凝胶和pH敏水凝胶,用于药物控释、生物大分子的分离以及酶的包埋,是极具潜力的智能响应材料。
纳米凝胶是具有三维网络结构的亚微米尺度粒子,与传统的块状水凝胶相比,纳米水凝胶具有尺寸小、比表面积大、易于穿透人体各种保护膜等优点,由于纳米凝胶独特的优点因而被越来越多的学者研究:Zhou等人将纳米凝胶应用于近红外成像协同定向光热化学治疗;Saunders等人采用甲基丙烯酸为单体合成了pH刺激响应型纳米水凝胶作为人体软组织修复的生物材料;Kikuchi等人将温敏型聚N-异丙基丙烯酰胺及其衍生物作为固定相用于分离生物活性物质,分离过程中只采用水作为流动相,没有任何引入其他有机溶剂。此外,有报道将纳米水凝胶与分子印迹技术相结合,Li和Song先后做了分子印迹纳米凝胶对蛋白质和玉米烯酮进行特异性识别,取得了很好的效果,可见基于分子印迹技术的纳米凝胶在目标分析物特异性检测方面有良好的应用前景。
碳量子点(carbon quantum dots,CQDs)是碳纳米材料中继二维的石墨烯,一维的碳纳米管之后出现的一种准零维的新型纳米荧光材料。Xu等在分离提纯一维纳米材料—碳纳米管时首次发现CQDs,自发现以来,由于其良好的性能(可控的发射波谱、良好的光稳定性、较大的斯托克斯位移、良好的生物相容性、较长的荧光寿命,良好的水溶性、易于表面修饰、抗光漂白)受到学者们的广泛关注和研究,近些年来CQDs在生物成像、传感、载药等领域的应用都取得了重大突破,具有无可比拟的优势。
发明内容
为此,本发明所要解决的技术问题在于提供一种基于分子印迹技术的纳米凝胶,以CQDs作为荧光探针,检测生物大分子溶酶菌的传感器。
为解决上述技术问题,本发明提供如下技术方案:
碳量子点分子印迹纳米凝胶荧光传感器的合成,包括如下步骤:
(1)碳量子点的制备;
(2)以碳量子点作为荧光单元,制备分子印迹纳米凝胶荧光传感器。
上述碳量子点分子印迹纳米凝胶荧光传感器的合成,在步骤(2)中,
(2-1)取步骤(1)中得到的碳量子点CQDs溶于水中,随后加入溶菌酶LYZ、N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体;
(2-2)常温下磁力搅拌,随后加入过硫酸铵APS和四甲基乙二胺TEMED,抽真空充氮气,进行反应,最后用超纯水透析除去未反应的物质;
(2-3)溶菌酶LYZ模板分子的洗脱:将步骤(2-2)的透析袋放入400mL的十二烷基硫酸钠SDS和乙酸HAc的混合液中进行透析,每隔24h用UV-vis分光光度计检测透析外液中的溶菌酶浓度变化,每次都以前一次检测的洗脱液作为空白基底参照,直至检测不到溶菌酶的变化,最后再用超纯水透析,除去十二烷基硫酸钠SDS和HAc,直至透析外液呈中性,取出透析袋中的分子印迹纳米凝胶,4℃保存以作备用,得到碳量子点分子印迹纳米凝胶荧光传感器。
上述碳量子点分子印迹纳米凝胶荧光传感器的合成,(2-1)取步骤(1)中得到的碳量子点CQDs用蒸馏水稀释定容至11.03g/L,然后取0.5mL的11.03g/L碳量子点CQDs溶于25mL水中,溶菌酶LYZ的加入量为0.025g,加入N-异丙基丙烯酰胺NIPAAm的物质的量占N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体三者总物质的量百分比为89.8%-94.0%,加入N,N’-亚甲基双丙烯酰胺MBA的物质的量占N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体三者总物质的量百分比为6.0%。
上述碳量子点分子印迹纳米凝胶荧光传感器的合成,所述辅助单体为丙烯酰胺AAm,甲基丙烯酸羟乙酯HEMA和甲基丙烯酸MAA中一种或两种的混合物。
上述碳量子点分子印迹纳米凝胶荧光传感器的合成,丙烯酰胺AAm的加入量占N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体三者总物质的量百分比为0-4.2%,甲基丙烯酸羟乙酯HEMA的加入量占N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体三者总物质的量百分比为0-2.1%,甲基丙烯酸MAA的加入量占N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体三者物质的量百分比为0-2.1%。
上述碳量子点分子印迹纳米凝胶荧光传感器的合成,在步骤(2-2)中:常温下磁力搅拌30min,随后加入0.014g过硫酸铵APS和0.05μL的四甲基乙二胺TEMED,抽真空充氮气,在40℃下反应3-12h,最后用超纯水透析除去未反应的物质,超纯水透析袋的截留分子量为15000Da。
上述碳量子点分子印迹纳米凝胶荧光传感器的合成,在步骤(2-3)中,所述十二烷基硫酸钠SDS和乙酸HAc的混合液中,十二烷基硫酸钠SDS的质量与乙酸HAc的体积二者的质量体积比为1:1g/mL,乙酸HAc的体积浓度为10%。
上述碳量子点分子印迹纳米凝胶荧光传感器的合成,在步骤(1)中,
(1-1)将0.5g聚乙烯亚胺BPEI和1.0g柠檬酸CA溶于10mL的40℃热水中,油浴控温加热,温度小于或等于200℃;
(1-2)待蒸发至粘稠状时,加入1mL 40℃热水继续加热;重复以上步骤直至溶液颜色变为橙色,停止加热;
(1-3)将制得的碳量子点CQDs用40℃的蒸馏水稀释至10mL,并且通过硅胶柱色谱以0.01mol/L的稀盐酸作为流动相进行纯化,最后用透析袋透析除去盐酸,透析袋的截留分子量500Da,在60℃下旋蒸得到纯CQDs
碳量子点分子印迹纳米凝胶荧光传感器的应用,将一定浓度1mL的溶菌酶溶液加入5mL离心管中,再加入1mL稀释15倍的权利要求1-8任一所述分子印迹纳米凝胶原液,放入摇床中控温25℃,以230rpm混合孵育5-35h,测定其荧光强度;荧光分光光度计工作参数:激发波长332nm,激发和发射的狭缝分别是10nm、10nm,光电倍增管电压为700V。
上述碳量子点分子印迹纳米凝胶荧光传感器的应用,所述孵育的时间为29h。
本发明的技术方案取得了如下有益的技术效果:
本文以CQDs为荧光信号,LYZ为模板分子,NIPAAm为温敏单体,AAm、HEMA、MAA为辅助单体,MBA为交联剂,APS为引发剂,以及TEMED为催化剂,合成了对模板分子LYZ具有选择性识别作用的分子印迹纳米凝胶荧光传感器。
1.采用扫描电子显微镜和紫外分光光度计进行了表征,证明溶菌酶分子印迹纳米凝胶的成功制备、印迹以及洗脱。
2.制备了含不同辅助单体组分的分子印迹纳米凝胶,经考察含有AAm辅助单体的纳米凝胶对模板分子溶菌酶表现出较好的选择性。
3.NIPAAm作为温敏单体,结果表明分子印迹纳米凝胶对溶菌酶的识别具有温度响应,因此纳米凝胶对溶菌酶的特异性识别可以通过温度进行有效调控
附图说明
图1本发明碳量子点分子印迹纳米凝胶荧光传感器的合成的制备路线;
图2a本发明碳量子点分子印迹纳米凝胶荧光传感器的LYZ分子印迹纳米凝胶在反应时间为3h时的扫描电镜图谱;图2b本发明碳量子点分子印迹纳米凝胶荧光传感器的LYZ分子印迹纳米凝胶在反应时间为9h时的扫描电镜图谱;图2c本发明碳量子点分子印迹纳米凝胶荧光传感器的LYZ分子印迹纳米凝胶在反应时间为12h时的扫描电镜图谱;
图3本发明碳量子点分子印迹纳米凝胶荧光传感器的洗脱液中模板分子LYZ紫外光谱图;
图4本发明碳量子点分子印迹纳米凝胶对模板分子LYZ的荧光图谱:(a)移除溶菌酶的分子印迹纳米凝胶,(b)再次结合溶菌酶的分子印迹纳米凝胶。
图5本发明碳量子点分子印迹纳米凝胶中模板分子LYZ和纳米凝胶孵育时间考察图:(a)CLYZ=0.026g/L,(b)CLYZ=0.34g/L;
图6在25℃时辅助单体为AAm的分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图7在25℃时辅助单体为AAm的非分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图8在25℃时辅助单体为AAm和HEMA的分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图9在25℃时辅助单体为AAm和HEMA的非分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图10在25℃时辅助单体为AAm和MAA的分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图11在25℃时辅助单体为AAm和MAA的非分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图12在45℃时辅助单体为AAm的分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图13在45℃时辅助单体为AAm的非分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图14在45℃时辅助单体为AAm和HEMA的分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图15在45℃时辅助单体为AAm和HEMA的非分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图16在45℃时辅助单体为AAm和MAA的分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图17在25℃时辅助单体为AAm和MAA的非分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图18在25℃时没有辅助单体的分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图19在25℃时没有辅助单体的非分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图20在45℃时没有辅助单体的分子印迹纳米凝胶对LYZ的荧光响应线性曲线;
图21在45℃时没有辅助单体的非分子印迹纳米凝胶对LYZ的荧光响应线性曲线。
具体实施方式
一、实验部分
1实验仪器和试剂
(1)实验仪器
表1实验仪器设备
仪器名称 | 型号 | 厂家 |
超声波清洗器 | KQ118 | 昆山超声仪器有限公司 |
扫描电子显微镜 | H-8010 | 日本日立 |
紫外分光光度计(UV-vis) | UV 2550 | 日本岛津 |
荧光光谱仪 | FL4500 | 日本日立 |
电子天平 | BSA1245-CW | 德国赛多利斯 |
高速离心机 | H1650 | 湖南湘仪 |
集热式恒温加热搅拌器 | 恒岩-1 | 郑州恒岩 |
磁力搅拌器 | HZ85-2型 | 北京中兴伟业有限公司 |
超纯水净化系统 | PLA-CAXXBIOM2 | 美国Casada BIO |
电热恒温鼓风干燥箱 | DHG-9070A | 上海柏欣仪器设备厂 |
控温摇床 | TS-200B | 上海天呈仪器厂 |
油泵 | SHB-Ⅲ | 郑州长城科工贸有限公司 |
(2)实验试剂
表2实验试剂
注:截留分子量500Da、15000Da透析袋购于MYM生物技术公司、实验用水为超纯水
2、分子印迹纳米凝胶的制备
2.1、首先制备CQDs,在BPEI存在的情况下,通过简单的一步低温热解CA来合成CQDs,具体合成步骤如下:将0.5g BPEI和1.0g柠檬酸CA溶于10mL的40℃热水中,油浴控温加热,温度不高于200℃。待蒸发至粘稠状时,加入1mL 40℃的热水继续加热。重复以上步骤直至溶液颜色变为橙色,停止加热。将制得的CQDs用40℃的蒸馏水稀释至10mL,并且通过硅胶柱色谱以0.01mol/L的稀盐酸作为流动相进行纯化,最后用透析袋(截留分子量500Da)透析除去盐酸,在60℃下旋蒸得到纯CQDs。
用蒸馏水稀释定容至11.03g/L,储存在4℃下以作备用。
2.2、分子印迹纳米凝胶的具体合成步骤:
(1)取0.5mL 11.03g/L CQDs溶于25mL水中,随后再分别加入LYZ(0.025g)、NIPAAm(89.8%-94.0%,n/n总)、MBA(6.0%,n/n总)以及辅助单体(AAm 0-4.2%、HEMA 0-2.1%、MAA0-2.1%,n/n总),加入后只是进行机械的搅拌,混合均匀,具体组分如表3(n总为N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体三者总物质的量)。
(2)常温下磁力搅拌30min,随后加入0.014g APS,0.05uL TEMED,抽真空充氮气,40℃反应12h,最后用超纯水透析(截留分子量15000Da)除去未反应的物质。
(3)LYZ模板分子洗脱过程:将上述透析袋放入400mL的SDS(10%,m/v)-HAc(10%,v/v)进行透析,每隔24h用UV-vis分光光度计检测透析外液中的溶菌酶浓度变化,每次都以前一次检测的洗脱液作为空白基底参照,直至检测不到溶菌酶的变化,最后再用超纯水透析,除去SDS和HAc,直至透析外液呈中性,取出透析袋中的分子印迹纳米凝胶,4℃保存以作备用。
非分子印迹纳米凝胶的合成过程与上述步骤类似,在制备过程中不加入LYZ模板分子。
表3单体组成成分
3、溶菌酶的荧光检测
将一定浓度1mL的溶菌酶溶液加入5mL离心管中,再加入1mL稀释15倍的分子印迹纳米凝胶或非分子印迹纳米凝胶原液,放入摇床中控温25℃,以230rpm混合一定时间,测定其荧光强度,为保证实验数据的准确性,每个样品测两次。荧光分光光度计工作参数:激发波长332nm,激发和发射的狭缝分别是10nm,10nm,光电倍增管电压为700V。
二、结果与讨论
1、制备路线
如图1所示为溶菌酶分子印迹纳米凝胶的制备路线。首先采用CQDs作为荧光信号,LYZ作为模板分子,NIPAAm作为温敏单体,AAm/HEMA/MAA作为辅助单体,MBA作为交联剂,APS作为催化剂,TEMED作为引发剂,通过化学交联合成分子印迹纳米凝胶,其中模板分子LYZ通过氢键作用与温敏单体NIPAAm、辅助单体结合形成纳米凝胶聚合物,在SDS-HAc洗脱液中透析洗脱模板分子LYZ后,留下具有特异性结合位点,形状、大小与LYZ一致的分子空穴。
2、合成时间优化
首先对LYZ分子印迹纳米凝胶的合成时间进行考察。如图2a、图2b和图2c所示,从SEM图我们可以看出:3h几乎看不到凝胶球的存在;9h凝胶球出现在视野里,但是粒径不均匀且产量较低;当反应时间达到12h时,效果较理想。因此合成时间选择为12h。
3、材料表征
(1)扫描电子显微镜
合成的分子印迹纳米凝胶采用SEM进行了表征,如图2c所示,聚合物纳米凝胶球大小约1μm。
(2)溶菌酶的洗脱
采用紫外分光光度法检测洗脱液中的模板分子LYZ,鉴别印迹是否成功以及是否成功洗脱。如图3所示,洗脱液的紫外吸收光谱图在波长280nm左右有峰,和LYZ的紫外吸收峰一致,证明印迹和洗脱成功,重复洗脱直至检测不到LYZ。
(3)荧光光谱分析
对LYZ与分子印迹纳米凝胶再吸附结合时荧光强度变化进行研究。如图4所示,分子印迹纳米凝胶对模板分子LYZ有明显的荧光响应,LYZ的结合会使分子印迹纳米凝胶的荧光明显增强。
4、分子印迹纳米凝胶与溶菌酶孵育时间考察
在LYZ的检测中,LYZ和分子印迹纳米凝胶的孵育时间是非常关键的参数,影响分子印迹纳米凝胶传感器对LYZ检测的准确性,因此我们对模板分子和纳米凝胶的孵育时间进行考察。如图5所示,我们考察了5-35h的孵育时间,结果表明:随着孵育时间的增加,分子印迹纳米凝胶的荧光强度首先明显增加,随着时间继续延长,分子印迹纳米凝胶的荧光强度趋于稳定,因此选取最佳孵育时间为29h。
5、分子印迹纳米凝胶组成对溶菌酶选择性考察
不同的辅助单体所含的官能团不同,与LYZ之间相互作用形成氢键,在检测过程中表现出不同程度的特异性,所以不同辅助单体的分子印迹纳米凝胶对LYZ的选择性有可能存在差异,因此我们合成了四个不同组分的分子印迹纳米凝胶(表3),考察了它们对模板分子LYZ的选择性。
如图6到图11以及图18和图19所示,分别是25℃时不同辅助单体组成的分子印迹纳米凝胶和非分子印迹纳米凝胶对LYZ的荧光响应线性曲线。
其中F0代表没有LYZ存在时分子印迹纳米凝胶的荧光强度,F代表加入溶菌酶后分子印迹纳米凝胶的荧光强度,C表示LYZ的浓度。
印迹因子IF用于评估纳米凝胶对溶菌酶的选择性,IF=K(MIP)/K(NIP)(K代表线性方程斜率),表3中成分1至成分4的印迹因子IF分别是:IF1=2.62、IF2=3.84、IF3=1.43、IF4=1.79,由此可知成分2仅含AAm辅助单体的分子印迹纳米凝胶传感器在25℃对LYZ的选择特异性较好。
6、温敏性能的研究
温敏单体NIPAAm的临界相转变温度为32℃,临界相转变温度以下凝胶球处于溶胀状态,该温度以上处于皱缩状态。基于此,我们研究了分子印迹纳米凝胶的温敏性能,分别探究了25℃和45℃下LYZ的检测效果。在45℃下四种纳米凝胶组分对LYZ的荧光响应的线性曲线如图12到图17以及图20和图21所示,表3中成分1至成分4的分子印迹纳米凝胶的印迹因子分别为:IF1=1、IF2=5.57、IF3=1.90、IF4=0.58,结果表明:仅含AAm辅助单体的分子印迹纳米凝胶传感器在45℃对LYZ的选择特异性较好。与25℃条件下的印迹因子进行比较,45℃的印迹因子均发生改变,表明分子印迹纳米凝胶具有温敏性质,故分子印迹纳米凝胶对LYZ的特异识别有望通过温度进行有效调控。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本专利申请权利要求的保护范围之中。
Claims (9)
1.碳量子点分子印迹纳米凝胶荧光传感器的合成,其特征在于,包括如下步骤:
(1)碳量子点的制备;
(2)以碳量子点作为荧光单元,制备分子印迹纳米凝胶荧光传感器;
(2-1)取步骤(1)中得到的碳量子点CQDs溶于水中,随后加入溶菌酶LYZ、N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体;
(2-2)常温下磁力搅拌,随后加入过硫酸铵APS和四甲基乙二胺TEMED,抽真空充氮气,进行反应,最后用超纯水透析除去未反应的物质;
(2-3)溶菌酶LYZ模板分子的洗脱:将步骤(2-2)的透析袋放入400 mL的十二烷基硫酸钠SDS和乙酸HAc的混合液中进行透析,每隔24 h用UV-vis分光光度计检测透析外液中的溶菌酶浓度变化,每次都以前一次检测的洗脱液作为空白基底参照,直至检测不到溶菌酶的变化,最后再用超纯水透析,除去十二烷基硫酸钠SDS和HAc,直至透析外液呈中性,取出透析袋中的分子印迹纳米凝胶,4℃保存以作备用,得到碳量子点分子印迹纳米凝胶荧光传感器。
2.根据权利要求1所述的碳量子点分子印迹纳米凝胶荧光传感器的合成,其特征在于,在步骤(2-1)中,取步骤(1)中得到的碳量子点CQDs用蒸馏水稀释定容至11.03g/L,然后取0.5mL的11.03g/L碳量子点CQDs溶于25mL水中,溶菌酶LYZ的加入量为0.025g,加入N-异丙基丙烯酰胺NIPAAm的物质的量占N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体三者总物质的量百分比为89.8%-94.0%,加入N,N’-亚甲基双丙烯酰胺MBA的物质的量占N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体三者总物质的量百分比为6.0%。
3.根据权利要求1所述的碳量子点分子印迹纳米凝胶荧光传感器的合成,其特征在于,所述辅助单体为丙烯酰胺AAm,甲基丙烯酸羟乙酯HEMA和甲基丙烯酸MAA中一种或两种的混合物。
4.根据权利要求3所述的碳量子点分子印迹纳米凝胶荧光传感器的合成,其特征在于,丙烯酰胺AAm的加入量占N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体三者总物质的量百分比为0-4.2%,甲基丙烯酸羟乙酯HEMA的加入量占N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体三者总物质的量百分比为0-2.1%,甲基丙烯酸MAA的加入量占N-异丙基丙烯酰胺NIPAAm、N,N’-亚甲基双丙烯酰胺MBA和辅助单体三者物质的量百分比为0-2.1%。
5.根据权利要求1所述的碳量子点分子印迹纳米凝胶荧光传感器的合成,其特征在于,在步骤(2-2)中:常温下磁力搅拌30min,随后加入0.014g过硫酸铵APS和0.05μL的四甲基乙二胺TEMED,抽真空充氮气,在40℃下反应3-12h,最后用超纯水透析除去未反应的物质,超纯水透析袋的截留分子量为15000 Da。
6.根据权利要求1所述的碳量子点分子印迹纳米凝胶荧光传感器的合成,其特征在于,在步骤(2-3)中,所述十二烷基硫酸钠SDS和乙酸HAc的混合液中,十二烷基硫酸钠SDS的质量与乙酸HAc的体积二者的质量体积比为1:1g/mL,乙酸HAc的体积浓度为10%。
7.根据权利要求1-6任一所述的碳量子点分子印迹纳米凝胶荧光传感器的合成,其特征在于,在步骤(1)中,
(1-1)将0.5 g聚乙烯亚胺BPEI和1.0 g柠檬酸CA溶于10 mL的40℃热水中,油浴控温加热,温度小于或等于200℃;
(1-2)待蒸发至粘稠状时,加入1 mL 40℃热水继续加热;重复以上步骤直至溶液颜色变为橙色,停止加热;
(1-3)将制得的碳量子点CQDs用40℃的蒸馏水稀释至10 mL,并且通过硅胶柱色谱以0.01 mol/L的稀盐酸作为流动相进行纯化,最后用透析袋透析除去盐酸,透析袋的截留分子量500 Da,在60℃下旋蒸得到纯CQDs。
8.碳量子点分子印迹纳米凝胶荧光传感器的应用,其特征在于,将一定浓度1 mL的溶菌酶溶液加入5 mL离心管中,再加入1 mL稀释15倍的权利要求1-7任一所述的碳量子点分子印迹纳米凝胶荧光传感器的合成制备的碳量子点分子印迹纳米凝胶荧光传感器,放入摇床中控温25℃,以230 rpm混合孵育5-35h,测定其荧光强度;荧光分光光度计工作参数:激发波长332 nm,激发和发射的狭缝分别是10 nm、10 nm,光电倍增管电压为700 V。
9.根据权利要求8所述的碳量子点分子印迹纳米凝胶荧光传感器的应用,其特征在于,所述孵育的时间为29h。
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