CN113655039A - 一种基于分子印迹技术构建的微囊藻毒素比率荧光传感器 - Google Patents
一种基于分子印迹技术构建的微囊藻毒素比率荧光传感器 Download PDFInfo
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Abstract
本发明公开一种基于分子印迹技术构建的微囊藻毒素比率荧光传感器,属于环境检测技术领域。本发明,通过合成以柠檬酸为碳源的碳量子点和二氧化硅负载异硫氰酸荧光素的纳米材料,它们分别对微囊藻毒素有荧光猝灭和荧光增强的特性,从而建立了一种比率荧光的方法,然后采用分子印迹方法,以二甲双胍为假模板,通过在复合纳米材料上留下印迹位点,对微囊藻毒素进行选择性识别,从而达到定量检测的效果。所得CQDS‑FITC‑APTES‑SiO2@MIP对微囊藻毒素RR/LR荧光相应度好,反应灵敏,荧光比率技术和分子印迹技术的结合,完全满足微囊藻毒素的快速检测需求,且成本低廉,适合各类场合使用。
Description
技术领域
本发明属于环境检测技术领域,具体涉及一种基于分子印迹技术构建的微囊藻毒素比率荧光传感器。
背景技术
微囊藻毒素(Microcystin,简称MC)是蓝藻产生的一类天然毒素,是一类环七肽缩氨酸肝毒素,具有强烈的促肝癌作用,通过食物网对水生生物、饮用水安全和人类健康构成巨大威胁。特别是,MC-LR、MC-RR作为毒性最强、最具普遍性的微囊藻毒素,正受到广泛关注。微囊藻有时会表现出超乎寻常的生命力,不论常规的自来水处理工艺,还是将水煮沸,都难以有效去除微囊藻毒素。研究显示,即使在300摄氏度高温下微囊藻毒素仍然可以保留一部分活性。因此,在生物和环境***的情况下,敏感和选择性地检测微囊藻毒素至关重要。
到目前为止,已经开发了许多检测微囊藻毒素的方法,包括高效液相色谱(HPLC)、酶联免疫吸附试验(ELISA)、电化学测量、蛋白磷酸酶抑制试验(PPIA)、和光学传感,即生化方法和仪器分析法。其中生化方法中主要采用酶联分析法,该方法简单、高效、快速,但是该方法一方面需要使用微囊藻毒素单克隆抗体,其制备困难导致需要进口,价格昂贵;另一方面该方法选择性较差,容易出现假阳性现象。仪器分析法主要采用高效液相色谱或高效液相色谱-串联质谱法。高效液相色谱法灵敏度低,需要高倍富集从而需要4-6个小时才能检测一批样品,不能够满足水华监测的快速及时分析。
因此,如何找到一种针对微囊藻毒素的快速检测方法,以快速反应水体中微囊藻毒素的存在,以及起到快速预警和控制,对于控制水体污染至关重要。
发明内容
本发明提供一种基于分子印迹技术构建的微囊藻毒素比率荧光传感器,其可实现微囊藻毒素的快速定性和定量检测,简单快捷,生物响应度高,
为实现上述技术目的,本发明所采用的技术方案为:
一种基于分子印迹技术构建的微囊藻毒素比率荧光传感器,制备方法为:
(1)合成荧光碳点CQDS:将0.3~0.6g无水柠檬酸溶解在10mL N-(β-氨基乙基)-γ-氨基丙基-甲基二甲氧基硅烷中,装入50mL特氟隆内衬的不锈钢高压釜中,并用氮气脱气20分钟;然后,将高压釜在240℃下保持2小时,冷却至室温;溶液用过滤膜过滤;再用石油醚洗涤三次,再将获得的产物分散在无水乙醇中,得到荧光碳点CQDS乙醇溶液,并储存在4℃的冰箱中以备进一步使用;根据每次获得产物的数量,产物和乙醇体积比是1:7左右;
(2)FITC-APTES-SiO2复合纳米粒子的制备:取4~10mg异硫氰酸荧光素FITC与10mL水混合,室温下磁力搅拌均匀,加入100μL3-氨丙基三乙氧基硅烷APTES,避光室温搅拌24h,制得FITC-APTES前驱体;取1.77mL Triton X-100、1.80mL正已醇、7.50mL环已烷混合,室温下磁力搅拌30min混合均匀,加入8~11mL的FITC-APTES前驱体作为分散相,继续搅拌,形成油包水微乳液;取正硅酸乙酯滴加到所述油包水微乳液中,两者体积比1:200,室温下磁力搅拌30min后,加入60μL氨水;持续室温搅拌24h后,加入10mL丙酮溶液破乳,高速离心后分别用无水乙醇、超纯水洗涤后真空干燥保存,得FITC-APTES-SiO2复合纳米粒子;
(3)CQDS-FITC-APTES-SiO2@MIP的合成:将100~200mg二甲双胍和40mL荧光碳点CQDS乙醇溶液加入到容器中;然后,在容器里加入步骤(2)所得FITC-APTES-SiO2复合纳米粒子,具体的是将FITC-APTES-SiO2复合纳米粒子配成0.5g/L乙醇溶液,与CQDS体积比1:6加入,再加入300~500μL3-氨丙基三乙氧基硅烷,1~2mL正硅酸乙酯,50~100mg过硫酸铵和800μL氨水搅拌均匀;混合物在室温下黑暗中静置24小时;离心收集所得产物,并用乙醇洗涤,得固体产物;
(4)再将固体产物用有机溶剂洗脱二甲双胍,然后在60℃的真空干燥得终产物比率荧光传感器。
进一步的,步骤(1)中所用滤膜为0.22μm滤膜。
进一步的,步骤(2)所用氨水质量浓度为25%。
进一步的,步骤(3)乙醇洗涤不少于3次。
进一步的,步骤(4)有机溶剂为无水乙醇和乙腈按照体积比4:1混合,洗脱不少于三次。
荧光比率分析法有线性动态范围宽、光谱干扰少等优点。比率荧光检测技术具有明显的优点:灵敏度高、响应速度快、不需要复杂的样品前处理、对样品无破坏、受环境的影响较小等优点。分子印迹技术是合成对某种分子具有特定识别位点的多孔材料的一种很有前途的技术。分子印迹聚合物(MIPs)具有选择性高、制备容易、化学稳定性好和成本低等优点,并已广泛应用于化学传感、光降解和分离。
因此,将荧光比率技术和分子印迹技术相结合,以开发一种适合检测微囊藻毒素的荧光传感器,实现对目标的高选择性和高灵敏度检测。
有益效果
本发明根据二甲双胍和微囊藻毒素均含有特异性胍基结构,以二甲双胍为假模板,后将二甲双胍去除,留下特异性分子印迹结合位点,可实现与微囊藻毒素的特异性结合,实现对微囊藻毒素的定性定量检测。所得CQDS-FITC-APTES-SiO2@MIP对微囊藻毒素RR/LR荧光相应度好,反应灵敏,荧光比率技术和分子印迹技术的结合,完全满足微囊藻毒素的快速检测需求,且成本低廉,适合各类场合使用。
附图说明
图1为本发明CQDS的TEM图;
图2为本发明CQDS的紫外吸收光谱以及荧光发射光谱;
图3为本发明FITC-APTES-SiO2复合纳米粒子透射电镜图;
图4为本发明FITC-APTES-SiO2复合纳米粒子扫描电镜图;
图5为本发明CQDS和FITC-APTES-SiO2纳米粒子的红外光谱对比图;
图6为本发明CQDS-FITC-APTES-SiO2@MIP扫描电镜图;
图7为CQDS-FITC-APTES-SiO2@MIP和CQDS-FITC-APTES-SiO2@NIP红外光谱图;
图8为CQDS-FITC-APTES-SiO2@MIP和CQDS-FITC-APTES-SiO2@NIP紫外光谱图;
图9为本发明CQDS-FITC-APTES-SiO2@MIP对微囊藻毒素-RR荧光响应图;
图10为本发明CQDS-FITC-APTES-SiO2@MIP对微囊藻毒素-LR荧光响应图;
图11为本发明CQDS-FITC-APTES-SiO2@NIP对微囊藻毒素-RR和微囊藻毒素-LR荧光响应图;
具体实施方式
下面结合具体实施例对本发明的技术方案做进一步说明,但不限于此。
实施例1
一种基于分子印迹技术构建的微囊藻毒素比率荧光传感器,制备方法为:
第一步:合成荧光碳点CQDS:将0.3g无水柠檬酸溶解在10mL N-(β-氨基乙基)-γ-氨基丙基-甲基二甲氧基硅烷中,装入50mL特氟隆内衬的不锈钢高压釜中,并用氮气脱气20分钟;然后,将高压釜在240℃下保持2小时,冷却至室温;溶液用过滤膜过滤;再用石油醚洗涤三次,再将获得的产物分散在无水乙醇中,得到荧光碳点CQDS乙醇溶液,并储存在4℃的冰箱中以备进一步使用;
第二步:FITC-APTES-SiO2复合纳米粒子的制备:取4mg异硫氰酸荧光素FITC与10mL水混合,室温下磁力搅拌均匀,加入100μL3-氨丙基三乙氧基硅烷APTES,避光恒温搅拌24h,制得FITC-APTES前驱体;取1.77mL Triton X-100、1.80mL正已醇、7.50mL环已烷混合,室温下磁力搅拌30min混合均匀,加入8mL的FITC-APTES前驱体作为分散相,继续搅拌,形成油包水微乳液;取100μL正硅酸乙酯滴加到所述油包水微乳液中,室温下磁力搅拌30min后,加入60μL氨水;持续室温搅拌24h后,加入10mL丙酮溶液破乳,高速离心后分别用无水乙醇、超纯水洗涤后真空干燥保存,得FITC-APTES-SiO2复合纳米粒子。
第三步:CQDS-FITC-APTES-SiO2@MIP的合成:将100mg二甲双胍和40mL荧光碳点CQDS乙醇溶液加入到容器中;然后,在容器里加入步骤(2)所得FITC-APTES-SiO2复合纳米粒子,300μL3-氨丙基三乙氧基硅烷,1mL正硅酸乙酯,50mg过硫酸铵和800μL氨水搅拌均匀;混合物在室温下黑暗中静置24小时;离心收集所得产物,并用乙醇洗涤,得固体产物;
再将固体产物用有机溶剂洗脱二甲双胍,洗脱不少于三次。然后在60℃的真空干燥得终产物比率荧光传感器。
实施例2
一种基于分子印迹技术构建的微囊藻毒素比率荧光传感器,制备方法为:
第一步:合成荧光碳点CQDS:将0.6g无水柠檬酸溶解在10mL N-(β-氨基乙基)-γ-氨基丙基-甲基二甲氧基硅烷中,装入50mL特氟隆内衬的不锈钢高压釜中,并用氮气脱气20分钟;然后,将高压釜在240℃下保持2小时,冷却至室温;溶液用过滤膜过滤;再用石油醚洗涤三次,再将获得的产物分散在无水乙醇中,得到荧光碳点CQDS乙醇溶液,并储存在4℃的冰箱中以备进一步使用;
第二步:FITC-APTES-SiO2复合纳米粒子的制备:取4~10mg异硫氰酸荧光素FITC与10mL水混合,室温下磁力搅拌均匀,加入100μL3-氨丙基三乙氧基硅烷APTES,避光恒温搅拌24h,制得FITC-APTES前驱体;取1.77mL Triton X-100、1.80mL正已醇、7.50mL环已烷混合,室温下磁力搅拌30min混合均匀,加入11mL的FITC-APTES前驱体作为分散相,继续搅拌,形成油包水微乳液;取100μL正硅酸乙酯滴加到所述油包水微乳液中,室温下磁力搅拌30min后,加入60μL氨水;持续室温搅拌24h后,加入10mL丙酮溶液破乳,高速离心后分别用无水乙醇、超纯水洗涤后真空干燥保存,得FITC-APTES-SiO2复合纳米粒子。
第三步:CQDS-FITC-APTES-SiO2@MIP的合成:将200mg二甲双胍和40mL荧光碳点CQDS乙醇溶液加入到容器中;然后,在容器里加入步骤(2)所得FITC-APTES-SiO2复合纳米粒子,500μL3-氨丙基三乙氧基硅烷,2mL正硅酸乙酯,100mg过硫酸铵和800μL氨水搅拌均匀;混合物在室温下黑暗中静置24小时;离心收集所得产物,并用乙醇洗涤,得固体产物;
再将固体产物用有机溶剂洗脱二甲双胍,洗脱不少于三次。然后在60℃的真空干燥得终产物比率荧光传感器。
实施例3
一种基于分子印迹技术构建的微囊藻毒素比率荧光传感器,制备方法为:
第一步:合成荧光碳点CQDS:将0.5g无水柠檬酸溶解在10mL N-(β-氨基乙基)-γ-氨基丙基-甲基二甲氧基硅烷中,装入50mL特氟隆内衬的不锈钢高压釜中,并用氮气脱气20分钟;然后,将高压釜在240℃下保持2小时,冷却至室温;溶液用过滤膜过滤;再用石油醚洗涤三次,再将获得的产物分散在无水乙醇中,得到荧光碳点CQDS乙醇溶液,并储存在4℃的冰箱中以备进一步使用;
所得荧光碳点CQDS的透射电镜图如图1所示。
碳点的合成产物包括无水柠檬酸的分解和热解,同时酰化反应和表面钝化同时发生。为了评价制备的碳点的光学性质,进行了紫外-可见吸收光谱和荧光光谱。在光谱图(图2)中有一个位于349nm的吸收峰。合成的碳点的荧光光谱在349nm激发下,在458nm处出现最大发射峰。
第二步:FITC-APTES-SiO2复合纳米粒子的制备:取4.3mg异硫氰酸荧光素FITC与10mL水混合,室温下磁力搅拌均匀,加入100μL3-氨丙基三乙氧基硅烷APTES,避光恒温搅拌24h,制得FITC-APTES前驱体;取1.77mL Triton X-100、1.80mL正已醇、7.50mL环已烷混合,室温下磁力搅拌30min混合均匀,加入10mL的FITC-APTES前驱体作为分散相,继续搅拌,形成油包水微乳液;取100μL正硅酸乙酯滴加到所述油包水微乳液中,室温下磁力搅拌30min后,加入60μL氨水;持续室温搅拌24h后,加入10mL丙酮溶液破乳,高速离心后分别用无水乙醇、超纯水洗涤后真空干燥保存,得FITC-APTES-SiO2复合纳米粒子。
进一步对FITC-APTES-SiO2复合纳米粒子进行透射电镜和扫描电镜的表征,TEM图如图3所示,SEM图如图4所示,图中可以看出,纳米粒子呈现边缘整齐而光滑的球型结构,形态良好。
进一步对合成荧光碳点CQDS和FITC-APTES-SiO2复合纳米粒子进行红外光谱分析,如图5所示,1565cm-1处的峰属于二级酰胺氮氢弯曲和碳氮拉伸,而1650cm-1处的峰属于二级酰胺碳氧拉伸,3291cm-1处的宽峰属于二级酰胺氮氢拉伸。这些情况揭示了酰胺键的形成,这是表明CQDS表面钝化反应成功的最典型特征。这些情况表明,CQDS是成功合成的。
1090cm-1和469cm-1附近处均有强的吸收峰,为氧化硅的特征振动峰,分别对应于Si-O-Si的不对称伸缩振动和弯曲振动。799cm-1处是Si-O对称伸缩振动峰,960cm-1为Si—OH的伸缩振动峰,1555cm-1是多了一个峰,为N—H弯曲振动的贡献,证明成功地将氨基官能团修饰到氧化硅材料中,进一步说明FITC已通过与APTES偶联,以化学键的形式连接到氧化硅纳米粒子中。
第三步:CQDS-FITC-APTES-SiO2@MIP的合成:将200mg二甲双胍和40mL荧光碳点CQDS乙醇溶液加入到容器中;然后,在容器里加入步骤(2)所得FITC-APTES-SiO2复合纳米粒子,350μL3-氨丙基三乙氧基硅烷,1.5mL正硅酸乙酯,50mg过硫酸铵和800μL氨水搅拌均匀;混合物在室温下黑暗中静置24小时;离心收集所得产物,并用乙醇洗涤,得固体产物;
再将固体产物用有机溶剂洗脱二甲双胍,洗脱不少于三次。然后在60℃的真空干燥得终产物比率荧光传感器。
图6为CQDS-FITC-APTES-SiO2@MIP扫描电镜图,图中可以看出,纳米粒子形貌明显发生改变,也可以说明,纳米粒子成分发生变化,有新物质附着于纳米粒子表面。
对比例1
CQDS-FITC-APTES-SiO2@NIP制备,即不使用二甲双胍进行分子印迹。
一种微囊藻毒素比率荧光传感器,制备方法为:
(1)合成荧光碳点CQDS:将0.5g无水柠檬酸溶解在10mL N-(β-氨基乙基)-γ-氨基丙基-甲基二甲氧基硅烷中,装入50mL特氟隆内衬的不锈钢高压釜中,并用氮气脱气20分钟;然后,将高压釜在240℃下保持2小时,冷却至室温;溶液用过滤膜过滤;再用石油醚洗涤三次,再将获得的产物分散在无水乙醇中,得到荧光碳点CQDS乙醇溶液,并储存在4℃的冰箱中以备进一步使用;
(2)FITC-APTES-SiO2复合纳米粒子的制备:取4.3mg异硫氰酸荧光素FITC与10mL水混合,室温下磁力搅拌均匀,加入100μL3-氨丙基三乙氧基硅烷APTES,避光恒温搅拌24h,制得FITC-APTES前驱体;取1.77mL Triton X-100、1.80mL正已醇、7.50mL环已烷混合,室温下磁力搅拌30min混合均匀,加入10mL的FITC-APTES前驱体作为分散相,继续搅拌,形成油包水微乳液;取100μL正硅酸乙酯滴加到所述油包水微乳液中,室温下磁力搅拌30min后,加入60μL氨水;持续室温搅拌24h后,加入10mL丙酮溶液破乳,高速离心后分别用无水乙醇、超纯水洗涤后真空干燥保存,得FITC-APTES-SiO2复合纳米粒子;
(3)CQDS-FITC-APTES-SiO2@NIP的合成:将40mL荧光碳点CQDS乙醇溶液加入到容器中;然后,在容器里加入步骤(2)所得FITC-APTES-SiO2复合纳米粒子,350μL3-氨丙基三乙氧基硅烷,1.5mL正硅酸乙酯,50mg过硫酸铵和800μL氨水搅拌均匀;混合物在室温下黑暗中静置24小时;离心收集所得产物,并用乙醇洗涤,得固体产物;然后在60℃的真空干燥得终产物比率荧光传感器。
本对比例与实施例1唯一的区别即为不使用二甲双胍印迹,其余步骤和原料选择配比均相同。
性能测试
CQDS-FITC-APTES-SiO2@MIP和CQDS-FITC-APTES-SiO2@NIP进行红外分析,谱图如图7所示,红外谱图中MIP,NIP具有相似的振动峰,说明模板分子二甲双胍被洗脱完全。同时对实施例洗脱前后进行分析,如图8所示,紫外谱图中,实施例1分子印迹聚合物在300-400nm范围内没有特征吸收峰,这表明它成功地去除了分子印迹聚合物纳米复合材料中的模板分子。
微囊藻毒素的检测
将实施例1得到的烘干的分子印迹涂层粉末CQDS-FITC-APTES-SiO2@MIP溶解在生理盐水中,制成分子印迹涂层工作溶液(400μg/mL)。3mL的工作溶液加入试管,然后加入一系列不同浓度的微囊藻毒素溶液。荧光测量在310nm的激发波长下进行。充分混合后立即测量荧光光谱。荧光响应图如图9-11所示,图中可以看出:CQDS-FITC-APTES-SiO2@MIP对两种微囊藻毒素具有良好的荧光响应,荧光传感器可以特异性识别藻毒素里面的胍基进而实现检测。而同一浓度的材料对同一浓度的两种藻毒素荧光响应大小是不一样的,以实现两种毒素的定性定量检测。
需要说明的是,上述实施例仅仅是实现本发明的优选方式的部分实施例,而非全部实施例。显然,基于本发明的上述实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的其他所有实施例,都应当属于本发明保护的范围。
Claims (5)
1.一种基于分子印迹技术构建的微囊藻毒素比率荧光传感器,其特征在于,制备方法为:
(1)合成荧光碳点CQDS:将0.3~0.6g水柠檬酸溶解在10mL N-(β-氨基乙基)-γ-氨基丙基-甲基二甲氧基硅烷中,装入50mL特氟隆内衬的不锈钢高压釜中,并用氮气脱气20分钟;然后,将高压釜在240℃下保持2小时,冷却至室温;溶液用过滤膜过滤;再用石油醚洗涤三次,再将获得的产物分散在无水乙醇中,得到荧光碳点CQDS乙醇溶液,并储存在4℃的冰箱中以备进一步使用;
(2)FITC-APTES-SiO2复合纳米粒子的制备:取4~10mg异硫氰酸荧光素FITC与10mL水混合,室温下磁力搅拌均匀,加入100μL 3-氨丙基三乙氧基硅烷APTES,避光室温搅拌24h,制得FITC-APTES前驱体;取1.77mL Triton X-100、1.80mL正已醇、7.50mL环已烷混合,室温下磁力搅拌30min混合均匀,加入8~11mL的FITC-APTES前驱体作为分散相,继续搅拌,形成油包水微乳液;取正硅酸乙酯滴加到所述油包水微乳液中,两者体积比1:200,室温下磁力搅拌30min后,加入60μL氨水;持续室温搅拌24h后,加入10mL丙酮溶液破乳,高速离心后分别用无水乙醇、超纯水洗涤后真空干燥保存,得FITC-APTES-SiO2复合纳米粒子;
(3)CQDS-FITC-APTES-SiO2@MIP的合成:将100~200mg二甲双胍和40mL荧光碳点CQDS乙醇溶液加入到容器中;然后,在容器里加入步骤(2)所得FITC-APTES-SiO2复合纳米粒子,300~500μL3-氨丙基三乙氧基硅烷,1~2mL正硅酸乙酯,50~100mg过硫酸铵和800μL氨水搅拌均匀;混合物在室温下黑暗中静置24小时;离心收集所得产物,并用乙醇洗涤,得固体产物;
(4)再将固体产物用有机溶剂洗脱二甲双胍,然后在60℃的真空干燥得终产物比率荧光传感器。
2.根据权利要求1所述基于分子印迹技术构建的微囊藻毒素比率荧光传感器,其特征在于,步骤(1)中所用滤膜为0.22μm滤膜。
3.根据权利要求1所述基于分子印迹技术构建的微囊藻毒素比率荧光传感器,其特征在于,步骤(2)所用氨水质量浓度为25%。
4.根据权利要求1所述基于分子印迹技术构建的微囊藻毒素比率荧光传感器,其特征在于,步骤(3)乙醇洗涤不少于3次。
5.根据权利要求1所述基于分子印迹技术构建的微囊藻毒素比率荧光传感器,其特征在于,步骤(4)有机溶剂为无水乙醇和乙腈按照体积比4:1混合。
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