CN106568748A - 一种基于壳核型上转换材料和二硫化钼发生荧光共振能量转移检测微囊藻毒素lr的方法 - Google Patents
一种基于壳核型上转换材料和二硫化钼发生荧光共振能量转移检测微囊藻毒素lr的方法 Download PDFInfo
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Abstract
一种基于壳核型上转换材料和二硫化钼发生荧光共振能量转移检测微囊藻毒素LR的方法，用于实际水样中微囊藻毒素LR(MC‑LR)的检测。制备壳核型上转换纳米材料(NaYF4:Yb,Tm@NaYF4:Yb)提高其荧光强度。将上转换材料与MC‑LR的核酸适配体连接后，核酸碱基与二硫化钼基底面通过范德华力吸附，上转换材料与二硫化钼间发生荧光共振能量转移现象，荧光淬灭。当检测中存在MC‑LR时，MC‑LR与其适配体特异性结合，适配体构象改变，从二硫化钼基底面分离，荧光恢复。监测361nm处的荧光强度，能定量检测MC‑LR，线性范围为0.01‑50ng/ml，检出限为0.002ng/ml。本发明用于MC‑LR检测灵敏度高，特异性强，稳定性好。并用于自来水及太湖水的检测，结果准确可靠。
Description
技术领域
一种基于壳核型上转换材料和二硫化钼发生荧光共振能量转移检测微囊藻毒素LR的方法，涉及纳米材料和分析化学技术领域，用于对水中微囊藻毒素LR的检测。
背景技术
微囊藻毒素(Microcystins，MCs)是由淡水蓝绿藻产生的一类毒性强、急性危害大的环七肽缩氨酸肝毒素，对水生生物、人类饮用水安全和人类健康产生严重影响。其中，微囊藻毒素-(亮氨酸-精氨酸)(Microcystin-(leucine-arginine)，MC-LR)是最常见、急性毒性最大的微囊藻毒素之一。其具有较强的肝毒性,并对心、肾、脾脏及胃肠也具有一定的毒性。目前，检测水体中MC-LR的传统方法中，高效液相色谱法、高效液相色谱-质谱联用技术检测效果较好，但其仪器设备价值昂贵、操作繁琐且对操作人员技术要求较高；植物细胞的生物测试法和蛋白磷酸酶抑制法灵敏度相对较低；酶联免疫吸附法灵敏度较高，但其线性范围较窄。所以，开发一种灵敏度高、特异性强，稳定性好，快速方便的新型检测方法很有必要。
核酸适配体(Aptamer)是通过指数富集系统配体进化(systematic evolution ofligands by exponential enrichment，SELEX)筛选获得的，与靶标具有高亲和力和特异性结合的核糖核酸(RNA)或单链脱氧核糖核酸(ssDNA)。这种寡核苷酸序列形成的高级结构能够识别与之对应的小分子、蛋白质、细胞、微生物等多种靶标。与抗体相比，适配体的靶分子范围广，体外筛选，易人工合成和修饰，分子小，对温度不敏感，稳定性好，容易保存。经过二十多年的发展，适配体已成为一种新型识别元件在医疗诊断与临床治疗、分析检测、分离纯化和食品安全等领域发挥广泛作用。
上转换发光纳米材料(Upconversion Nanoparticles)是一类吸收长波长、低能量光子，发射短波长高能量光子的新型荧光探针材料，具有许多独特的优点，检测背景低，穿透性强，光化学稳定，可以选择不同的基质材料和掺杂离子来调节上转换发光，并且可以实现同一激发，不同发射。鉴于上述优点，上转换发光纳米材料已成为一种优秀的生物标记材料。而壳核型上转换材料更是克服了由于材料表面缺陷和表面配体而导致荧光淬灭，大大提高了荧光强度。二硫化钼(Molybdenum Disulfide)作为一种新型的二维层状纳米材料，近年来以其独特的物理、化学性质而成为新兴的研究热点。层状的二硫化钼材料被广泛地应用于光催化剂、电化学生物传感器、锂离子电池的电极材料、超级电容器及光学器件等方面的应用。
本发明首先制备壳核型上转换纳米材料(NaYF4:Yb,Tm@NaYF4:Yb)提高其荧光强度。同时二硫化钼材料具有良好的荧光淬灭能力，并且对单链DNA有较强的吸附能力，可以作为荧光受体。将上转换材料与微囊藻毒素LR的核酸适配体连接后，核酸碱基与二硫化钼基底面通过范德华力吸附，使得上转换纳米材料与二硫化钼之间的距离拉近，发生荧光共振能量转移现象，从而实现荧光淬灭。当检测中存在微囊藻毒素LR时，微囊藻毒素LR与核酸适配体特异性结合，适配体空间构象发生改变，从而与二硫化钼基底面分离，荧光恢复。监测361nm处的荧光恢复值，在一定范围内，微囊藻毒素LR对数浓度与上转换荧光信号恢复值成正比，建立标准曲线，达到对微囊藻毒素LR检测的目的。
发明内容
一种基于壳核型上转换发光材料和二硫化钼发生荧光共振能量转移检测微囊藻毒素LR的方法：制备了壳核型上转换发光纳米材料NaYF4:Yb,Tm@NaYF4:Yb，对其进行表面改性并与亲和素(Avidin)偶联，上转换发光纳米材料通过亲和素(Avidin)与生物素(Biotin)修饰的适配体DNA特异性结合。以其为能量供体，与能量受体二硫化钼经过一段时间孵育，核酸碱基与二硫化钼基底面通过范德华力吸附，使得上转换纳米材料与二硫化钼之间的距离拉近，发生荧光共振能量转移现象，从而实现淬灭荧光。利用980nm激光激发，此时的荧光信号为最小值。当检测中存在微囊藻毒素LR时，微囊藻毒素LR与核酸适配体特异性结合，适配体空间构象发生改变，从而与二硫化钼基底面分离，荧光信号增强。监测361nm处的荧光恢复值，在一定范围内，微囊藻毒素LR对数浓度与上转换荧光信号恢复值成正比，建立标准曲线，达到对微囊藻毒素LR检测的目的。步骤为：
(1)通过高温热解法技术，制备NaYF4:Yb,Tm上转换发光颗粒。称取一定量的YCl3·6H2O，YbCl3·6H2O，TmCl3·6H2O(Ln:79.5mol％Y3+，20mol％Yb3+，0.5mol％Tm3+，共计1mmol)于100ml三口烧瓶中，加入4mL油酸以及16mL 1-十八烯。磁力搅拌条件下，逐渐升高温度至160℃保持半小时，自然冷却至室温。准确称取0.1g氢氧化钠和0.148g氟化铵，溶于10mL甲醇溶液混合均匀。将上述溶液缓慢加入到三口烧瓶中，升至50℃磁力搅拌30min。之后温度升至80℃保持30min缓慢蒸发甲醇，脱气后，将混合液加热到300℃，持续1h。反应结束后，自然冷却到室温，用环己烷和乙醇洗涤三次，分散在5ml环乙烷中备用。
(2)通过高温热解法技术，制备NaYF4:Yb,Tm@NaYF4:Yb上转换发光颗粒。称取一定量的YCl3·6H2O，YbCl3·6H2O(Ln:80mol％Y3+，20mol％Yb3+，共计1mmol)于100ml三口烧瓶中，加入4mL油酸以及16mL 1-十八烯。磁力搅拌条件下，逐渐升高温度至160℃保持半小时，自然冷却至室温。将上述环己烷分散的NaYF4:Yb,Tm纳米材料滴加入三口烧瓶中，之后加入10ml甲醇分散的0.1g氢氧化钠和0.148g氟化铵，温度升至50℃磁力搅拌30min。之后温度升至80℃保持45min缓慢蒸发甲醇和环乙烷，脱气后，将混合液加热到300℃，持续1h。反应结束后，自然冷却到室温，用环己烷和乙醇洗涤三次，干燥以待进一步表征。
(3)利用配体交换法对上转换发光纳米颗粒进行表面羧基化修饰。取30mg油酸包覆的NaYF4:Yb,Tm@NaYF4:Yb上转换纳米材料，分散在5ml氯仿和甲苯混合溶液中，体积比为2:3。再将其加入到10ml溶解有200mg聚丙烯酸的水溶液中，搅拌24h。通过离心获得聚丙烯酸纳米材料，水清洗三次。
(4)利用EDC/NHS法将羧基化上转换发光纳米材料与亲和素(Avidin)偶联。称取2mg聚丙烯酸修饰的上转换纳米材料分散于2mL PBS(pH 7.4)缓冲液中，超声15min，加入0.16mL 2mg/mL EDC溶液和0.08mL 2mg/mL NHS溶液，37℃摇床中活化2h。离心收集材料，用PBS冲液清洗三次，然后分散于1.8ml PBS中，加入200μL 1mg/mL亲和素溶液，在37℃摇床中反应2h。反应结束后清洗数次，弃上清。
(5)通过亲和素(Avidin)与生物素(Biotin)之间的特异性结合将表面修饰亲和素(Avidin)的上转换发光纳米颗粒与生物素(Biotin)修饰的微囊藻毒素LR适配体DNA单链连接。具体方法为：将亲和素修饰的上转换纳米材料分散于2mL PBS缓冲液中，加入一定量的生物素化微囊藻毒素LR适配体，在37℃摇床中孵育12h，离心收集材料和上清，用磷酸缓冲液清洗三次，分散于Tris-HCl缓冲液中(pH 7.4,100mM NaCl,10mM MgCl2)
(6)通过核酸碱基与二硫化钼基底面间的范德华力吸附，上转换探针与二硫化钼距离拉近，发生荧光共振能量转移现象，从而实现淬灭荧光。具体方法为：取终浓度为0.1mg/ml微囊藻毒素LR适配体标记的上转换发光纳米颗粒溶液与终浓度为0.45mg/ml二硫化钼溶液混合，在Tris-HCl缓冲液中37℃反应10min，形成上转换纳米颗粒与二硫化钼的复合物。
(7)对微囊藻毒素LR标准品进行检测，建立标准曲线。配制不同浓度的微囊藻毒素LR标准品加入到上述复合物体系中，37℃孵育30min，在980nm激光激发下得到361nm处的上转换发光信号，空白组检测得到的淬灭发光信号(F0)最小，随着微囊藻毒素LR的浓度增加，荧光光信号(F)逐步增加。根据发光差值(△F＝F-F0)与对应的微囊藻毒素LR标准品浓度建立标准曲线，实验结果在0.01-50ng/mL区间内得到良好线性关系。
(8)对微囊藻毒素LR样品进行检测：对样品做简单前的处理后，在上述纳米复合物体系中加入不同浓度的微囊藻毒素LR37℃孵育30min后，直接在980nm激光下激发得到361nm处的上转换发光信号，从标准曲线中求得对应的微囊藻毒素LR的浓度。
本发明的优点是：
(1)上转换纳米材料表面包覆壳，减弱了由于配体及表面缺陷发生的荧光淬灭，增强了上转换荧光。
(2)利用适配体对被检测物质实现特异性捕获，有效提高了检测的稳定性和特异性。而且可以通过适配体的更换用于其他目标物的检测。
(3)利用二硫化钼对上转换纳米材料实现荧光淬灭，并且由于其吸收峰范围广，可以作为不同发光光谱的上转换纳米材料的淬灭剂。分析检测过程不需要进行分离，检测时间短，使分析检测过程简化。
附图说明
图1：基于壳核型上转换发光材料和二硫化钼之间发生荧光共振能量转移检测微囊藻毒素LR的实验原理图
图2：NaYF4:Yb,Tm上转换发光纳米材料电镜图(a)；NaYF4:Yb,Tm@NaYF4:Yb上转换发光纳米材料电镜图(b)；聚丙烯酸修饰NaYF4:Yb,Tm@NaYF4:Yb上转换发光纳米材料电镜图(c)
图3：二硫化钼电镜图(a)；二硫化钼原子力显微镜图(b)
图4：NaYF4:Yb,Tm与NaYF4:Yb,Tm@NaYF4:Yb上转换纳米材料荧光对比图(a)；二硫化钼紫外吸收图与NaYF4:Yb,Tm@NaYF4:Yb上转换纳米颗粒发光光谱(b)
图5：上转换荧光强度随微囊藻毒素LR浓度变化叠加图(a)；微囊藻毒素LR检测标准曲线图(b)，浓度范围在0.01-50ng/mL
具体实施方式
本发明包括但不限于以上实施例，凡是在本发明的精神和原则下进行的任何等同替换或者局部改进，都将视为在本发明的保护范围之内。
实施例1：自来水及太湖水实际样品中微囊藻毒素LR检测标准曲线建立及检测样品预处理：收集的自来水及太湖水样依次经过离心，过滤，过膜的方法进行前处理。将澄清的液体收集备用。利用本发明方法和高效液相色谱法分别测定其中微囊藻毒素LR的含量，结果见表一。两种方法检测结果一致，无明显差异。
表一：实际样品检测，本发明方法与HPLC方法对比
注：ND为未检出
实施例2：水实际样品中微囊藻毒素LR的检测及加标回收率实验样品预处理同实施例1。
以实施例1得到的6组微囊藻毒素LR浓度数据为本底值，分别向其中加入三种不同浓度的微囊藻毒素LR标准品，同样利用本发明方法再次检测其中微囊藻毒素LR的含量，得到检测值。回收率％＝(检测值-本底值)/添加量×100％。
从表二数据可以看到回收率在94％～112％，说明本发明稳定，灵敏，准确，适用于实际水样品中微囊藻毒素LR的检测。
表二：实际样品中微囊藻毒素LR的检测及加标回收率
Claims (6)
1.一种基于壳核型上转换材料和二硫化钼发生荧光共振能量转移检测微囊藻毒素LR的方法，其特征在于：制备壳核型上转换纳米材料(NaYF4:Yb,Tm@NaYF4:Yb)提高其荧光强度。将上转换材料与微囊藻毒素LR的核酸适配体连接后，核酸碱基与二硫化钼基底面通过范德华力吸附，使得上转换纳米材料与二硫化钼之间的距离拉近，发生荧光共振能量转移现象，从而实现荧光淬灭。当检测中存在微囊藻毒素LR时，微囊藻毒素LR与核酸适配体特异性结合，适配体空间构象发生改变，从而与二硫化钼基底面分离，荧光恢复。监测361nm处的荧光恢复值，在一定范围内，微囊藻毒素LR对数浓度与上转换荧光信号恢复值成正比，建立标准曲线，达到对微囊藻毒素LR检测的目的。
2.如权利要1所述的一种基于壳核型上转换材料和二硫化钼发生荧光共振能量转移检测微囊藻毒素LR的方法，其特征在于：合成NaYF4:Yb,Tm@NaYF4:Yb壳核型上转换纳米颗粒，结合平均厚度为0.8nm的单层二硫化钼用于微囊藻毒素LR检测。
3.如权利要1所述的一种基于壳核型上转换材料和二硫化钼发生荧光共振能量转移检测微囊藻毒素LR的方法，其特征在于：微囊藻毒素LR适配体与壳核型上转换纳米材料偶联形成探针。
4.如权利要1所述的一种基于壳核型上转换材料和二硫化钼发生荧光共振能量转移检测微囊藻毒素LR的方法，其特征在于：利用EDC/NHS作为偶联剂连接聚丙烯酸修饰的上转换发光纳米材料和生物素化的微囊藻毒素LR适配体。
5.如权利要1所述的一种基于壳核型上转换材料和二硫化钼发生荧光共振能量转移检测微囊藻毒素LR的方法，其特征在于：微囊藻毒素LR的适配体序列为：5’-biotin-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCGC-3’。
6.如权利要1所述的一种基于壳核型上转换材料和二硫化钼发生荧光共振能量转移检测微囊藻毒素LR的方法，其特征在于：所述方法能够用于水样中微囊藻毒素LR的检测。
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