CN117347336A - 一种检测卡那霉素的荧光适配体生物传感器及其制备方法和应用 - Google Patents
一种检测卡那霉素的荧光适配体生物传感器及其制备方法和应用 Download PDFInfo
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Abstract
一种检测卡那霉素的荧光适配体生物传感器及其制备方法和应用，涉及生物传感器技术领域，该荧光适配体生物传感器通过合成以适配体为模板的荧光铜银纳米团簇DNA‑Cu/AgNCs作为能量供体，同时制备锆金属有机框架NH2–UiO–66作为能量受体，本发明的构建适配体荧光传感器中做为能量供体的DNA‑Cu/AgNCs具有高的荧光和更好的稳定性以及更高的量子产率，该体系中做为能量受体的NH2‑UiO‑66，具有立体多孔隙结构，比表面积大，孔隙结构规则，粒径孔隙可调节性等特点，而且容易制造，具有成本效益，高效且环保的优点；本发明的构建适配体荧光传感器检测卡那霉素速度快，加入卡那霉素目标后，响应时间仅需2min，整个检测体系在25min内即可完成。
Description
技术领域
本发明涉及生物传感器技术领域，具体涉及一种检测卡那霉素的荧光适配体生物传感器及其制备方法和应用。
背景技术
现有技术中开发了各种技术来检测卡那霉素，例如比色法、电化学、光电化学、酶联免疫吸附测定(ELISA)、高效液相色谱(HPLC)、毛细管电泳(CE)、气相色谱-质谱联用、表面增强拉曼散射(SERE)，以上方法各有优缺点。比色法灵敏度有限；电化学方法，电极修饰繁琐，稳定性不好：酶联免疫方法由于经济问题，其需要抗体参与，成本较高；其余高效液相色谱、毛细管电泳、表面增强拉曼散射大多耗时，操作复杂，仪器设备复杂，综上所述，为了克服传统检测方法的缺点，需要开发简单、稳定、经济的快速检测方法，即采用荧光适配体生物传感器来检测抗生素卡那霉素。
发明内容
本发明提出一种检测卡那霉素的荧光适配体生物传感器。
技术方案如下：该荧光适配体生物传感器通过合成以适配体为模板的荧光铜银纳米团簇DNA-Cu/AgNCs作为能量供体，同时制备锆金属有机框架NH2–UiO–66作为能量受体。
另一技术方案，荧光适配体生物传感器的制备方法；
DNA-Cu/AgNCs的合成步骤为：首先将适配体纳米团簇序列，即DNA(Cu/AgNCs)溶解在PBS溶液中，将AgNO3和(Cu(NO3)2·3H2O加入到含有适配体纳米团簇序列的PBS溶液中，然后进行冰浴；之后继续加入新鲜配置的NaBH4溶液中剧烈震荡，将适配体纳米团簇序列、AgNO3、Cu(NO3)2、NaBH4的混合物在室温下孵育后，得到淡黄色的DNA-Cu/AgNCS溶液，然后置于4℃冰箱避光储存；
所述适配体纳米团簇序列为：(5'-3')CCCTTAATCCCC TGGGGGTTGAGGCTAAGCCGA；
其中，NH2-UiO-66的合成步骤为：称取ZrCl4和H2ATA溶解在DMF中，超声至分散均匀，然后加入苯甲酸，搅拌至溶解，转移至聚四氟乙烯反应釜中反应，之后取出过夜冷却至室温，然后用DMF和乙醇洗涤，之后干燥过夜。
优选地，所述DNA-Cu/AgNCs的合成步骤中：所述适配体纳米团簇序列浓度为100μmol/L；PBS溶液浓度为40mmol/L、pH＝7；AgNO3浓度为1mmol/L；(Cu(NO3)2·3H2O浓度为1mmol/L；冰浴时间为15min；NaBH4浓度为2mmol/L；震荡时间为1min；孵育时间为90min。
优选地，所述DNA-Cu/AgNCs的合成步骤中：所述适配体纳米团簇序列、AgNO3、Cu(NO3)2、NaBH4的终浓度之比为1:3:8:11。
优选地，所述NH2-UiO-66的合成步骤中：ZrCl4质量为140mg；H2ATA质量为49.25mg；DMF的体积为20mL；超声30min至分散均匀；苯甲酸的质量为366.5mg；在聚四氟乙烯反应釜中120℃反应48h。
另一技术方案：检测卡那霉素的荧光适配体生物传感器的应用，该荧光适配体生物传感器用于检测卡那霉素，具体步骤为：首先在离心管中加PBS缓冲溶液，取NH2-UiO-66加入离心管与PBS充分混合；然后混合液中加入DNA-Cu/AgNCs,将混合液在黑暗条件下孵育，待荧光猝灭恢复到室温后，加入待检测的卡那霉素，室温条件下2min荧光恢复，使用荧光光谱仪对上述体系进行荧光测定。
优选地，PBS缓冲溶液的浓度为40mmol/L、pH＝7；黑暗条件下孵育的温度为37℃，孵育时间20min。
与现有技术相比，本发明的有益效果：
基于DNA-Cu/AgNCs和NH2–UiO–66的荧光适配体传感器用来检测残留在牛奶中的卡那霉素，通过合成以适配体为模板的荧光铜银纳米团簇，作为能量供体，同时制备锆金属有机框架NH2–UiO–66作为能量受体，由于NH2–UiO–66与DNA-Cu/AgNCs之间的π-π堆积，氢键和配位作用，NH2–UiO–66可以吸附并猝灭DNA-Cu/AgNCs的荧光，当存在卡那霉素时，由于适配体与卡那霉素亲和力更强，适配体识别并结合卡那霉素，降低NH2–UiO–66与单链DNA之间的π-π堆积作用，导致从其表面解析，在560nm处荧光恢复，且目标与适配体结合，荧光恢复仅需要两分钟，达到即时检测的效果，然后通过荧光强度与卡那霉素浓度之间的关系来定量对其进行检测。
(1)：本发明的构建适配体荧光传感器，由以适配体为模板纳米团簇与锆金属有机框架形成的新的检测体系，该体系中做为能量供体的DNA-Cu/AgNCs具有高的荧光和更好的稳定性以及更高的量子产率，该体系中做为能量受体的NH2-UiO-66，具有立体多孔隙结构，比表面积大，孔隙结构规则，粒径孔隙可调节性等特点，而且容易制造，具有成本效益，高效且环保的优点；
(2)：本发明的构建适配体荧光传感器检测卡那霉素速度快，加入卡那霉素目标后，响应时间仅需2min，整个检测体系在25min内即可完成。
附图说明
图1：本发明提供实施例1方法制备的DNA-Cu/AgNCs和NH2-UiO-666材料的电镜表征及粒径图，其中(A)(B)为DNA-Cu/AgNCs及粒径直方图、(C)(D)为NH2-UiO-66。
图2：本发明提供实施例1和对比例1不同物质的荧光光谱，470nm激发、560nm发射。
图3：DNA-Cu/AgNCS和RhB的荧光光谱。
图4：本发明提供实施例2中对不同浓度(0.5-10μM)卡那霉素进行检测的荧光响应图(A)DNA-AgNCs和(B)DNA-Cu/AgNCs在不同浓度(0.5-10μM)卡那霉素的荧光光谱。
具体实施方式
下面将结合本发明实施例中的附图，对发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：一种检测卡那霉素的荧光适配体生物传感器，该荧光适配体生物传感器通过合成以适配体为模板的荧光铜银纳米团簇DNA-Cu/AgNCs作为能量供体，同时制备锆金属有机框架NH2–UiO–66作为能量受体。
该荧光适配体生物传感器的制备方法如下：
DNA-Cu/AgNCs的合成步骤为：首先将适配体纳米团簇序列即DNA(Cu/AgNCs)溶解在PBS溶液(40mmol/L、pH＝7)中，将AgNO3(浓度为1mmol/L)和(Cu(NO3)2·3H2O(浓度为1mmol/L)加入到含有适配体纳米团簇序列的PBS溶液中，然后进行冰浴15min；之后继续加入新鲜配置的NaBH4(浓度为2mmol/L)溶液中剧烈震荡1min，适配体纳米团簇序列、AgNO3、Cu(NO3)2、NaBH4的终浓度之比为1:3:8:11，将适配体纳米团簇序列、AgNO3、Cu(NO3)2、NaBH4的混合物在室温下孵育90min后，得到淡黄色的DNA-Cu/AgNCS溶液，然后置于4℃冰箱避光储存。
其中，所述适配体纳米团簇序列浓度为100μmol/L；所述适配体纳米团簇序列为：(5'-3')CCCTTAATCCCC TGGGGGTTGAGGCTAAGCCGA；其中，实验所用的DNA序列均由生工生物工程(上海)股份有限公司合成并纯化，序列(5'－3')为：CCC TTAATC CCC TGG GGG TTGAGG CTAAGC CGA命名为DNA-Cu/AgNCs，根据以下序列复合而成，
如铜银模板序列CCCTTAATCCCCu/Ag-Aptamer(DNA-Cu/AgNCs)CCCTTAATCCCCTGGGGGTTGAGGCTAAGCCG。
NH2-UiO-66的合成步骤为：称取氯化锆(ZrCl4)(140mg)和2-氨基对苯二甲酸(H2ATA)(49.25mg)溶解在体积为20mL的2-氨基对苯二甲酸(DMF)中，超声30min至分散均匀，然后加入苯甲酸(366.5mg)，搅拌至溶解，转移至聚四氟乙烯反应釜中120℃反应48h，之后取出过夜冷却至室温，然后用DMF和乙醇洗涤数次，之后120℃干燥过夜。
对采用实施例1方法制备的DNA-Cu/AgNCs和NH2-UiO-66进行材料表征，结果如图1所述，对所制备的DNA-Cu/AgNCs的形貌进行了TEM表征，如图1-A所示，用TEM对DNA-Cu/AgNCs荧光探针进行形貌和粒径分析，结果如图1-A所示。从TEM图可看出，DNA-Cu/AgNCs分散均匀，均呈球状。分别对图中出现的粒子进行粒径测定绘成粒径分布直方图，可以看出的平均粒径约为5.5nm，根据TEM表征表明，DNA-Cu/AgNCs具有较为均一的尺寸。
同时，通过TEM对NH2-UiO-66的形貌进行了表征。如图1-C、D所示，可以清楚地显示表面形貌，显示相对均匀的八面体NH2-UiO-66(Zr)纳米晶体结构，平均粒径在40-50nm范围内。
对比例1：合成DNA-AgNCs，与实施例1中DNA-Cu/AgNCs合成的区别在于除去加入Cu(NO3)2，其他反应条件一致。
荧光强度：对比两个纳米团簇荧光强度，470nm激发条件下，DNA-Cu/AgNCs、DNA-AgNCs 560nm荧光发射强度对比。如图2很明显铜银纳米团簇荧光强度更高。
DNA-AgNCs和DNA-Cu/AgNCs量子产率对比：
量子产率(QY)的大小可以表示物质的发光能力，QY值越大，说明荧光材料的荧光性能越好。本实验采用参比法测定合成的DNA-NCs的QY。如图3所示，DNA-Cu/AgNCs和标准物质RhB(1μg/mL，无水乙醇为溶剂)的荧光发射光谱(注意：先将标准品跟样品稀释到吸光度<0.05)。然后按照下式计算待测物质的QY：
其中：Yu为待测物质的QY；Ys为参比物质的QY；Fu为待测物质的积分荧光强度；Fs为参比物质的积分荧光强度；Au为待测物质在同一激发波长下的吸光值；As为参比物质在同一激发波长的吸光值。
根据上述公式算出，DNA-Cu/AgNCs量子产率QY＝58.13％。
据相关文献记载同样合成方法DNA-AgNCs量子产率为11.5％，对比DNA-AgNCs量子产率，DNA-Cu/AgNCs荧光量子产率明显更高。
实施例2：对比例1合成的DNA-AgNCs、实施例1合成的DNA-Cu/AgNCs与猝灭材料NH2-UiO-66的体系猝灭效率对比。
如图4，对不同浓度(0.5-10μM)卡那霉素进行检测的荧光响应图，通过计算猝灭率＝△F/F初始得到DNA-AgNCs、DNA-Cu/AgNCs的荧光的猝灭率分别(45.78％、58.96％)，以及恢复率△F/F0荧光恢复率(1μM卡那霉素为14.97％、23.61％)，所以明显看看出DNA-Cu/AgNCs猝灭及恢复效果更好。
实施例3：采用实施例1对卡那霉素进行检测
首先在离心管中加80.5uLPBS缓冲溶液中(40mmol/L、pH7)中，取6μL(0.5mg/mL)NH2-UiO-66加入离心管与PBS充分混合。然后混合液中加入53.5μL的DNA-Cu/AgNCs,将混合液在37℃(600rpm)黑暗条件下孵育20min，使得荧光猝灭恢复到室温后，加入10μL不同浓度的卡那霉素，室温条件下2min荧光恢复，使用荧光光谱仪对上述体系进行荧光测定记为F，同时空白对照组即未加入卡那霉素，其他操作条件不变，记录荧光猝灭，用荧光光谱仪对上述体系进行荧光测定记为F0，测定结果如图5，不同浓度(0.05、0.1、0.5、1、2、4、8、10、12μM)的卡那霉素荧光响应。本实施例采用构建适配体荧光传感器检测卡那霉素速度快，加入卡那霉素目标后，响应时间仅需2min，整个检测体系在25min内即可完成。
综上所述，本发明的构建适配体荧光传感器，由以适配体为模板纳米团簇与锆金属有机框架形成的新的检测体系，体系中做为能量供体DNA-Cu/AgNCs具有高的荧光和更好的稳定性以及更高的量子产率，该体系中做为能量受体的NH2-UiO-66，具有立体多孔隙结构，比表面积大，孔隙结构规则，粒径孔隙可调节性等特点，而且容易制造，具有成本效益，高效且环保的优点。且构建适配体荧光传感器检测卡那霉素速度快，加入卡那霉素目标后，响应时间仅需2min，整个检测体系在25min内即可完成。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围。
Claims (7)
1.一种检测卡那霉素的荧光适配体生物传感器，其特征在于，该荧光适配体生物传感器通过合成以适配体为模板的荧光铜银纳米团簇DNA-Cu/AgNCs作为能量供体，同时制备锆金属有机框架NH2–UiO–66作为能量受体。
2.权利要求1所述的荧光适配体生物传感器的制备方法，其特征在于，
DNA-Cu/AgNCs的合成步骤为：首先将适配体纳米团簇序列，即DNA(Cu/AgNCs)溶解在PBS溶液中，将AgNO3和(Cu(NO3)2·3H2O加入到含有适配体纳米团簇序列的PBS溶液中，然后进行冰浴；之后继续加入新鲜配置的NaBH4溶液中剧烈震荡，将适配体纳米团簇序列、AgNO3、Cu(NO3)2、NaBH4的混合物在室温下孵育后，得到淡黄色的DNA-Cu/AgNCS溶液，然后置于4℃冰箱避光储存；
所述适配体纳米团簇序列为：(5'-3')CCCTTAATCCCC TGGGGGTTGAGGCTAAGCCGA；
其中，NH2-UiO-66的合成步骤为：称取ZrCl4和H2ATA溶解在DMF中，超声至分散均匀，然后加入苯甲酸，搅拌至溶解，转移至聚四氟乙烯反应釜中反应，之后取出过夜冷却至室温，然后用DMF和乙醇洗涤，之后干燥过夜。
3.根据权利要求2所述的荧光适配体生物传感器的制备方法，其特征在于，所述DNA-Cu/AgNCs的合成步骤中：所述适配体纳米团簇序列浓度为100μmol/L；PBS溶液浓度为40mmol/L、pH＝7；AgNO3浓度为1mmol/L；
(Cu(NO3)2·3H2O浓度为1mmol/L；冰浴时间为15min；NaBH4浓度为2mmol/L；震荡时间为1min；孵育时间为90min。
4.根据权利要求2所述的荧光适配体生物传感器的制备方法，其特征在于，所述DNA-Cu/AgNCs的合成步骤中：所述适配体纳米团簇序列、AgNO3、Cu(NO3)2、NaBH4的终浓度之比为1:3:8:11。
5.根据权利要求2所述的荧光适配体生物传感器的制备方法，其特征在于，所述NH2-UiO-66的合成步骤中：ZrCl4质量为140mg；H2ATA质量为49.25mg；DMF的体积为20mL；超声30min至分散均匀；苯甲酸的质量为366.5mg；在聚四氟乙烯反应釜中120℃反应48h。
6.权利要求1制备的检测卡那霉素的荧光适配体生物传感器的应用，其特征在于，该荧光适配体生物传感器用于检测卡那霉素，具体步骤为：首先在离心管中加PBS缓冲溶液，取NH2-UiO-66加入离心管与PBS充分混合；然后混合液中加入DNA-Cu/AgNCs,将混合液在黑暗条件下孵育，待荧光猝灭恢复到室温后，加入待检测的卡那霉素，室温条件下2min荧光恢复，使用荧光光谱仪对上述体系进行荧光测定。
7.根据权利要求6制备的检测卡那霉素的荧光适配体生物传感器的应用，其特征在于，PBS缓冲溶液的浓度为40mmol/L、pH＝7；黑暗条件下孵育的温度为37℃，孵育时间20min。
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