CN104449696B - 一种荧光探针及其制备方法、牛奶中土霉素的检测方法 - Google Patents
一种荧光探针及其制备方法、牛奶中土霉素的检测方法 Download PDFInfo
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Abstract
本发明公开了一种荧光探针及其制备方法、牛奶中土霉素的检测方法,荧光探针,由以单宁酸为碳源合成的表面酚羟基化的碳点与Fe3+溶液中的Fe3+结合组成。通过分别检测加入盐酸土霉素标准溶液前后荧光探针的荧光强度,根据加入盐酸土霉素标准溶液前后荧光探针的荧光强度比值和盐酸土霉素的浓度建立线性公式,再在相同条件下检测加入牛奶前后探针的荧光强度,根据线性公式计算出牛奶中土霉素的具体含量。
Description
技术领域
本发明属于化学检测领域,具体涉及一种荧光探针及其制备方法、牛奶中土霉素的检测方法。
背景技术
土霉素,一种四环素类抗生素,是一种广谱抗菌素。它对多种细菌和芽孢杆菌有抗菌作用,用来治疗上呼吸道感染、胃肠道感染、支原体感染和衣原体感染疾病等。
给农场动物饲喂土霉素能预防相关疾病,如对猪的副伤寒、附红细胞体、炭疽病、喘气病、痢疾、猪肺疫疗效显著,对雏禽痢疾、衣原体病有很好的疗效,还可以用来缓解应激反应、提高产蛋量、促进幼禽增重等。水产业应用其治疗鱼类弧病菌、脱磷病、烂鳃病、鳗鱼受德化氏病、鳟鱼疮病、鳗赤鳍病等。但过量的土霉素可以导致其在动物食品中的大量积累,包括肉类和牛奶。这种积累可能会对人体健康产生危害。
传统方法中,色谱法和电泳法,包括色谱质谱连用法、高效液相色谱法、液相色谱一串联质谱法等已经用来探测土霉素。但是,这些方法都需要复杂而昂贵的仪器,专业的技术,而且实验步骤复杂而耗时。此外,光学分析法,包括分光光度法、光纤荧光法和化学发光法等,由于操作简单而且成本廉价,也用来探测土霉素。但是,这种方法样品提取步骤复杂而且实验所用部分试剂有毒。
发明内容
为解决上述技术问题,本发明提供一种荧光探针及其制备方法、牛奶中土霉素的检测方法。利用荧光碳点检测克服了传统检测方法的成本高、实验步骤复杂而耗时以及实验所用部分试剂有毒等缺点,具有灵敏度高、选择性和稳定性好、检测成本低、方法操作和样品处理简单等优点。
为了实现上述目的,本发明采用的技术方案如下:
一种荧光探针,由以单宁酸为碳源合成的表面酚羟基化的碳点与Fe3+溶液中的Fe3+结合组成。
一种荧光探针的制备方法,包括以下步骤:
(1)将单宁酸溶解在蒸馏水中,加热至180℃反应2h,冷却后,透析除杂,真空干燥,得到黑色粉末状碳点,溶于二次蒸馏水,获得碳点溶液;
(2)向pH为7的柠檬酸-NaHPO4缓冲溶液中加入碳点溶液和Fe3+溶液,制备得到荧光探针。
步骤(2)中碳点在制备得到的荧光探针体系中浓度为0.5±0.02mg/mL;
步骤(2)中所用Fe3+溶液中含有Fe3+的浓度为10-4M。
步骤(1)制备碳点溶液的具体步骤为:
称取0.1035g单宁酸,加40mL蒸馏水溶解,转移到聚四氟乙烯反应釜内衬中并装好钢套,于烘箱中逐渐加热到180℃并恒温2h,自然冷却至室温,用切割分子量为2000的透析袋于二次水中透析1天除去杂质,透析后的碳点溶液在12000r/min的转速下离心15min,除去聚集的大颗粒,收集滤液,最后于真空干燥箱中干燥48h,即得到黑色粉末状碳点,称重并溶于二次水中,即得质量浓度的碳点溶液;
步骤(2)制备荧光探针的具体步骤为:
在5mL干净的具塞试管中依次加入300μLpH为7的柠檬酸-NaHPO4缓冲溶液,200μL上述合成的碳点溶液,10-4M的Fe3+溶液350μL,定容至5ml,制备得到荧光探针,碳点在该体系中浓度为0.5±0.02mg/mL。
一种牛奶中土霉素的检测方法,包括以下步骤:
平行取上述荧光探针若干份,加入pH值为7.0的柠檬酸-NaHPO4缓冲溶液,再加入不同浓度的盐酸土霉素标准溶液,分别检测加入盐酸土霉素标准溶液前后荧光探针的荧光强度,根据加入土霉素前后体系的发射峰处荧光强度的增加值与未加土霉素时体系的发射峰处荧光强度的比值和盐酸土霉素的浓度建立线性公式;在相同条件下检测加入牛奶前后探针的荧光强度,根据线性公式计算出牛奶中土霉素的具体含量。
由于碳点性质受pH值变化影响较大,本发明使用的碳点在pH为7的条件下具有很好的荧光性能,且pH为7时Fe3+对CDs的猝灭效率也最好,而土霉素在中性条件下具有很强的络合作用。所以利用缓冲液调节pH值为7最为合适。碳点发射峰位置的选择决定了检测体系是否具有灵敏的荧光强度变化,为了实现对土霉素浓度的灵敏响应,将碳点的激发波长设定在440nm。
本发明方法线性范围为0-3.5μmol/L,检测限为2.28×10-8M,回收率介于92.5%到103.0%之间。
本发明涉及的方法具有高的灵敏度、选择性和稳定性,且检测成本低;方法操作和样品处理简单,整个检测过程只需要二十分钟;利用线性公式还可以很好的对牛奶中土霉素的含量进行定量检测。
附图说明
图1为不同浓度的土霉素对CDs/Fe3+体系的荧光回升作用;
图2为土霉素(3μM)、寡聚糖(165μM)、强力霉素(180μM)、四环素(105μM)青霉素、链霉素、红霉素、氯霉素(300μM)对CNP/Fe3+体系的干扰情况;
图3为土霉素(3μM),VA、VD3、VD2、VB1、VC、VB2、VB6、Choline(1.5mM)对CNP/Fe3+体系的干扰情况;
图4荧光的回升效率与土霉素浓度之间的线性关系;
图5碳点的XPS谱图;
图6碳点的C1s谱图;
图7碳点的O1s谱图;
图8碳点的红外吸收光谱。
具体实施方式
实施例1
一种荧光探针,由以单宁酸为碳源合成的表面酚羟基化的碳点与Fe3+溶液中的Fe3+结合组成。
一种荧光探针的制备方法,包括以下步骤:
(1)、用电子天平称取0.1035g单宁酸,加40mL蒸馏水溶解,并将上述溶液转移到聚四氟乙烯反应釜内衬中并装好钢套,于烘箱中逐渐加热到180℃并恒温2h,自然冷却至室温。最后将反应好的溶液用切割分子量为2000的透析袋于二次水中透析1天除去杂质。透析后的碳点溶液在12000r/min的转速下离心15min,除去聚集的大颗粒,收集滤液,最后于真空干燥箱中干燥48h,即得到黑色粉末状碳点,称重并溶于10ml二次蒸馏水中,即得质量浓度的碳点溶液;
(2)、在5mL干净的具塞试管中依次加入300μLpH为7的柠檬酸-NaHPO4缓冲溶液,200μL上述合成的碳点溶液,10-4M的Fe3+溶液350μL,定容至5ml,制备得到荧光探针,碳点在该体系中浓度为0.5mg/mL。
对碳点的表面组成和元素分析做了XPS表征(如图5。碳点的XPS光谱在282.2eVand533.9eV处的俩个峰,分别是C1sandO1s产生的。C1s谱(如图6显示284.4eV,285.9eV和288.5eV三个峰,分别是由C=C(45.55%),C-O(38.89%)和C=O(15.55%)产生的。O1s谱(如图7显示531.3eV和532.7eV俩个峰,分别是由C=O(15.4%)和C-OH/C-O-C(84.6%)产生的。
利用红外吸收光谱确定了碳点表面的官能团,如图8。3300cm-1和1050cm-1处的吸收峰是-OH的伸缩振动的特征吸收,证明碳点表面有大量的羟基。1400cm-1处的吸收峰是-COOH的对称伸缩振动的吸收。还观测到-C=O的伸缩振动在1700cm-1处的特征吸收峰,和C-OH的伸缩振动在1200cm-1处的吸收峰。
一种牛奶中土霉素的检测方法,在5mL干净的具塞试管中取上述荧光探针,加入pH值为7.0的缓冲溶液,再加入不同浓度的盐酸土霉素标准溶液(0到3.5μM),分别检测加入盐酸土霉素标准溶液前后荧光探针的荧光强度,根据加入土霉素前后体系的发射峰处荧光强度的增加值与未加土霉素时体系的发射峰处荧光强度的比值和盐酸土霉素的浓度建立线性公式,建立的线性公式如下:y=0.0003+0.2635x(R2=0.9968),其中y为加入土霉素前后体系的荧光强度的增加值与未加土霉素时体系的荧光强度的比值,即ΔF/F0,x为盐酸土霉素的浓度c。
在5mL干净的具塞试管中依次加入300μLpH为7的citricacid-NaHPO4缓冲溶液,200μLCDs,7μMFe3+,然后再加入一定量的经处理好的牛奶样品,用二次水稀释、摇匀。二十分钟后,进行荧光信号检测,激发波长设置为440nm。根据线性公式计算出牛奶中土霉素的具体含量。
根据相关文献报导,牛奶中土霉素的含量在0.1μM左右。本发明的实验样品为超市随机购买的几种新鲜牛奶。
从本发明的实验结果看,测得的几种牛奶样品中土霉素的含量均在0.1μM左右,与相关文献报导一致,且回收率介于92.5%到103.0%之间,表明本发明的方法具有一定的实用性和重现性。
综上所述,本发明涉及的方法具有灵敏度高、选择性和稳定性好、检测成本低、方法操作和样品处理简单等优点。在牛奶中土霉素的检测中有很大的应用价值。
Claims (6)
1.一种荧光探针的制备方法,其特征在于,所述制备方法包括以下步骤:
(1)将单宁酸溶解在蒸馏水中,加热至180℃反应2h,冷却后,透析除杂,真空干燥,得到黑色粉末状碳点,溶于二次蒸馏水,获得碳点溶液;
(2)向pH为7的柠檬酸-NaHPO4缓冲溶液中加入碳点溶液和Fe3+溶液,制备得到荧光探针。
2.根据权利要求1所述的制备方法,其特征在于,步骤(2)中碳点在制备得到的荧光探针体系中浓度为0.5±0.02mg/mL。
3.根据权利要求1所述的制备方法,其特征在于,步骤(2)中所用Fe3+溶液中含有Fe3+的浓度为10-4M。
4.根据权利要求1或2所述的制备方法,其特征在于,步骤(1)制备碳点溶液的具体步骤为:
称取0.1035g单宁酸,加40mL蒸馏水溶解,转移到聚四氟乙烯反应釜内衬中并装好钢套,于烘箱中逐渐加热到180℃并恒温2h,自然冷却至室温,用切割分子量为2000的透析袋于二次水中透析1天除去杂质,透析后的碳点溶液在12000r/min的转速下离心15min,除去聚集的大颗粒,收集滤液,最后于真空干燥箱中干燥48h,即得到黑色粉末状碳点,称重并溶于二次水中,即得质量浓度的碳点溶液。
5.根据权利要求1或3所述的制备方法,其特征在于,步骤(2)制备荧光探针的具体步骤为:
在5mL干净的具塞试管中依次加入300μLpH为7的柠檬酸-NaHPO4缓冲溶液,200μL上述合成的碳点溶液,10-4M的Fe3+溶液350μL,定容至5ml,制备得到荧光探针,碳点在该体系中浓度为0.5±0.02mg/mL。
6.一种牛奶中土霉素的检测方法,其特征在于,采用权利要求1-5任一项所制备的荧光探针进行检测,所述检测方法包括以下步骤:
平行取荧光探针若干份,加入pH值为7.0的柠檬酸-NaHPO4缓冲溶液,再加入不同浓度的盐酸土霉素标准溶液,分别检测加入盐酸土霉素标准溶液前后荧光探针的荧光强度,根据加入土霉素前后体系的发射峰处荧光强度的增加值与未加土霉素时体系的发射峰处荧光强度的比值和盐酸土霉素的浓度建立线性公式;在相同条件下检测加入牛奶前后探针的荧光强度,根据线性公式计算出牛奶中土霉素的具体含量。
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