CN113533271B - 一种近红外光激发检测环丙沙星的方法 - Google Patents
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Abstract
本发明涉及一种近红外光激发检测环丙沙星的方法，利用水溶性镧系稀土上转换材料为探针，铜化合物作为荧光淬灭剂和环丙沙星识别单元，铜化合物中的铜离子可通过能量共振转移淬灭水溶性镧系稀土上转换材料，通过检测加入环丙沙星后，环丙沙星与铜离子形成螯合物，使铜离子与水溶性镧系稀土上转换材料的距离增加，使荧光恢复，荧光恢复的程度与加入环丙沙星的量呈线性，从而实现环丙沙星特异性定量检测。本发明采用的水溶性镧系稀土上转换材料可利用红外光作为激发光能有效避免高能量光的光损伤及生物背景发光强的缺陷，同时该材料具有荧光寿命长、发射峰多且发射带尖锐，特别适合作为比率型荧光探针。本发明的方法简单、所需样品少、检测速度快。
Description
技术领域
本发明属于化学检测领域，具体涉及一种近红外光激发检测环丙沙星的方法。
背景技术
环丙沙星是第三代喹诺酮类抗菌药物，抗菌谱广、抗菌力强、价格低廉，被广泛应用于兽医临床感染性疾病。由于药物不规范的使用，使得部分动物性产品或水中兽药残留超标。如果长期摄食低剂量药物残留的食品，由于蓄积对人体健康具有潜在危害，如对人体中枢神经系统、循环系统、消化系统、泌尿系统、呼吸系统等造成损害或诱导人类致病菌对该药耐药性的增强。因此，建立了快速、灵敏测定各类食品样品及水体中环丙沙星残留的方法十分重要。
现有检测环丙沙星的方法主要有色谱及联用法、酶联免疫分析法和毛细管电泳法。高效液相色谱法对样品的前处理时间较长，检测过程较为复杂，不能满足大批量检测的需求；同时受样品基质的影响较大。液相色谱-质谱联用法所需仪器昂贵，操作复杂难以普及；同样存在丰富的基质效应，影响定量和选择性能。酶联免疫分析法易受到抗原影响。毛细管电泳法使用的毛细管直径小、光路短、灵敏度较低, 且电渗会因样品组成而变化, 从而影响分离重现性。
荧光分析法相比于传统分析法，方法简便、选择性好、灵敏度高、成本低、检测速度快等。传统的下转换荧光材料（有机染料、荧光蛋白、含有重金属的半导体量子点）只能采用紫外光激发，荧光寿命短，通常只有单一发射峰，容易受生物体内自发荧光的干扰，有一定毒性，且传统的荧光分光光度计所需样品量相对较多。
发明内容
发明目的：本发明的目的是提供一种近红外光激发检测环丙沙星的方法，解决采用下转换荧光材料作为探针等缺点，并减小荧光分析法检测样品用量。
技术方案：
1）检测环丙沙星的标准曲线：将水溶性镧系稀土上转换材料、铜化合物和不同浓度的环丙沙星混匀后室温下孵育，将混合液置于外置980nm或808nm激发光的荧光光谱仪中检测荧光强度；利用不加环丙沙星与加环丙沙星654nm/528nm荧光强度的比值对环丙沙星的浓度作图，获得标准曲线，拟合得到标准曲线方程；
2）待测样品环丙沙星的检测：水溶性镧系稀土上转换材料、铜化合物和待测溶液，混匀后室温下孵育，将混合液置于外置980nm或808nm激发光的荧光光谱仪中检测荧光强度；根据不加环丙沙星与加待测溶液654nm/528nm荧光强度的比值和标准曲线方程，算出待测溶液中环丙沙星的含量。
优选的，所述的水溶性镧系稀土上转换材料包括去修饰的镧系稀土上转换材料或者是柠檬酸、PAA、PEI、PEO、PVP、PEG、PLGA、PVA、甲壳素中的一种或多种包覆的镧系稀土上转换材料。
优选的，所述镧系稀土上转换材料包括NaYF4：Yb，Er；NaYF4：Yb，Nd，Er；NaGdF4：Yb，Nd，Er；NaGdF4：Yb，Er；NaYF4：Yb，Er@NaYF4；NaYF4：Yb，Er@NaYF4：Nd；NaYbF4：Nd@NaYF4：Er、NaYF4：Yb，Er，Nd@NaYF4：Nd。
优选的，所述的水溶性镧系稀土上转换材料浓度为0.01-50 mg/mL；铜化合物浓度为0.01M-0.5M。
优选的，所述的铜化合物包括柠檬酸铜、氯化铜、硫酸铜、硝酸铜。
有益效果：本方法采用的水溶性镧系稀土上转换材料作为荧光探针，该探针可利用红外光作为激发光能有效避免高能量光的光损伤及生物背景发光强的缺陷，同时该材料具有荧光寿命长、发射峰多且发射带尖锐，特别适合作为比率型荧光探针。利用铜化合物作为荧光淬灭剂和环丙沙星识别单元，铜化合物中的铜离子可通过能量共振转移淬灭水溶性镧系稀土上转换材料，加入环丙沙星后，环丙沙星与铜离子形成螯合物，使铜离子与水溶性镧系稀土上转换材料的距离增加，使荧光恢复。本发明的检测方法简单、所需样品少、检测速度快、能特异性检测环丙沙星。
附图说明
图1 本发明所述近红外光激发检测环丙沙星的检测机理；
图2 为不同浓度环丙沙星加入后的荧光光谱；
图3 为本发明所述一种近红外激发检测环丙沙星的标准曲线；
图4 为本发明所述近红外激发检测环丙沙星的选择性检测结果；
图5 为本发明所述一种近红外激发检测环丙沙星的特异性检测结果。
具体实施方式
本发明所述近红外光激发检测环丙沙星的检测机理如说明书图1所示，利用水溶性镧系稀土上转换材料为探针，铜化合物作为荧光淬灭剂和环丙沙星识别单元，铜化合物中的铜离子可通过能量共振转移淬灭水溶性镧系稀土上转换材料，通过检测加入环丙沙星后，环丙沙星与铜离子形成螯合物，使铜离子与水溶性镧系稀土上转换材料的距离增加，使荧光恢复，荧光恢复的程度与加入环丙沙星的量呈线性，从而实现环丙沙星特异性定量检测。
下面通过说明书附图及具体实施例对本发明进一步说明
实施例1：
通过本发明提供的一种近红外激发检测环丙沙星的方法检测医疗废水中的环丙沙星：
1）检测环丙沙星的标准曲线：
将1μL 20 mg/mL NaYF4：Yb，Er，Nd@NaYF4：Nd、1μL 0.1 M氯化铜和5μL一系列不同浓度（0mg/mL、1mg/mL、5mg/mL、10mg/mL、20mg/mL）的环丙沙星加入到直径为3 mm、高度为0.5 mm的微杯中，混匀后室温下孵育5-10分钟，将微杯置于外置808nm激发光的荧光光谱仪中检测荧光强度如图2；不加环丙沙星与加环丙沙星654nm/528nm荧光强度的比值对环丙沙星的浓度作图，见图3，获得标准曲线，拟合得到标准曲线方程:y=0.0326x+3.95。
2）待测样品环丙沙星的检测：
1μL 20 mg/mL NaYF4：Yb，Er，Nd@NaYF4：Nd、1μL 0.1 M氯化铜和5μL医疗废水待测溶液，混匀后室温下孵育5-10分钟，将微杯置于外置808nm激发光的荧光光谱仪中检测荧光强度；根据不加环丙沙星与5μL医疗废水654nm/528nm荧光强度的比值和标准曲线方程，算出待测溶液中环丙沙星的含量。
实施例2选择性检测试验：
1μL 20 mg/mL NaYF4：Yb，Er，Nd@NaYF4：Nd、1μL 0.1 M氯化铜、20mg/L环丙沙星分别与200mg/L氯化钠、200mg/L氯化钾、200mg/L氯化镁、200mg/L葡萄糖、200mg/L L-半胱氨酸混匀后，室温下孵育5-10分钟，将微杯置于外置808nm激发光的荧光光谱仪中检测荧光强度；不加环丙沙星与上诉溶液654nm/528nm荧光强度的比值见图4。可见存在食品中常见的一些离子并不影响环丙沙星的检测。
实施例3特异性检测试验
1μL 20 mg/mL NaYF4：Yb，Er，Nd@NaYF4：Nd、1μL 0.1 M氯化铜分别与20mg/L恩诺沙星、20mg/L达氟沙星、20mg/L噁喹酸混匀后，室温下孵育5-10分钟，将微杯置于外置808nm激发光的荧光光谱仪中检测荧光强度；不加环丙沙星与上述溶液654nm/528nm荧光强度的比值见图5。可见该方法可特异性检测环丙沙星。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (2)
1.一种近红外光激发检测环丙沙星的方法，其特征在于，所述方法包括如下步骤：
1）检测环丙沙星的标准曲线：将水溶性镧系稀土上转换材料、铜化合物和不同浓度的环丙沙星混匀后室温下孵育，将混合液置于外置808nm激发光的荧光光谱仪中检测荧光强度；利用不加环丙沙星与加环丙沙星654nm/528nm荧光强度的比值对环丙沙星的浓度作图，获得标准曲线，拟合得到标准曲线方程；其中，镧系稀土上转换材料为NaYF4：Yb，Er，Nd@NaYF4：Nd；铜化合物为氯化铜
2）待测样品环丙沙星的检测：水溶性镧系稀土上转换材料、铜化合物和待测溶液，混匀后室温下孵育，将混合液置于外置808nm激发光的荧光光谱仪中检测荧光强度；根据不加环丙沙星与加待测溶液654nm/528nm荧光强度的比值和标准曲线方程，算出待测溶液中环丙沙星的含量。
2.根据权利要求1所述一种近红外光激发检测环丙沙星的方法，其特征在于，所述的水溶性镧系稀土上转换材料浓度为0.01-50mg/mL；铜化合物浓度为0.01M-0.5M。
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