CN113201577A - 一种基于酶抑制法-碳量子点荧光淬灭效应的有机磷农残快速检测方法 - Google Patents
一种基于酶抑制法-碳量子点荧光淬灭效应的有机磷农残快速检测方法 Download PDFInfo
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Abstract
本发明属于农药残留快速检测技术领域,针对目前农残检测灵敏度低的问题,提供了一种基于酶抑制法‑碳量子点荧光淬灭效应的有机磷农残快速检测方法。农残与乙酰胆碱酯酶混合反应25 min;然后加入乙酰胆碱反应15 min;反应体系中同时加入Ag+溶液、碳量子点及OPD,反应5 min;记录加入不同浓度农残情况下,碳量子点荧光发射峰460nm处与2,3‑二氨基吩嗪DAP荧光发射峰558nm处的荧光强度,并计算二者比例响应I558/I460,建立农残浓度与I558/I460比值之间的线性关系。该方法检测速度快,5min之后就可以测得反应结果;检测灵敏度高,最低检测限为0.31ng/mL。
Description
技术领域
本发明属于农药残留快速检测技术领域,具体涉及一种基于酶抑制法-碳量子点荧光淬灭效应的有机磷农残快速检测方法。
背景技术
近年来,农药广泛应用于蔬菜、水果等农作物的害虫防治,但农药的不合理使用导致农产品中的农药残留超标,使其直接或间接进入到人体内,影响消费者食用安全,严重时会造成消费者致病、发育不正常,甚至直接导致中毒死亡。因此,农残检测成为重中之重,为了更有力地保障人们的健康,研究与开发适用于现场使用的、方便快捷、灵敏高效的农残快速检测技术对于有效的控制与减少食品中农药残留具有重要的意义。
目前,市场上常用的农残检测方法主要分为两种:一种是基于传统大型精密仪器的检测方法,如气相色谱法、高分离度液相色谱法和毛细管电泳法等。这些传统的精密仪器检测方法一般具有检测精度高、用量较少等优点,广泛应用于大型检测机构或政府部门,但是在检测时耗时长、样本前处理过程较复杂、需要专业的技术人员进行操作,对于具有检测食品中农残需求的普通生产者以及一些需要现场即时快速检测的情境并不适用。
基于此,农残快速检测方法就成为了一种常用的检测方法,其中,酶抑制方法是农残快速检测领域最为常用的方法。1961年,Ellman 等人(Ellman G L, Courtney K D,Andres V Jr, et al. A new and rapid colorimetric determination ofacetylcholinesterase activity, Biochemical Pharmacology, 1961, 7: 88-95.)采用二硫代双硝基苯甲酸根离子(DTNB)作为显色剂,硫代乙酰胆碱在乙酰胆碱酯酶的作用下的水解产物进行反应生成黄色耦合物,观测吸光度变化值以测定血液中乙酰胆碱酯酶的酶活性。因此,在Ellman 等人的研究基础上建立起来的酶抑制法就成为常用的有机磷类农残的快速检测。2015年,Guo 等人(Guo J, Wong J X H, Cui C E, et al., A smartphonereadable barcode assay for the detection and quantitation of pesticideresidues, Analyst, 2015, 140(16): 5518-5525.)将智能手机的扫码功能和酶抑制法结合,将设计的透明PDMS通道嵌入打印的条形码,利用智能手机中的扫码功能和图像分析软件分别实现了马拉硫磷的快速定性和定量检测。2016年,Hou等人(Hou J , Dong G , TianZ , et al., A sensitive fluorescent sensor for selective determination ofdichlorvos based on the recovered fluorescence of carbon dots-Cu(II) system,Food Chemistry, 2016, 202, 81–87.)设计了灵敏度高、选择性好的乙酰胆碱酯酶抑制型碳点荧光探针,依据乙酰胆碱与金属阳离子(Cu2+ )之间的氧化还原反应,实现了对敌敌畏的定量检测。2019年,Jin等人(Jin R , Kong D , Yan X , et al., IntegratingTarget-Responsive Hydrogels with Smartphone for On-Site ppb-LevelQuantitation of Organophosphate Pesticides, ACS Applied Materials &Interfaces, 2019, 11(31): 27605−27614.)开发了一种基于硅量子点淬灭,以水凝胶为反应基底的有机磷农残快速检测方法,利用智能手机获取荧光信号,并通过ImageJ软件分析转换为与有机磷浓度成线性比例的色相参数,实现有机磷农残的定量检测。
近年来,碳量子点由于具有强荧光、低毒性、荧光波长可调、无光闪烁等特点,并且碳量子点的制备方法多样化,合成过程快捷、简便;使其在生化传感、环境检测等领域中得到了广泛应用。目前,碳量子点已被认为是一种较为理想的荧光标记或检测材料之一。
发明内容
本发明针对目前农残检测灵敏度低的问题,提供了一种基于酶抑制法-碳量子点荧光淬灭效应的有机磷农残快速检测方法。在反应体系中,农残作为乙酰胆碱酯酶的抑制剂可防止水解产生硫代胆碱,从而阻止了与Ag+形成金属聚合物的形成,进一步触发了邻苯二胺OPD的氧化,生成黄色的2,3-二氨基吩嗪(DAP),碳量子点的荧光强度通过荧光内滤效应被DAP淬灭,产生了典型的荧光比例响应。通过建立农残浓度与I558/I460比值之间的线性关系,实现农残的快速、高灵敏定量检测。
本发明由如下技术方案实现的:一种基于酶抑制法-碳量子点荧光淬灭效应的有机磷农残快速检测方法,农残与乙酰胆碱酯酶混合反应25 min;然后加入乙酰胆碱反应15min;反应体系中同时加入Ag+溶液、碳量子点及领苯二胺OPD,反应5 min;记录加入不同浓度农残情况下,碳量子点荧光发射峰460nm处与2,3-二氨基吩嗪DAP荧光发射峰558nm处的荧光强度,并计算二者比例响应I558/I460,建立农残浓度与I558/I460比值之间的线性关系。
具体步骤为:
(1)制备农残定量检测反应体系:农残与乙酰胆碱酯酶避光条件下混合均匀后反应25 min,加入乙酰胆碱混合均匀后37℃避光条件下反应15 min,反应体系中同时加入Ag+溶液、碳量子点及OPD,混匀后37℃避光条件下静置5 min,DAP荧光增强,碳量子点荧光淬灭,形成反应体系;
(2)定量检测农残:荧光光谱仪检测558 nm处及460 nm处的荧光强度,计算二者的比值I558/I460,建立农残浓度与I558/I460的线性关系,通过线性关系对农残定量检测。
步骤(1)中农残体积:乙酰胆碱酯酶体积:乙酰胆碱体积为25:1:1;控制反应体系中:Ag+的浓度为700 μM,碳量子点的浓度50 μg/mL,OPD的浓度为25 mM。
所述OPD溶液配制时置于冰超纯水中配制,超声10分钟使其充分溶解。
制备浓度为25 mM的OPD溶液时需要将其放置在冰水中,超声快速溶解,防止OPD溶液被氧化。
在反应体系中,农残作为乙酰胆碱酯酶的抑制剂可防止水解产生硫代胆碱,从而阻止了与Ag+形成金属聚合物的形成,进一步触发了OPD的氧化,生成黄色的2,3-二氨基吩嗪(DAP),碳量子点的荧光强度通过荧光内滤效应被DAP淬灭,碳量子点与DAP产生了典型的荧光比例响应。
本发明中碳量子点直接从商家购买,生产厂家:星紫(上海)新材料技术开发有限公司;产品型号:WA11144-100mg。
本发明的有益效果如下:
本发明首次将碳量子点荧光淬灭与酶抑制法相结合,用于有机磷农药残留的快速检测;以碳量子点与OPD为指示剂,利用Ag+对OPD的氧化作用生成DAP,碳量子点的荧光强度通过荧光内滤效应被DAP淬灭,产生了典型的荧光比例响应,通过建立农残浓度与I558/I460比值之间的线性关系,实现农残的定量检测。本发明所述检测方法简单快速、检测灵敏度高;最低检测限为0.31ng/mL,远低于国内及欧盟食品中农药残留标准。
本发明的检测速度快,在有机磷农残与乙酰胆碱酯酶、乙酰胆碱反应的基础上,一步式加入Ag+、OPD及碳量子点,5min之后就可以测得反应结果;检测灵敏度高,与传统的酶抑制法显色反应相比(检测灵敏度为μg/mL级别),本发明的检测限为0.31ng/mL,远低于国内及欧盟食品中有机磷农残残留标准。
附图说明
图1为本发明实施例中Ag+浓度优化的光谱图及分散点图,其中,A为光谱图,B为分散点图;
图2为本发明实施例中加入不同浓度乙酰甲胺磷溶液后,碳量子点(460nm)及DAP(558nm)处发射光谱图和分散点图,其中,A为发射光谱图,B为分散点图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明的一部分实施例,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1:一种基于酶抑制法-碳量子点荧光淬灭效应的有机磷农残快速检测方法,农残与乙酰胆碱酯酶混合反应25 min;然后加入乙酰胆碱反应15 min;反应体系中同时加入Ag+溶液、碳量子点及OPD,反应5 min;记录加入不同浓度农残情况下,碳量子点荧光发射峰460nm处与2,3-二氨基吩嗪DAP荧光发射峰558nm处的荧光强度,并计算二者比例响应I558/I460,建立农残浓度与I558/I460比值之间的线性关系。
具体步骤如下:
(1)Ag+溶液的配制:称取0.014 g AgNO3粉末溶解于10 mL的去离子水中,配制成8500 μM的Ag+溶液备用;
(2)不同浓度乙酰甲胺磷的配制:将100 μg/mL的乙酰甲胺磷溶液稀释成浓度为0.5 、1.0、1.2、1.5、2.0 ng/mL备用。
(3)碳量子点溶液的配制:量取50μL 50mg/mL 的碳量子点加入9.95mL去离子水,配制成10 mL 250 μg/mL的碳量子点备用。碳量子点直接从商家购买,生产厂家:星紫(上海)新材料技术开发有限公司;产品型号:WA11144-100mg。
(4)OPD溶液的配制:称取0.01352 g OPD粉末溶解于5mL 冰去离子水中,在超声波清洗机中超声10分钟,直至OPD粉末充分溶解。
(5)Ag+溶液浓度的优化:量取不同体积的8500 μM的Ag+溶液,加入不同体积的去离子水,配制成浓度分别为0μM、17μM、34μM、68μM、85μM、170μM、340μM、510μM、680μM、850μM、1020μM、1190μM、1360μM、1530μM、1700μM的Ag+溶液;取300 μL不同浓度的Ag+溶液分别加入到100 μL 25mM的OPD溶液和100 μL 250 μg/mL的碳量子点的混合体系中,使用快速混匀器混匀,混匀后在避光条件下静置反应5 min,如图1所示,当Ag+溶液浓度为700μM时,碳量子点淬灭达到最大值,所以反应体系中优化后的Ag+溶液浓度为700μM。
(6)乙酰甲胺磷的定量检测:首先,将25μL不同浓度的乙酰甲胺磷溶液与1μL的乙酰胆碱酯酶混合,常温下反应25min,随后,加入1μL乙酰胆碱混合均匀后37℃避光条件下反应15 min,最后,反应体系中同时加入95μL 700μM 的Ag+溶液、283μL 250μg/mL的碳量子点及95μL 25mM 的OPD溶液,混匀后37℃避光条件下静置5 min,使得DAP荧光增强,碳量子点荧光淬灭,使用荧光光谱仪进行检测。记录460nm处(碳量子点荧光发射峰)与558nm处(DAP荧光发射峰)荧光强度,并计算二者比例响应I558/I460,通过建立有机磷农残浓度与I558/I460比值之间的线性关系,实现乙酰甲胺磷的定量检测。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (4)
1.一种基于酶抑制法-碳量子点荧光淬灭效应的有机磷农残快速检测方法,其特征在于:农残与乙酰胆碱酯酶混合反应25 min;然后加入乙酰胆碱反应15 min;反应体系中同时加入Ag+溶液、碳量子点及OPD,反应5 min;记录加入不同浓度农残情况下,碳量子点荧光发射峰460nm处与2,3-二氨基吩嗪DAP荧光发射峰558nm处的荧光强度,并计算二者比例响应I558/I460,建立农残浓度与I558/I460比值之间的线性关系。
2.根据权利要求1所述的一种基于酶抑制法-碳量子点荧光淬灭效应的有机磷农残快速检测方法,其特征在于:具体步骤为:
(1)制备农残定量检测反应体系:农残与乙酰胆碱酯酶避光条件下混合均匀后反应25min,加入乙酰胆碱混合均匀后37℃避光条件下反应15 min,反应体系中同时加入Ag+溶液、碳量子点及OPD,混匀后37℃避光条件下静置5 min,DAP荧光增强,碳量子点荧光淬灭,形成反应体系;
(2)定量检测农残:荧光光谱仪检测558 nm处及460 nm处的荧光强度,计算二者的比值I558/I460,建立农残浓度与I558/I460的线性关系,通过线性关系对农残定量检测。
3.根据权利要求2所述的一种基于酶抑制法-碳量子点荧光淬灭效应的有机磷农残快速检测方法,其特征在于:步骤(1)中农残体积:乙酰胆碱酯酶体积:乙酰胆碱体积为25:1:1;控制反应体系中:Ag+的浓度为700 μM,碳量子点的浓度50 μg/mL,OPD的浓度为25 mM。
4.根据权利要求3所述的一种基于酶抑制法-碳量子点荧光淬灭效应的有机磷农残快速检测方法,其特征在于:所述OPD溶液配制时置于冰超纯水中配制,超声10分钟使其充分溶解。
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