CN114774120B - 一种基于掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法及其应用 - Google Patents
一种基于掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法及其应用 Download PDFInfo
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- CN114774120B CN114774120B CN202210321791.2A CN202210321791A CN114774120B CN 114774120 B CN114774120 B CN 114774120B CN 202210321791 A CN202210321791 A CN 202210321791A CN 114774120 B CN114774120 B CN 114774120B
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Abstract
本发明公开了一种基于掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法及其应用,该生物传感器包括Fe3O4@PDA纳米颗粒和适配体功能化的掺铁碳点原溶液,制备为:将Fe3O4粉末和盐酸多巴胺混合加入到缓冲液中搅拌,分离洗涤产物,然后真空干燥过夜得到Fe3O4@PDA纳米颗粒粉末;将1‑乙基‑(3‑二甲基氨基丙基)碳二亚胺盐酸盐和N‑羟基琥珀酰亚胺溶解在掺铁碳点水溶液中,加入适配体搅拌,透析除去游离的适配体得到适配体功能化的掺铁碳点原溶液。本发明生物传感器灵敏度高且特异性高,检测范围宽,可以同时快速检测乳制品中的抗生素、亚硝酸盐等安全危害因子,实现对乳制品中抗生素及亚硝酸盐的定性及定量检测。
Description
技术领域
本发明为生物传感器技术领域,具体涉及一种基于掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法及应用。
背景技术
乳制品已经成为全民营养膳食中不可或缺的一部分,其质量安全问题一直备受关注,直接关系到人类的生命健康。“有抗奶”、“含汞奶”等乳制品安全事故给消费者和乳制品行业都带来了巨大的冲击。抗生素类兽药残留以及污染物是乳制品中的重要危害因子。卡那霉素是一种氨基糖苷类广谱抗生素,已作为兽药和饲料添加剂被广泛用于治疗微生物感染。然而,卡那霉素的不合理使用极易导致在食源性动物体内的残留,通过食物链在人体中积累,威胁人体健康,产生耳毒性、肝肾毒性以及过敏反应甚至抗生素耐药性。至于乳制品中常见的污染物,奶牛可以通过喂养和供水来摄入亚硝酸盐。国际癌症研究机构已将亚硝酸盐列为人类GROUP 2A致癌物。长期或大量摄入亚硝酸盐会导致高铁血红蛋白血症。此外,在胃酸环境下亚硝酸盐与食物中的酰胺等反应生成强致癌物亚硝胺。这对发育尚未完全、免疫力差但乳制品摄入量又大的婴儿来说无疑是个潜在的危险。综上所述,有效控制乳制品中抗生素和亚硝酸盐的含量具有重要的现实意义。
现有技术的理化检测(如液相色谱-质谱联用、电化学法、离子色谱法、分光光度法等)、微生物抑制法及免疫分析法等已广泛用于抗生素或亚硝酸盐的检测。然而,操作繁琐、试剂消耗大、成本高、耗时长等是以上检测技术无法避免的问题。并且,大部分检验方法是针对乳制品中某一大类物质,但不能满足日常监测中快速高效地同时检测多类物质的需要。
碳点(CDs)是一种尺寸小于10nm的零维新型碳基纳米材料,由于其优异的光学性能、催化性能和水分散性,使其在多重检测方面拥有巨大的潜力。然而,CDs的类酶催化效率会受到结构内部的电子转移效率的限制,这种情况可以通过杂原子掺杂来改善。在类过氧化物酶催化过程中,铁掺杂会导致更高的活性中心利用率,且铁掺杂碳纳米酶表现出比其他金属(如Mn、Co、Ni、Cu)掺杂的碳纳米酶更高的模拟酶活性。
基于荧光的生物传感策略中,通常会通过应用一些纳米材料来降低背景信号或放大荧光强度来获得较高的灵敏度和稳定性。这些材料包括氧化石墨烯、金纳米颗粒、过渡金属二硫化物纳米片、碳纳米管和聚合物或聚合物涂层纳米材料等。聚多巴胺纳米颗粒是一种由多巴胺聚合而来的聚合物纳米材料,具有优异的荧光猝灭性能以及对不同构象单链DNA的选择性吸附能力,这不仅使它在荧光生物传感技术中广泛应用,也可以与适配体生物传感器相结合。
发明内容
发明目的:针对现有技术存在的问题,本发明提供了一种操作简单、试剂消耗小、成本低、耗时短、高灵敏度、高特异性的基于掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法;
本发明的另一目的是提供一种基于掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法以及所制备的掺铁碳点结合磁性Fe3O4@PDA生物传感器在检测抗生素和亚硝酸盐中的应用。
技术方案:为了解决现有技术中的问题,本发明提供了一种基于掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法,生物传感器包括Fe3O4@PDA纳米颗粒和卡那霉素适配体功能化的掺铁碳点原溶液,其中Fe3O4@PDA纳米颗粒的制备为将Fe3O4粉末和盐酸多巴胺混合加入到缓冲液中搅拌,分离洗涤产物,然后真空干燥过夜;适配体功能化的掺铁碳点原溶液的制备为将1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺溶解在掺铁碳点水溶液中,加入适配体搅拌,透析除去游离的适配体。
其中,所述适配体功能化的掺铁碳点原溶液中的适配体为卡那霉素适配体、链霉素适配体、四环素适配体、黄曲霉毒素M1适配体中的任意一种。
作为优选,所述适配体功能化的掺铁碳点原溶液为卡那霉素适配体功能化的掺铁碳点原溶液,卡那霉素适配体核苷酸序列如5’NH2-TGGGGGTTGAGGCTAAGCCGA所示。
进一步地,制备Fe3O4@PDA纳米颗粒的具体方法为:将三氯化铁和柠檬酸钠溶于乙二醇中,磁力搅拌溶解混匀,加无水乙酸钠搅拌,高温加热后冷却到室温,真空干燥过夜得Fe3O4粉末;将盐酸多巴胺和上述所得Fe3O4粉末加入到缓冲液中搅拌,分离洗涤产物,真空干燥过夜得Fe3O4@PDA粉末。
进一步地,制备适配体功能化的掺铁碳点原溶液的具体方法为:将柠檬酸、乙二胺和三氯化铁溶于超纯水中,将混合均匀后的溶液转移到聚四氟乙烯不锈钢高压釜中,高温加热,然后冷却至室温,用滤膜过滤棕黑色的悬浮液,用去离子水透析后冷冻干燥得到掺铁碳点粉末,将1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺溶解在掺铁碳点水溶液中,将溶液在37℃下搅拌1h,加入适配体搅拌,透析除去游离的适配体,得到适配体功能化的掺铁碳点原溶液。
本发明所述基于掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法以及所制备的掺铁碳点结合磁性Fe3O4@PDA生物传感器。
本发明所述的基于掺铁碳点结合磁性Fe3O4@PDA生物传感器在同时或分别检测抗生素和亚硝酸盐中的应用。
其中,所述抗生素包括卡那霉素、链霉素、庆大霉素、四环素等。
其中,所述亚硝酸盐包括要亚硝酸钠、亚硝酸钾。
进一步地,生物传感器同时检测抗生素和亚硝酸盐的具体方法为:将卡那霉素溶液、亚硝酸钠溶液和适配体功能化的掺铁碳点原溶液混合,加入4-羟乙基哌嗪乙磺酸缓冲液共孵育,再加入Fe3O4@PDA溶液共孵育,对反应溶液磁分离后测定上清液的荧光光谱以测定抗生素的含量;将上清液与TMB、过氧化氢溶液加入到缓冲溶液中室温孵育,孵育结束后测定溶液的紫外可见吸收光谱,以测定亚硝酸盐的含量。
进一步地,生物传感器分别检测抗生素和亚硝酸盐的具体方法为:将卡那霉素溶液、亚硝酸盐溶液分别和适配体功能化的掺铁碳点原溶液混合,混合后分别加入4-羟乙基哌嗪乙磺酸缓冲液共孵育,再加入Fe3O4@PDA纳米颗粒溶液共孵育,分别对反应溶液磁分离取上清液,测定含有抗生素上清液的荧光光谱以测定抗生素的含量;取含有亚硝酸钠的上清液与TMB、过氧化氢溶液加入到缓冲溶液中室温孵育,孵育结束后测定溶液的紫外可见吸收光谱,以测定亚硝酸钠的含量。
作为优选,抗生素和亚硝酸盐同时或分别检测的时间为10-20min。
作为优选,检测卡那霉素浓度为0.01ng/mL-200ng/mL;检测亚硝酸钠浓度为5-5000μM。
发明机理:将具有类过氧化物酶活性的荧光掺铁碳点与磁性Fe3O4@PDA纳米颗粒结合,用于乳制品中抗生素和亚硝酸盐的同时快速检测;抗生素兽药残留例如以卡那霉素为例,污染物亚硝酸盐以亚硝酸钠为例;掺铁碳点与卡那霉素适配体共价结合,得到适配体功能化的掺铁碳点作为识别元件;在适配体功能化的掺铁碳点识别并结合卡那霉素后,适配体的构象发生改变,Fe3O4@PDA对不同构象的适配体表现出不同的亲和力,引起了卡那霉素浓度依赖性的荧光强度变化;Fe3O4@PDA纳米颗粒发挥了对单链DNA的特异性识别和样品快速分离的作用;Fe-CDs的类过氧化物酶活性应用于亚硝酸盐的比色检测,在3,3',5,5'-四甲基联苯胺(TMB)-过氧化氢体系中,掺铁碳点可以在酸性条件下催化过氧化氢对TMB的氧化作用,氧化型TMB(ox-TMB)会与亚硝酸盐反应,生成独特的绿色重氮化产物,以其吸光度值监测亚硝酸盐的浓度。
有益效果:与现有技术相比,本发明具有如下显著优点:
(1)本发明基于掺铁碳点结合磁性Fe3O4@PDA生物传感器灵敏度高、特异性高、检测范围宽,可以快速分别或同时检测乳制品中的抗生素和亚硝酸盐。
(2)本发明基于掺铁碳点结合磁性Fe3O4@PDA生物传感器以抗生素卡那霉素作为乳制品中抗生素检测的模式兽药,例如最低可检测到的卡那霉素浓度为0.00053ng/mL,可检测到的亚硝酸钠浓度为0.54μM。
(3)本发明实现了用一个生物传感器同时或分别检测抗生素和亚硝酸盐,在17min内可以同时完成抗生素和亚硝酸盐的检测,在12min内可以单独完成抗生素的检测,检测速度快。
(4)本发明通过改变抗生素适配体,可以实现对乳制品中不同的抗生素兽药残留的检测,且对目标抗生素检测迅速,检测结果安全可靠;同时,利用TMB-过氧化氢纳米酶比色体系,实现对乳制品中亚硝酸盐的定性及定量检测。
附图说明
图1为本发明生物传感器的荧光强度分析示意图;
图2为本发明生物传感器的裸眼比色观测图;
图3为本发明生物传感器的比色紫外可见吸收光谱分析示意图;
图4为本发明的Fe3O4@PDA纳米颗粒的红外光谱图;
图5为本发明的Fe3O4@PDA纳米颗粒的溶液磁分离图;
图6为本发明的掺铁碳点的透射电镜图;
图7为本发明掺铁碳点的荧光性能图;
图8为本发明掺铁碳点的类过氧化物酶活性验证图;
图9为本发明卡那霉素适配体功能化的掺铁碳点的红外光谱图;
图10为本发明基于具有类过氧化物酶活性的荧光掺铁碳点与磁性Fe3O4@PDA纳米颗粒结合的生物传感器的九个检测参数优化图;
图11为采用本发明的检测方法,卡那霉素检测的标准曲线图;
图12为采用本发明的检测方法,亚硝酸钠检测的标准曲线图;
图13为本发明生物传感器检测四种食物基质中(超高温加工牛奶、巴氏杀菌奶、发酵奶、婴幼儿奶粉)的卡那霉素含量的结果表;
图14为本发明生物传感器检测四种食物基质中(超高温加工牛奶、巴氏杀菌奶、发酵奶、婴幼儿奶粉)的亚硝酸钠含量的结果表。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。卡那霉素适配体核苷酸序列由上海生工合成。
实施例1
掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法
(1)Fe3O4@PDA纳米颗粒的制备过程:将1.5g三氯化铁和0.2g柠檬酸钠溶于20mL乙二醇中,磁力搅拌溶解混匀,加入2.8g无水乙酸钠,将混合物剧烈搅拌30min,然后密封在聚四氟乙烯不锈钢高压釜中,在200℃下加热2h,然后冷却到室温。所得产物用乙醇和去离子水洗涤三次,然后真空干燥过夜得Fe3O4纳米颗粒的粉末。将20mg盐酸多巴胺和20mg Fe3O4纳米颗粒加入到40mL 10mM Tris-HCl缓冲液(pH 8.5)中。室温下搅拌12h后,磁分离洗涤产物,去离子水和乙醇交替洗涤三次,然后真空干燥过夜得Fe3O4@PDA粉末。
(2)掺铁碳点的制备过程:将1.0507g柠檬酸、335μL乙二胺和1.0507g三氯化铁溶于10mL超纯水,混合均匀得溶液转移到聚四氟乙烯不锈钢高压釜中,在200℃加热5h,然后冷却到室温。用0.22μm的滤膜过滤棕黑色的悬浮液,用去离子水透析12h(透析袋截留分子量500Da),然后冷冻干燥得到掺铁碳点粉末以备后用。
(3)卡那霉素适配体功能化的掺铁碳点的制备过程:25mg的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和25mg的N-羟基琥珀酰亚胺溶解在5mL的0.5mg/mL掺铁碳点水溶液中,然后将溶液在37℃下搅拌1h,加入50μL的10μM卡那霉素适配体,在37℃下继续搅拌4h。透析2h除去游离的卡那霉素适配体(透析袋截留分子量10000Da),得到掺铁碳点浓度为0.5mg/mL的卡那霉素适配体功能化的掺铁碳点原溶液,使用前储存在4℃。
实施例2
检测卡那霉素、亚硝酸钠的具体方法
将50μL卡那霉素标准溶液(0.01、0.05、0.1、0.2、0.5、1、2、5、10、20、50、100、200、500ng/mL)和100μL亚硝酸钠标准溶液(0、1、2、5、10、20、50、100、200、500、1000、2000、5000μM)与40μL实施例1制备的卡那霉素适配体功能化的掺铁碳点原溶液混合,再加入10mM 4-羟乙基哌嗪乙磺酸缓冲液(2mM Ca2+,pH 7.0)使总体积为300μL,在37℃下孵育10min。之后,加入100μL的实施例1制备的Fe3O4@PDA水溶液(1mg/mL),并室温孵育2min。对反应混合溶液磁分离,未反应的Fe3O4@PDA和Fe3O4@PDA-Apt-Fe-CDs通过磁力分离,而反应的Fe-CDs-Apt-KAN和游离亚硝酸钠保留在上清液中。取上清液在360-650nm处测定上清液的荧光光谱以测定卡那霉素的含量,样品中卡那霉素的浓度与上清液中检测到的荧光强度呈正相关。
取150μL上清液、50μL 15mM TMB和50μL 1mM过氧化氢溶液加入到1.8mL醋酸钠-醋酸缓冲溶液(pH 4.0)中。室温孵育5min后,在400-750nm处测定溶液的紫外可见吸收光谱以测定亚硝酸钠,通过445nm和652nm处的吸光度比来定量亚硝酸钠。
实施例3
将50μL纯水和50μL卡那霉素标准溶液(0.53pg/mL)作为对照组和实验组,分别与40μL实施例1制备的卡那霉素适配体功能化的掺铁碳点原溶液混合,加入10mM 4-羟乙基哌嗪乙磺酸缓冲液(2mM Ca2+,pH 7.0)使总体积为300μL,在37℃下孵育10min。之后,加入100μL实施例1制备的Fe3O4@PDA溶液(1mg/mL),并室温孵育2min。对反应溶液磁分离(方法同实施例2),在360-650nm处测定上清液的荧光光谱。如图1所示,为卡那霉素适配体功能化的掺铁碳点结合磁性Fe3O4@PDA生物传感器用于检测卡那霉素的荧光强度图,当待测样品不含有卡那霉素时,荧光强度较低;当待测样品中含有卡那霉素时,荧光强度较高。
实施例4
将100μL不同浓度亚硝酸钠标准溶液(0、1、2、5、10、20、50、100、200、500、1000、2000、5000μM)与40μL实施例1制备的卡那霉素适配体功能化的掺铁碳点原溶液混合,加入10mM 4-羟乙基哌嗪乙磺酸缓冲液(2mM Ca2+,pH7.0)使总体积为300μL,在37℃下孵育10min。之后,加入100μL的Fe3O4@PDA(1mg/mL),并室温孵育2min。对反应溶液磁分离(方法同实施例2),取上清液150μL,再向溶液中加入50μL 15mM TMB和50μL 1mM过氧化氢溶液,补充醋酸钠-醋酸缓冲溶液(pH 4.0)使总体积为2mL,室温孵育5min,观察溶液颜色,并在400-750nm处测定溶液的紫外可见吸收光谱。如图2所示,为该生物传感器在不同浓度亚硝酸盐检测的裸眼比色结果图,当样品中亚硝酸盐的浓度依次为0、1、2、5、10、20、50、100、200、500、1000、2000、5000μM时,随着亚硝酸钠浓度由低到高,溶液颜色发生由浅及深、由蓝到黄绿的颜色变化。
实施例5
将100μL纯水和100μL亚硝酸钠标准溶液(0.54μM)作为对照组和实验组,分别与实施例1制备的40μL卡那霉素适配体功能化的掺铁碳点原溶液混合,加入10mM 4-羟乙基哌嗪乙磺酸缓冲液(2mM Ca2+,pH 7.0)使总体积为300μL,在37℃下孵育10min。之后,加入100μL实施例1制备的Fe3O4@PDA(1mg/mL),并室温孵育2min。对反应溶液磁分离,取150μL的上清液,向溶液中加入50μL 15mM TMB和50μL 1mM过氧化氢溶液,补充醋酸钠-醋酸缓冲溶液(pH4.0)使总体积为2mL,室温孵育5min,在400-750nm处测定溶液的紫外可见吸收光谱。如图3所示,为该生物传感器用于亚硝酸盐检测的紫外可见吸收光谱结果分析示意图,当待测样品中不含有亚硝酸盐时,溶液在652nm处有特征吸收峰;当待测样品中含有亚硝酸盐时,溶液在652nm处的吸收峰下降,产生在445nm处的特征吸收峰。
实施例6
分别取10mg盐酸多巴胺、聚多巴胺(PDA)、Fe3O4纳米颗粒(实施例1制备)、Fe3O4@PDA纳米颗粒粉末(实施例1制备)用傅立叶变换红外吸收光谱仪在4000-400cm-1的波数范围内扫描红外图谱。如图4所示,为制备的Fe3O4@PDA纳米颗粒的红外光谱图,在580cm-1处出现了Fe-O伸缩振动带,在1604cm-1和1506cm-1处的吸收峰归因于与PDA的苯环中的C=C有关的振动带,在1287cm-1处出现了酚羟基中的C-O的特征峰,这表明PDA在Fe3O4纳米颗粒表面的成功包覆。
其中聚多巴胺制备过程为:取盐酸多巴胺20mg加入到20mL Tris-HCl缓冲液(pH8.5,10mM)和8mL异丙醇中溶解,搅拌72小时,反应完毕后用纯水洗涤,60℃真空干燥得聚多巴胺的粉末。
实施例7
取10mg Fe3O4@PDA纳米颗粒用纯水溶解后施加外磁场测定其磁分离能力。如图5所示,为施加外磁场后Fe3O4@PDA纳米颗粒的溶液磁分离情况,在1min内该溶液可以实现良好的分离效果,表明所获得的Fe3O4@PDA纳米粒子具有很高的磁性。
实施例8
取10μL实施例1制备的掺铁碳点的水溶液(0.5mg/mL)滴加到铜网上,待自然晾干后用透射电镜观察。如图6所示,为制备的掺铁碳点的透射电镜图,所制备的掺铁碳点为球形,分散均匀,无明显聚集,颗粒直径为2-5nm,表明掺铁碳点的成功合成。
实施例9
取0.5mg/mL实施例1制备的掺铁碳点水溶液,用荧光分光光度计测定激发波长分别为300、320、330、336、350、360、380nm时,掺铁碳点在350-650nm范围内的荧光发射光谱。如图7所示,为掺铁碳点的荧光性能图,所制备的掺铁碳点具有明显的激发波长依赖性,且最大激发波长为336nm,最大发射波长为438nm。
实施例10
以下组别均采用TMB浓度为25mM,体积为50μL;过氧化氢为10mM,体积为50μL;Fe-CDs浓度为0.5mg/mL,体积为20μL。设置TMB+过氧化氢、Fe-CDs+过氧化氢、Fe-CDs+TMB、CDs+TMB+过氧化氢和Fe-CDs+TMB+过氧化氢5个组,加入醋酸钠-醋酸缓冲溶液(pH 4.0)使总体积为2mL,室温孵育5min后在400-750nm处测定溶液的紫外可见吸收光谱。如图8所示,为掺铁碳点的类过氧化物酶活性验证图,通过对比TMB+过氧化氢、Fe-CDs+过氧化氢、Fe-CDs+TMB、CDs+TMB+过氧化氢和Fe-CDs+TMB+过氧化氢这5个组,表明当过氧化氢存在时,掺铁碳点会催化过氧化氢氧化TMB,产生652nm处的特征吸收峰,且它的类过氧化物酶活性来源于铁的掺杂。
实施例11
分别取10mg实施例1制备的掺铁碳点粉末和卡那霉素适配体功能化的掺铁碳点粉末,用傅立叶变换红外吸收光谱仪在4000-400cm-1的波数范围内扫描其红外图谱。如图9所示,为卡那霉素适配体功能化的掺铁碳点的红外光谱图,位于1705cm-1和1605cm-1的振动带分别代表酰胺中的C=O伸缩振动和N-H的弯曲振动,位于2720cm-1和1240cm-1的特征峰分别归因于磷酸分子中的-OH伸缩振动和P=O伸缩振动,这证明了掺铁碳点与适配体的成功连接。如图10所示,以50ng/mL的卡那霉素标准溶液,100μM的亚硝酸钠标准溶液进行参数优化,分别对荧光强度、445nm处与652nm处的吸光度之比作为判断标准,以最大荧光强度以及最大吸光度之比所对应的参数作为最佳实验条件。实验采用10mM 4-羟乙基哌嗪乙磺酸缓冲液为最佳检测溶液体系(图10中A所示),其中所含Ca2+浓度为2mM(图10中C所示),pH为7.0(图10中B所示);卡那霉素适配体与卡那霉素共孵育时间为10min(图10中D所示);加入Fe3O4@PDA终浓度为250μg/mL(图10中E所示);Fe3O4@PDA加入后反应时间为2min(图10中F所示);亚硝酸钠检测时间为5min(图10中G所示);亚硝酸钠检测体系中所用的TMB终浓度为0.36mM(图10中H所示),过氧化氢浓度为0.02mM(图10中I所示)。
实施例12
将50μL卡那霉素标准溶液(0.01,0.05,0.1,0.2,0.5,1,2,5,10,20,50,100,200,500ng/mL)和100μL亚硝酸钠标准溶液(100μM)与40μL实施例1制备的卡那霉素适配体功能化的掺铁碳点原溶液混合,加入10mM 4-羟乙基哌嗪乙磺酸缓冲液(2mM Ca2+,pH 7.0)使总体积为300μL,在37℃下孵育10min。之后,加入100μL的Fe3O4@PDA(1mg/mL),并室温孵育2min。对反应溶液磁分离(方法同实施例2),在360-650nm处测定上清液的荧光光谱以测定卡那霉素的含量。
如图11所示,为该检测方法用于卡那霉素检测的标准曲线图,表明卡那霉素的线性检测范围是0.01ng/mL-200ng/mL,R2=0.9916,计算得检出限为0.00053ng/mL。
实施例13
将50μL卡那霉素标准溶液(100ng/mL)和100μL亚硝酸钠标准溶液(0,1,2,5,10,20,50,100,200,500,1000,2000,5000μM)与40μL实施例1制备卡那霉素适配体功能化的掺铁碳点原溶液混合,加入10mM 4-羟乙基哌嗪乙磺酸缓冲液(2mM Ca2+,pH 7.0)使总体积为300μL,在37℃下孵育10min。之后,加入100μL的Fe3O4@PDA(1mg/mL),并室温孵育2min。对反应溶液磁分离(方法同实施例2),取150μL上清液,加入50μL 15mM TMB和50μL 1mM过氧化氢溶液,然后加入醋酸钠-醋酸缓冲溶液(pH 4.0)使总体积为2mL。室温孵育5min后,在400-750nm处测定溶液的紫外可见吸收光谱,以测定亚硝酸钠的含量。
如图12所示,为该检测方法用于亚硝酸钠检测的标准曲线图,表明亚硝酸钠的线性检测范围是5-5000μM,R2=0.9985,计算得检出限为0.54μM。
实施例14
分别将4.0g超高温加工牛奶、巴氏杀菌奶、发酵奶样品稀释在20mL纯水中,并通过滴加乙酸(40%,v/v)将混合物调至pH 4.6,以使蛋白质变性并沉淀。将样品在10,000rpm下离心20min,将上清液稀释10倍后用0.22μm滤膜过滤。将4.0g婴幼儿奶粉样品分散在20mL纯水中,然后以与牛奶样品相同的方式处理。在四个预处理过的样品中加入设定的不同浓度的卡那霉素KAN和亚硝酸钠标准溶液(0.5、1.0、5.0μg/kg的卡那霉素和100、200、500μM的亚硝酸钠),得到最终的样品溶液,按实施例2的检测方法进行回收率测定实验。本实施例含卡那霉素样品溶液和含亚硝酸钠样品溶液是根据国家食品安全标准的常用检测方法的检出限参考设定的标准液值浓度。
如图13所示,为本生物传感器按实施例2的检测方法对四种食物基质中(超高温加工牛奶、巴氏杀菌奶、发酵奶、婴幼儿奶粉)的卡那霉素含量的回收率测定实验结果,回收率为92.8-108.6%,相对标准偏差为1.62%-8.60%,表明该方法用于食物基质中卡那霉素检测的准确性。
如图14所示,为本生物传感器按实施例2的检测方法对四种食物基质中(超高温加工牛奶、巴氏杀菌奶、发酵奶、婴幼儿奶粉)的亚硝酸钠含量的回收率测定实验结果,回收率为90.3-107.2%,相对标准偏差为1.05%-7.07%,表明该方法用于食物基质中亚硝酸钠检测的准确性。
Claims (10)
1.一种基于掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法,其特征在于,所述生物传感器包括Fe3O4@PDA纳米颗粒和适配体功能化的掺铁碳点原溶液;其中所述Fe3O4@PDA纳米颗粒的制备为将Fe3O4粉末和盐酸多巴胺混合加入到缓冲液中搅拌,分离洗涤产物,然后真空干燥过夜;所述适配体功能化的掺铁碳点原溶液的制备为将1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺溶解在掺铁碳点水溶液中加入适配体搅拌透析除去游离的适配体。
2.根据权利要求1所述的基于掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法,其特征在于,所述适配体功能化的掺铁碳点原溶液中的适配体为卡那霉素适配体、链霉素适配体、四环素适配体、黄曲霉毒素M1适配体中的任意一种。
3.根据权利要求1所述的基于掺铁碳点结合磁性Fe3O4@PDA生物传感器的制备方法,其特征在于,所述适配体功能化的掺铁碳点原溶液为卡那霉素适配体功能化的掺铁碳点原溶液,所述卡那霉素适配体核苷酸序列为5’NH2-TGGGGGTTGAGGCTAAGCCGA。
4.根据权利要求1所述的生物传感器的制备方法,其特征在于,所述制备Fe3O4@PDA纳米颗粒的具体方法为:将三氯化铁和柠檬酸钠溶于乙二醇中,混匀后加无水乙酸钠搅拌,高温加热后冷却到室温,真空干燥过夜得Fe3O4粉末;将盐酸多巴胺和上述所得Fe3O4粉末加入到缓冲液中搅拌,分离洗涤产物,真空干燥过夜得Fe3O4@PDA纳米颗粒粉末。
5.根据权利要求1所述的生物传感器的制备方法,其特征在于,所述适配体功能化的掺铁碳点原溶液的制备方法为:将柠檬酸、乙二胺和三氯化铁溶于超纯水中,混匀后高温加热,然后冷却至室温,用滤膜过滤悬浮液,用去离子水透析后冷冻干燥得到掺铁碳点粉末,将1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺溶解在掺铁碳点水溶液中,加入适配体搅拌,透析除去游离的适配体。
6.一种利用权利要求1所述生物传感器的制备方法所制备的掺铁碳点结合磁性Fe3O4@PDA生物传感器。
7.一种权利要求6所述生物传感器在同时或者分别检测抗生素和检测亚硝酸盐中的应用。
8.根据权利要求7所述同时或者分别检测抗生素和亚硝酸盐中的应用,其特征在于,所述生物传感器同时检测抗生素和亚硝酸盐的具体方法为:将抗生素溶液、亚硝酸盐溶液和适配体功能化的掺铁碳点原溶液混合,加入4-羟乙基哌嗪乙磺酸缓冲液共孵育,再加入Fe3O4@PDA纳米颗粒溶液共孵育,对反应溶液磁分离后测定上清液的荧光光谱以测定抗生素的含量;取所述上清液与3,3’ ,5,5’-四甲基联苯、过氧化氢溶液加入到缓冲溶液中室温孵育,孵育结束后测定溶液的紫外可见吸收光谱,以测定亚硝酸盐的含量。
9.根据权利要求7所述同时或者分别检测抗生素和亚硝酸盐中的应用,其特征在于,所述生物传感器分别检测卡那霉素和亚硝酸盐的具体方法为:将抗生素溶液、亚硝酸盐溶液分别和适配体功能化的掺铁碳点原溶液混合,混合后分别加入4-羟乙基哌嗪乙磺酸缓冲液共孵育,再加入Fe3O4@PDA纳米颗粒溶液共孵育,分别对反应溶液磁分离取上清液,测定含有抗生素上清液的荧光光谱以测定抗生素的含量;取含有亚硝酸盐的上清液与3,3’ ,5,5’-四甲基联苯、过氧化氢溶液加入到缓冲溶液中室温孵育,孵育结束后测定溶液的紫外可见吸收光谱,以测定亚硝酸盐的含量。
10.根据权利要求8或者9所述的应用,其特征在于,同时或者分别检测抗生素和亚硝酸盐的时间为10-20min。
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