CN110548864B - 一种荧光丝胶铂纳米簇及其制备方法和应用 - Google Patents
一种荧光丝胶铂纳米簇及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供的一种丝胶蛋白包裹的发青光的铂纳米簇及其制备方法及应用。本发明的制备方法为将丝胶蛋白和高价铂配合物以一定比例混合,涡旋器充分混匀后,加NaOH调节pH值,涡旋器再次混匀。水浴若干小时,得到铂纳米簇的聚合物。本发明制备方法工艺简单、绿色、高效,可行性高,具有广泛、实际的应用价值,本发明的纳米簇既可应用于物质中的氯吡硫磷的检测,应用范围包括环境水样、土样、及水果蔬菜样品中;又可以应用于样品中氯霉素含量的检测。本发明的检测方法简便高效,特异性强,灵敏度高,同时也为丝胶蛋白的使用提供了新思路。
Description
技术领域
本发明属于功能纳米荧光材料的制备和应用领域,具体涉及一种荧光丝胶铂纳米簇及其制备方法和应用。
背景技术
金属纳米簇是由几个至几十个原子组成的具有荧光性、水溶性的分子聚集体。它因特有的量子尺寸效应、生物相容性、光稳定性强、反聚合能力强等特点,使其在生物标记、环境监测等领域有着广泛的应用前景。其中,铂纳米簇具有良好的荧光特性,光稳定性,低毒性,生物相容性,表面易修饰等众多优势,受到研究者越来越广泛的关注。但是光稳定性强,生物相容性好,并且简单易重复的铂纳米簇难以合成。
丝胶蛋白(Sericin)由18种氨基酸组成,包括甘氨酸、丝氨酸、天门冬氨酸等,其中包括大量的氨基、羟基、羧基等极性基团。丝胶蛋白具有良好的抗氧化性、生物相容性、生物降解性等性能。一直以来,由于人们对丝胶蛋白认识不足,导致其每年被大量浪费,对环境造成了严重污染。怎样将其科学合理地应用到纳米簇的制备中,也是目前需要解决的一个难题。
中国是农产品生产大国,但在农产品种植过程中存在农药使用不合理或者滥用等行为,导致农产品常被检测出农残超标问题。氯吡硫磷(又名毒死蜱,氯蜱硫磷)是一种可以应用于果树、蔬菜、茶树等多个地方的硫代磷酸酯类农药,因其具有高效、低毒等优势而被广泛应用于农产品的病虫害防治。但其具有的触杀、胃毒作用,损害人体的神经中枢系统,若人长期服用氯吡硫磷超标的农产品可引起一系列中毒现象甚至死亡。农产品农药残留引起的质量安全问题是近年来国内外普遍关注的焦点,欧盟制定了严格的氯吡硫磷农残限量标准,规定在蔬菜和坚果中氯吡硫磷的最大残留限量(MRLs)不能超过0.05mg/kg。但是近年来用于氯吡硫磷农残检测的高效液相色谱法、酶联免疫法、酶抑制法等方法存在前期处理繁琐、仪器昂贵、耗时长等缺陷。
抗生素在人类感染性疾病的治疗和动物的健康保护或生长促进方面被广泛使用和滥用。抗生素由于其对环境和人类健康的严重威胁而在全球范围内受到重视。畜禽养殖场所是抗生素污染的重灾区,大量的抗生素污染物通过食物链、直接或间接接触进入到环境和人体。因此,急需拓展合理的抗生素检测方法。
发明内容
为了解决上述技术问题,本发明提供了一种丝胶蛋白(Sericin)作为模板形成铂纳米簇,本发明制备的荧光丝胶铂纳米簇(Sericin-Pt NCs)光稳定性强,生物相容性好。本发明还提供了此纳米簇的的制备方法,制备方法是以生物来源的丝胶蛋白酶为原料采用环境友好的“一锅煮法”合成。本发明还提供了此种纳米簇的应用,其既可应用于农产品和土壤及水样中氯吡硫磷的检测,又可以应用于样品中氯霉素的检测,检测方法快速、高效、绿色,有可观的社会经济效益和环保效益。
本发明的一种荧光丝胶铂纳米簇,其在铂纳米材料外表面包覆有丝胶蛋白。
优选所述荧光丝胶铂纳米簇平均粒径为2nm,其荧光最大激发波长为320nm,最大发射波长为440nm。
本发明所述的荧光丝胶铂纳米簇的制备方法,其包括如下步骤:
(1)将丝胶蛋白水溶液和高价铂配合物的水溶液进行混合,涡旋器充分混匀5~10min;
(2)加NaOH溶液调节pH值为8~12,涡旋器再次混匀5~10min;
(3)将所制备的样品进行水浴后即得。
优选所述高价铂配合物为六氯铂酸(H2PtCl6)、六氯铂酸盐、四氯铂酸或四氯铂酸盐中的任意一种或多种组合。进一步优选所述高价铂配合物为六氯铂酸,其水溶液浓度优选为25~100mM,最优选为25mM。
优选步骤(1)中丝胶蛋白水溶液的浓度为25~50mg/mL,进一步优选为50mg/mL,这样合成的效果最佳,化学反应最完全,产率最高,荧光效果最明显。
优选步骤(2)中的pH值为8~12,进一步优选为pH为11,此时荧光效果最明显。
优选步骤(3)中水浴时间为8~16h,进一步优选为12h,此时化学反应最完全。
优选步骤(3)中水浴温度为37℃~65℃,进一步优选为60℃,此时丝胶蛋白和铂结合的效果最佳。
最优选本发明所述的荧光丝胶铂纳米簇的制备方法为:将5mL的50mg/mL丝胶蛋白的水溶液和1mL的25mM六氯铂酸的水溶液混合,涡旋器充分混匀5min,加入1M的NaOH溶液调节pH值为11,加水补足至10mL,涡旋器再次充分混匀5min;将所制备的样品在60℃下水浴12h,得到铂纳米簇的聚合物,5,000rpm/min离心10min后,取上清液并置于4℃冰箱避光保存备用。
本发明所述的荧光丝胶铂纳米簇在氯吡硫磷检测中的应用。本发明发现氯吡硫磷能够使本发明制备的纳米簇的荧光淬灭。通过对氯吡硫磷进行浓度梯度检测发现,Sericin-Pt NCs对氯吡硫磷具有特异的选择性和高敏感性。
本发明对氯吡硫磷检测采用荧光分光光度法,首先向铂纳米簇体系中加入一系列不同浓度的氯吡硫磷溶液,优选检测标准体系为1mL,其中不同浓度的氯吡硫磷50μL,25mg/mL的Sericin-Pt NCs 50μL,加水补足至1mL,25℃水浴5min后,在激发光波长为320nm的条件下,检测溶液在440nm的发射光的峰高。
优选其检测应用范围为用于环境水、土壤或者农产品包括水果及蔬菜中的氯吡硫磷的检测。优选其检测氯吡硫磷的线性范围为25μM-350μM。
本发明所述的荧光丝胶铂纳米簇在氯霉素检测中的应用。本发明通过实验发现氯霉素能够使本发明的纳米簇的荧光淬灭。通过对氯霉素进行浓度梯度检测发现,Sericin-Pt NCs对氯霉素具有特异的选择性和高敏感性,可广泛应用于猪肉、牛肉、羊肉、鸡肉等畜禽类肉质中氯霉素的检测。
有益效果
本发明以生物来源的丝胶蛋白酶为原料采用环境友好的“一锅煮法”合成了一种新的荧光铂纳米材料,此纳米材料光稳定性强,生物相容性好,制作方法简单科学。本发明的荧光丝胶纳米簇对氯吡硫磷、氯霉素具有高敏感性,可应用于农产品和土壤、水样中氯吡硫磷的检测以及样品中氯霉素的检测,检测方法具有简便高效,特异性强,样品用量少,检测成本低等优点,可带来可观的社会经济效益和环保效益。
附图说明
图1为实施例1所制的Sericin-Pt NCs的透射电镜图。
图2为实施例1所制的Sericin-Pt NCs的紫外-可见吸收光谱图。
图3为实施例1所制的Sericin-Pt NCs的红外-可见吸收光谱图。
图4为实施例1所制的Sericin-Pt NCs的荧光光谱图。
图5为实施例1所制的Sericin-Pt NCs应用于检测氯吡硫磷的荧光光谱图。
图6为实施例1所制的Sericin-Pt NCs应用于检测氯吡硫磷的线性关系图。
图7为实施例1所制的Sericin-Pt NCs应用于检测氯霉素的荧光光谱图。
图8为实施例1所制的Sericin-Pt NCs应用于检测氯霉素的线性关系图。
具体实施方式
下面结合说明书附图介绍本发明的较佳实施例,举例证明本发明可以实施,通过向本领域中的技术人员完整介绍本发明,使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现,其保护范围并非仅限于文中提到的实施例,本文的附图和说明本质上是举例说明而不是限制本发明。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
其它所用的原材料、试剂和设备等,如无特殊说明,均可从商业途径得到或已公开。
下面结合实施例对本发明做详细的说明。
实施例1:Sericin-Pt NCs的制备
(1)取250mg丝胶蛋白加入5mL纯水中制备成50mg/mL的丝胶蛋白溶液,取此溶液5mL在其中加入1mL 25mM的H2PtCl6水溶液,涡旋器充分混匀5min。
(2)加入1M的NaOH溶液于上一步的溶液中,使pH值为11。加水补足至10mL,涡旋器再次混匀5min。
(3)将EP管在避光的条件下置于60℃水浴锅中12h,得到铂纳米簇的聚合物,5,000rpm离心10min,取上清液置于4℃冰箱避光保存备用。
实施例2
(1)取125mg丝胶蛋白加入5mL纯水中制备成25mg/mL的丝胶蛋白溶液,取此溶液5mL在其中加入1mL 50mM的H2PtCl6水溶液,涡旋器充分混匀10min。
(2)加入1M的NaOH溶液于上一步的溶液中,使pH值为10。加水补足至10mL,涡旋器再次混匀5min。
(3)将EP管在避光的条件下置于37℃水浴锅中8h,得到铂纳米簇的聚合物,5,000rpm离心10min,取上清液置于4℃冰箱避光保存备用。
实施例3
(1)取250mg丝胶蛋白加入5mL纯水中制备成50mg/mL的丝胶蛋白溶液,取此溶液5mL在其中加入1mL 100mM的H2PtCl6水溶液,涡旋器充分混匀10min。
(2)加入1M的NaOH溶液于上一步的溶液中,使pH值为12。加水补足至10mL,涡旋器再次混匀5min。
(3)将EP管在避光的条件下置于50℃水浴锅中16h,得到铂纳米簇的聚合物,5,000rpm离心10min,取上清液置于4℃冰箱避光保存备用。
实施例4
(1)取250mg丝胶蛋白加入5mL纯水中制备成50mg/mL的丝胶蛋白溶液,取此溶液5mL在其中加入1mL 50mM的Na2PtCl6·6H2O水溶液,涡旋器充分混匀10min。
(2)加入1M的NaOH溶液于上一步的溶液中,使pH值为11。加水补足至10mL,涡旋器再次混匀5min。
(3)将EP管在避光的条件下置于60℃水浴锅中14h,得到铂纳米簇的聚合物,5,000rpm离心10min,取上清液置于4℃冰箱避光保存备用。
实施例5
(1)取250mg丝胶蛋白加入5mL纯水中制备成50mg/mL的丝胶蛋白溶液,取此溶液5mL在其中加入1mL 100mM的K2PtCl6水溶液,涡旋器充分混匀10min。
(2)加入1M的NaOH溶液于上一步的溶液中,使pH值为9。加水补足至10mL,涡旋器再次混匀5min。
(3)将EP管在避光的条件下置于37℃水浴锅中16h,得到铂纳米簇的聚合物,5,000rpm离心10min,取上清液置于4℃冰箱避光保存备用。
实施例6
(1)取125mg丝胶蛋白加入5mL纯水中制备成25mg/mL的丝胶蛋白溶液,取此溶液5mL在其中加入1mL 50mM的N2H8PtCl6水溶液,涡旋器充分混匀10min。
(2)加入1M的NaOH溶液于上一步的溶液中,使pH值为12。加水补足至10mL,涡旋器再次混匀5min。
(3)将EP管在避光的条件下置于50℃水浴锅中12h,得到铂纳米簇的聚合物,5,000rpm离心10min,取上清液置于4℃冰箱避光保存备用。
实施例7:Sericin-Pt NCs样品形貌表征
取实施例1所制的Sericin-Pt NCs(在冰箱中放置的备用的上清液)用去离子水稀释20倍后,取10μL滴于铜网,美国FEI Tecnai G-20型透射电子显微镜(TransmissionElectron Microscope,TEM),加速电压100kV。结果显示铂纳米簇均匀分布(图1),其平均粒径约为2nm,该铂纳米簇在水溶液中具有良好的分散性。
实施例8:Sericin、Sericin-Pt NCs紫外光谱的表征
分别取实施例1制备的Sericin水溶液及Sericin-Pt NCs放入比色皿中,使用紫外分光光度仪UV-1700检测紫外光谱,结果表明Sericin在280nm处有紫外吸收,而Sericin-PtNCs在此处没有最大吸收,且其在300-400nm范围具有较宽的吸收图谱(图2),证明实施例1所制的Sericin-Pt NCs和Sericin本身不是同一种物质。
实施例9:Sericin、Sericin-Pt NCs红外光谱的表征
分别取纯的丝胶蛋白(Sericin)及实施例1所制的Sericin-Pt NCs使用红外分析光谱检测红外光谱,结果表明Sericin和的Sericin-Pt NCs在多处有明显不同(图3),证明实施例1所制的Sericin-Pt NCs和Sericin本身不是同一种物质。
实施例10:Silksericin-Pt NCs荧光光谱的表征
分别取实施例1制备的Sericin水溶液及Sericin-Pt NCs置于EP管中,采用暗箱式四用紫外分析仪,观察它们的的荧光特性,结果表明在365nm紫外灯照射下,Sericin-PtNCs溶液发射出强烈的青色荧光,可见光下Sericin-Pt NCs溶液为浅黄色,而Sericin水溶液在可见光下为无色。
取实施例1所制的Sericin-Pt NCs放入比色皿中,使用RF-5301荧光分光光度计测量Sericin-Pt NCs的最大激发光谱和最大发射光谱,结果表明该物质最大激发光谱和最大发射光谱分别为320nm和440nm(图4)。
实施例11:实施例1所制的Sericin-Pt NCs应用于氯吡硫磷的检测
向铂纳米簇体系中加入一系列不同浓度的氯吡硫磷溶液,检测标准体系为1mL,其中不同浓度的氯吡硫磷溶液50μL,25mg/mL的Sericin-Pt NCs 50μL,加水补足至1mL,25℃水浴5min后,在激发光波长为320nm的条件下,检测溶液在440nm的发射光的峰高。结果显示,Sericin-Pt NCs荧光淬灭程度随氯吡硫磷浓度的增大而增强(图5),其相对荧光强度线性检测曲线为y=0.00161x+0.99783(R2=0.993)。所构建的检测氯吡硫磷的线性范围为25μM-350μM,其检测限为2.26μM(图6)。结果表明,实施例1所制的Sericin-Pt NCs对氯吡硫磷具有高度选择性,因此本发明所制的铂纳米簇荧光探针同样可用于分析检测实际样品中氯吡硫磷的含量。
实施例12:实施例1所制的Sericin-Pt NCs对实际水样中氯吡硫磷的监测
为了更清楚的说明本发明实施例的应用方案,证实Sericin-Pt NCs作为探针检测氯吡硫磷的实用性,下面将详细介绍氯吡硫磷对实际水样检测过程。从芜湖市采集了长江水、镜湖水和自来水样本。将取自不同环境中的水样10,000rpm离心10min,0.22μm滤膜抽滤,并储存在4℃冰箱中备用。
(1)实际水样的检测:
检测体系为1mL,其中25mg/mL的Sericin-Pt NCs溶液50μL,待测的环境水样950μL;将此体系于25℃水浴5min,在激发光波长为320nm的条件下,检测溶液在440nm的发射光的峰高;将测试结果带入实施例11绘制的标准曲线中计算。
将芜湖市长江水,镜湖水,自来水分别按照上述方法进行检测,实验独立重复3次。三种水样的检测结果均为0μM。
(2)检测加标回收率:
在三个环境水样中分别添加浓度为25μM、50μM和100μM的氯吡硫磷溶液标样,进行检测并计算各水样的氯吡硫磷回收率。检测标准体系为1mL,其中25mg/mL的Sericin-PtNCs溶液50μL,环境水样900μL,不同浓度的氯吡硫磷50μL,将此体系于25℃水浴5min,在激发光波长为320nm的条件下,检测溶液在440nm的发射光的峰高。实验独立重复3次。表1是标准加入不同浓度系列的氯吡硫磷后的结果,可以看出,回收率可以达到95%以上。说明该方法在实际水样中的应用效果良好。
表1 Sericin-Pt NCs对各水样中氯吡硫磷的检测
实施例13:实施例1所制的Sericin-Pt NCs对实际土样中氯吡硫磷的检测
为了更清楚的说明本发明实施例的应用方案,证实Sericin-Pt NCs作为探针检测氯吡硫磷的实用性,下面将详细介绍氯吡硫磷对实际土样检测过程。从芜湖市采集葡萄种植园的土样、农田土样以及安徽师范大学赭山校区的土样。将不同环境中的土样置于60℃烘箱烘干2h,加去离子水稀释10倍溶解后10,000rpm离心10min,并储存在4℃冰箱中备用。
(1)实际土样的检测:
检测体系为1mL,其中50μL 25mg/mL的Sericin-Pt NCs溶液,950μL待测的土样稀释液;将此体系于25℃水浴5min,在激发光波长为320nm的条件下,检测溶液在440nm的发射光的峰高;将测试结果带入实施例11绘制的标准曲线中计算。
将三种土壤样品分别按照上述方法进行检测,实验独立重复3次。三种土样的检测结果均为0μM。
(2)检测加标回收率:
检测标准体系为1mL,50μL 25mg/mL的Sericin-Pt NCs溶液,900μL的土样稀释液,50μL不同浓度的氯吡硫磷溶液,25℃水浴5min,在激发光波长为320nm的条件下,检测溶液在440nm的发射光的峰高,实验独立重复3次,同时对实际土样的回收率进行计算。为了计算实际土样中氯吡硫磷的回收率,分别在25μM、50μM和100μM的氯吡硫磷浓度下,对三种实际土样样品进行加标样中氯吡硫磷的回收率计算。表2是标准加入不同浓度系列的氯吡硫磷后的结果,可以看出,回收率可以达到95%以上。说明该方法在实际土样中的应用效果良好。
表2 Sericin-Pt NCs对各土样中氯吡硫磷的检测
实施例14:实施例1所制的Sericin-Pt NCs对水果蔬菜样品中氯吡硫磷的检测
为了更清楚的说明本发明实施例的应用方案,证实Sericin-Pt NCs作为探针检测氯吡硫磷的实用性,下面将详细介绍氯吡硫磷对水果蔬菜样品检测过程。分别从芜湖市商品市场购买了苹果、生菜和菠菜。将不同样品放置于研钵中研磨5min,充分研磨得到植物提取液,移取植物提取液200μL至1.5mL的EP管中,其中苹果研磨液稀释50倍后10,000rpm离心10min,并储存在4℃冰箱中备用。生菜和菠菜研磨液稀释600倍后10,000rpm离心10min,并储存在4℃冰箱中备用。
(1)水果蔬菜的检测:
检测体系为1mL,其中50μL 25mg/mL的Sericin-Pt NCs溶液,950μL待测的样品稀释液;将此体系于25℃水浴5min,在激发光波长为320nm的条件下,检测溶液在440nm的发射光的峰高;将测试结果带入实施例11绘制的标准曲线中计算。
将待测样品分别按照上述方法进行检测,实验独立重复3次。待测样品的检测结果均为0μM。
(2)检测加标回收率:
检测标准体系为1mL,50μL 25mg/mL的Sericin-Pt NCs溶液,900μL待测样品溶液,50μL不同浓度的氯吡硫磷溶液,25℃水浴5min,在激发光波长为320nm的条件下,检测溶液在440nm的发射光的峰高,实验独立重复3次,同时对各样品的回收率进行计算。为了计算实际样品中氯吡硫磷的回收率,分别在25μM、50μM和100μM的氯吡硫磷浓度下,对苹果、生菜、菠菜样品进行加标样中氯吡硫磷的回收率。表3是标准加入不同浓度系列的氯吡硫磷后的结果,可以看出,回收率可以达到95%以上。说明该方法在实际水果蔬菜样品中的应用效果良好。
表3 Sericin-Pt NCs对各水果蔬菜样品中氯吡硫磷的检测
实施例15:实施例1所制的Sericin-Pt NCs应用于氯霉素的检测
向铂纳米簇体系中加入一系列不同浓度的氯霉素溶液,检测标准体系为1mL,其中不同浓度的氯霉素50μL,25mg/mL的Sericin-Pt NCs 50μL,加水补足至1mL,25℃水浴5min后,在激发光波长为320nm的条件下,检测溶液在440nm的发射光的峰高。结果显示,Sericin-Pt NCs荧光淬灭程度随氯霉素浓度的增大而增强(图7),其相对荧光强度线性检测曲线为y=1.63763x+0.93013(R2=0.99673),所构建的检测氯霉素的线性范围为100μM-1100μM,其检测限为12μM(图8)。结果表明,实施例1所制的Sericin-Pt NCs对氯霉素具有高度选择性,因此本发明所制的铂纳米簇荧光探针同样可用于分析检测实际样品中氯霉素的含量。
Claims (1)
1.一种荧光丝胶铂纳米簇在氯吡硫磷检测中的应用,其特征是,所述荧光丝胶铂纳米簇在铂纳米材料外表面包覆有丝胶蛋白;
所述的荧光丝胶铂纳米簇的制备方法为:将5mL的50mg/mL丝胶蛋白的水溶液和1mL的25mM六氯铂酸的水溶液混合,涡旋器充分混匀5min,加入1M的NaOH溶液调节pH值为11,加水补足至10mL,涡旋器再次充分混匀5min;将所制备的样品在60℃下水浴12h,得到铂纳米簇的聚合物,5000rpm/min离心10min后,取上清液并置于4℃冰箱避光保存备用;
对氯吡硫磷检测时采用荧光分光光度法,首先向荧光丝胶铂纳米簇体系中加入一系列不同浓度的氯吡硫磷溶液,检测标准体系为1mL,其中不同浓度的氯吡硫磷50μL,25mg/mL的荧光丝胶铂纳米簇50μL,加水补足至1mL,25℃水浴5min后,在激发光波长为320nm的条件下,检测溶液在440nm的发射光的峰高,其相对荧光强度线性检测曲线为y=0.00161x+0.99783,R2=0.993,其中横坐标x为氯吡硫磷的浓度,单位为μM,纵坐标y为相对荧光强度,R为相关系数,所构建的检测氯吡硫磷的线性范围为25μM-350μM,其检测限为2.26μM。
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