CN113292987B - 双发射金团簇比率荧光探针及其制备方法和强力霉素的检测方法 - Google Patents
双发射金团簇比率荧光探针及其制备方法和强力霉素的检测方法 Download PDFInfo
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Abstract
一种双发射金团簇比率荧光探针及其制备方法和强力霉素的检测方法,该双发射金团簇材料,采用牛血清白蛋白修饰金团簇制备得到,其在390nm激发下在480nm处和640nm处分别具有荧光发射峰。该双发射金团簇材料作为比率型荧光探针应用于强力霉素的高选择性、高灵敏性检测。本发明还公开了该双发射金团簇材料在选择性检测强力霉素方面的应用。该检测方法能够有效消除系统误差,具有检测限低,准确性好,简单快速检测等优点。
Description
技术领域
本发明涉及生物传感技术领域,具体涉及一种双发射比率荧光探针及其制备方法,以及该荧光探针用于检测食品中强力霉素的方法。
背景技术
近些年来,抗生素大量应用于农业、畜牧业、水产养殖业和制药行业,它作为一种新兴的污染物,一旦残留过量,将对土壤、水和食品质量安全造成潜在的风险,然后以生态循环等途径进一步危害人体健康和生态环境。自从抗生素被批准可以作为动物饲料添加剂后,全世界抗生素的产量和使用量都急速增长。强力霉素是由土霉素6-位上脱氧而制成的半合成四环素类抗生素。据报道,2018年我国四环素类抗生素兽用使用量达13664.822t,占全部抗菌药的比例45.9%,其中强力霉素使用量在1000t以上。强力霉素是一种高效且广谱的半合成四环素抗生素,被广泛用作预防和治疗动物感染的饲料添加剂。当饲料中过量添加抗生素后,如果抗生素未完全代谢,它们将残留在动物组织或副产品中,人体长期食用会危害身体健康。
目前国内外已研究了多种检测强力霉素的方法,包括高效液相色谱法,液相色谱-质谱法,酶连锁免疫吸附测定,毛细管电泳,电化学。这些方法可以提供灵敏且准确的多种抗生素的分析检测,但这些技术通常很复杂且耗时,并且需要昂贵的仪器和经过专业培训的人员来进行操作。因此,这些方法可能会限制快速和实时检测强力霉素。
金团簇由于其特殊的物理和化学特性,已被广泛用作检测生物和化学系统中某些物质的分析探针。Zhang等报道了一种基于螯合增强荧光的功能化金团簇,并检测了人血清中的磷酸盐。Zhao等通过金团簇的骨架增强荧光检测三硝基甲苯。目前已经报道了多种合成金纳米团簇的方法。例如,Song等使用D-His作为还原剂和稳定剂来合成D-His@Au NCs,D-His@Au NCs的催化活性由于Cu2+诱导的聚集而大大降低,但在添加强力霉素后恢复,基于此达到检测强力霉素的目的。Jain等人报道了用BSA作为还原剂和稳定剂合成的Au NCs用于检测肝细胞中过氧化氢的水平,方法是通过活性氧(ROS)淬灭BSA-Au NCs的651nm处的单发射峰。到目前为止,金团簇已经通过嵌入,组装或共轭其他荧光纳米材料达到了单发射或比例荧光检测的目的。Yang等开创性地发现用谷胱甘肽合成的金纳米颗粒分别在610nm和810nm处具有固有的双发射峰,在此之前很少有自身具有的双发射金团簇用于分析传感的报道。目前尚未有研究报道自身具有双发射峰的BSA-Au NC作为分析传感中的比例荧光探针。
1.Zhang文献出处:Zhang,Z.P.,Feng,J.Y.,Huang,P.C.,Li,S.,&Wu,F.Y.(2019).Ratiometric fluorescent detection of phosphate in human serum withfunctionalized gold nanoclusters based on chelation-enhancedfluorescence.Sensors and Actuators B-Chemical,298,6.https://doi.org/10.1016/j.snb.2019.126891.
2.Zhao文献出处:Zhao,Y.,Pan,M.,Liu,F.,Liu,Y.H.,Dong,P.,Feng,J.,...Liu,X.Q.(2020).Highly selective and sensitive detection of trinitrotoluene byframework-enhanced fluorescence of gold nanoclusters.Analytica Chimica Acta,1106,133-138.https://doi.org/10.1016/j.aca.2020.01.055
3.Song文献出处:Song,Y.Y.,Qiao,J.,Liu,W.,&Qi,L.(2020).Colorimetricdetection of serum doxycycline with d-histidine-functionalized goldnanoclusters as nanozymes.Analyst,145(10),3564-3568.https://doi.org/10.1039/d0an00297f.
4.Jain文献出处:Jain,V.,Bhagat,S.,&Singh,S.(2021).Bovine serum albumindecorated gold nanoclusters:A fluorescence-based nanoprobe for detection ofintracellular hydrogen peroxide.Sensors and Actuators B-Chemical,327.https://doi.org/10.1016/j.snb.2020.128886.
5.Yang文献出处:Yang,Y.,Lu,L.Q.,Tian,X.K.,Li,Y.,Yang,C.,Nie,Y.L.,&Zhou,Z.X.(2019).Ratiometric fluorescence detection of mercuric ions by soleintrinsic dual-emitting gold nanoclusters.SensorsandActuators B-Chemical,278,82-87.https://doi.org/10.1016/j.snb.2018.09.072.
发明内容
本发明的目的是针对现有技术存在的问题而提供一种具有高灵敏度和更好选择性的一种双发射金团簇比率荧光探针。
本发明的目的是这样实现的:一种双发射金团簇比率荧光探针,荧光探针采用牛血清白蛋白和四氯金酸为原材料制备而成,该荧光探针在390nm激发下,在480nm和640nm处显示出两个不同的发射峰,这两个发射峰分别来自牛血清白蛋白和金纳米团簇。荧光探针在高分辨率透射电子显微镜下的形貌为分布均匀的单分散准球形,直径为0.5~3.5nm,平均值为2.06nm。
本发明的第二目的提供上述比率荧光探针的制备方法。
本发明第二目的是这样实现的:一种双发射金团簇比率荧光探针的制备方法,包括以下步骤:
(1)在剧烈搅拌下将5mL,10mM,37℃的HAuCl4·4H2O溶液添加到5mL,50mg/mL,37℃的牛血清白蛋白溶液中;
(2)2min后,逐滴加入1M的NaOH溶液,使混合液的pH调节至12;
(3)在37℃水浴条件下持续搅拌12h,最终得到产物BSA-Au NCs。
本发明的第三目的是提供采用上述比率荧光探针检测强力霉素的方法。
本发明的第三目的是这样实现的:一种双发射金团簇比率荧光探针检测强力霉素的方法,按照下述步骤进行:
(1)分别配制浓度梯度为0、0.2、2、5、10、15、20、30、40、60μM的强力霉素标准溶液;
(2)然后将不同浓度的强力霉素标准溶液与BSA-Au NCs检测溶液按体积比1:1混合,用1M的NaOH溶液调节混合液的pH调节至12,然后孵育2min,使用390nm作为激发波长,随着强力霉素浓度的增加,记录510nm和640nm处的荧光发射峰,通过510nm和640nm处的荧光强度的比值与加入的强力霉素的浓度进行线性拟合得到线性方程,建立强力霉素检测的标准曲线。
本发明的第四目的是提供上述比率荧光探针检测食品中强力霉素的方法。
本发明的第四目的是这样实现的:一种双发射金团簇比率荧光探针检测食品中强力霉素的制备方法,具体步骤如下:
(1)用1M NaOH和HCl调节0.01M PBS缓冲液的pH至12。
(2)分别将2mL牛奶、1g鸡蛋和1g蜂蜜放入10mL烧杯中,加入5mL,pH=12,0.01M的PBS缓冲液和1mL的5%三氯乙酸,涡旋10min并以10,000rpm离心10min后,取上清液,用1MNaOH将上清液的pH值调节至12,再以10,000rpm离心10min后,用0.22μm滤膜过滤所得的上清液,分别获得牛奶、鸡蛋、蜂蜜待测样品液。
(3)分别将1g猪肉和1g鸡肝放入10mL烧杯中,加入5mL,pH=12,0.01M的PBS缓冲液,使用细胞粉碎仪搅拌至匀浆,然后加入1mL的5%三氯乙酸,涡旋10min并以10,000rpm离心10min,取上清液,用1M NaOH将上清液的pH值调节至12,再以10,000rpm离心10min后,用0.22μm滤膜过滤所得的上清液,分别获得猪肉、鸡肝待测样品液。
(4)将不同浓度的强力霉素加入到上述上清液中得到待测样品液中;采用本发明检测强力霉素的方法,扫描荧光光谱,根据检测出的510nm和640nm处的荧光强度的比值,并结合强力霉素检测的标准曲线拟合出线性方程,计算出实际食品样品中的强力霉素浓度。
与现有技术相比,本发明具有以下有益效果:
(1)本发明提供的金团簇合成方法采用一步水热法,反应条件温和、绿色、简便。
(2)本发明提供的金团簇具有稳定光学性能和良好的水溶性。
(3)本发明提供的金团簇由牛血清白蛋白修饰合成,具有双发射峰。
(4)本发明提供的双发射金团簇可作为比率荧光探针直接检测强力霉素。
(5)本发明提供的双发射金团簇检测强力霉素的方法与传统检测方法相比,不需要昂贵的仪器和专业培训的人员来进行操作即可检测,同时灵敏度高,选择性好,具有省时省力,检测限低,准确性好,简单快速检测的优点。
本发明采用的比率荧光将两个不同荧光峰处的荧光强度之比作为响应信号,与单发射荧光探针相比,比率荧光法的样品应用范围更广,并且具有更高的灵敏度和更好的选择性。
本发明可定性和定量检测食品中的强力霉素,且该检测方法具有快速,可操作性强,选择性好和灵敏度高,反应迅速,动态线性范围宽的技术特点。
本发明采用一步水热法来合成BSA-Au NCs,该合成方法简单且环保。合成的BSA-Au NCs在390nm激发下具有双发射峰。此外,随着添加的强力霉素浓度的增加,绿色荧光(I510)增加,而红色荧光(I640)保持不变。在此基础上,设计了一种利用比率荧光检测强力霉素的简便方法。所开发的测定方法成功进一步用于食品中强力霉素的分析检测。
附图说明
图1为BSA-Au NCs的HRTEM图像和BSA-Au NCs的尺寸范围分布;
图2为BSA-Au NCs的FT-IR光谱;
图3为BSA-Au NCs的XPS光谱;
图4为BSA-Au NCs的Au4f的XPS光谱;
图5为BSA-Au NCs光学性质图谱;
图6为BSA-Au NCs在310nm-410nm的激发下的荧光发射光谱;
图7为BSA-Au NCs检测不同浓度强力霉素的荧光发射光谱图
图8为BSA-Au NCs检测不同浓度强力霉素的线性关系图
图9为不同溶液pH与BSA-Au NCs检测强力霉素的关系图;
图10为不同反应时间与BSA-Au NCs检测强力霉素的关系图;
图11为BSA-Au NCs检测强力霉素选择性柱状图;
图12为BSA-Au NCs检测强力霉素干扰性柱状图;
具体实施方式
下面结合实施例对本发明做详细说明,实施例给出了详细的实施方案和具体操作过程,但本发明的保护范围不限于下述的实施例。
材料
牛血清白蛋白(BSA)和HAuCl4·4H2O购自麦克林生物公司。包含强力霉素在内的所有抗生素均购自阿拉丁化学公司。盐酸、硝酸、乙醇、金属盐试剂,氨基酸购自成都科龙有限公司。蛋白质购自上海源叶生物技术有限公司。所有化学药品均为分析纯,所有溶液均用双蒸馏水稀释。所有的荧光光谱都是从F-4500日立荧光分光光度计(日本东京)测得。
BSA-Au NCs的合成
在剧烈搅拌下将5mL,10mM,37℃的HAuCl4·4H2O溶液添加到5mL,50mg/mL,37℃的牛血清白蛋白溶液中。2min后,逐滴加入0.5mL,1M的NaOH溶液,使混合液的pH调节至12。在37℃水浴条件下持续搅拌12h,最终得到产物BSA-Au NCs。随着搅拌时间的延长,化合物的颜色从浅黄色变为红棕色,并且溶液在紫外光下显示出强烈的红色荧光。
荧光测量
分别配制浓度梯度为0、0.2、2、5、10、15、20、30、40、60μM的强力霉素标准溶液。然后将不同浓度的强力霉素标准溶液与BSA-Au NCs溶液按体积比1:1混合,用1M的NaOH溶液调节混合液的pH调节至12,然后孵育2min,使用390nm作为激发波长,随着强力霉素浓度的增加,记录510nm和640nm处的荧光发射峰,通过510nm和640nm处的荧光强度的比值与加入的强力霉素的浓度进行线性拟合得到线性方程,建立强力霉素检测的标准曲线。
食品样品测定
选择猪肉,鸡肝,牛奶,鸡蛋和蜂蜜作为测试样品。因为强力霉素被广泛用作上述食品的物质添加剂,这可能会导致在这些动物源性食品中残留有强力霉素。所有样品均从当地一家超市购买。对样品进行预处理:首先,将2mL牛奶或1g其他样品放入10mL烧杯中,并用5mLPBS缓冲液(pH=12,0.01M)稀释。然后将1mL的5%三氯乙酸加入到烧杯中。涡旋10分钟并以10,000rpm离心10min后,获得混合物的上清液。用1M NaOH将上清液的pH值调节至12,并以10,000rpm离心10min,然后稀释上清液并根据测定程序进行加标回收率分析。
实施例1
双发射金团簇比率荧光探针的制备方法,包括以下步骤:
(1)在剧烈搅拌下将5mL,10mM,37℃的HAuCl4·4H2O溶液添加到5mL,50mg/mL,37℃的牛血清白蛋白溶液中;
(2)2min后,逐滴加入1M的NaOH溶液,使混合液的pH调节至12;
(3)在37℃水浴条件下持续搅拌12h,最终得到产物BSA-Au NCs。
实施例2
将实施例1制备的双发射金团簇比率荧光探针进行HRTEM、FT-IR和XPS表征(图1-4),结果表明双发射金团簇比率荧光探针在高分辨率透射电子显微镜下的形貌为分布均匀的单分散准球形,直径为0.5-3.5nm,平均值为2.06nm。通过FT-IR和XPS表征可以看出牛血清白蛋白作为保护剂和还原剂,成功将金离子合成金团簇。
实施例3
分别扫描实施例1制备的双发射金团簇比率荧光探针紫外光谱、荧光最佳激发光谱和最佳激发波长下所对应的荧光发射光谱(图5)。如图5所示,当激发波长为400nm时,BSA-Au NCs在480nm和640nm处显示双发射峰。
实施例4
在不同激发下(310nm-410nm)分别扫描实施例1制备的双发射金团簇的发射光谱(图6),记录两个发射峰的位置和荧光强度。观察BSA-Au NCs是否表现出依赖于激发的荧光行为,同时获得与最强荧光强度对应的最佳激发波长。结果显示,BSA-Au NCs具有激发依赖性,后续实验选择390nm作为激发波长。
实施例5
将实施例1制备的双发射金团簇材料比率检测强力霉素,步骤为:
(1)分别配制浓度梯度为0、0.2、2、5、10、15、20、30、40、60μM的强力霉素标准溶液;
(2)然后将不同浓度的强力霉素标准溶液与BSA-Au NCs溶液按体积比1:1混合,用1M的NaOH溶液调节混合液的pH调节至12,然后孵育2min,使用390nm作为激发波长,随着强力霉素浓度的增加,记录510nm和640nm处的荧光发射峰,通过510nm和640nm处的荧光强度的比值与加入的强力霉素的浓度进行线性拟合得到线性方程,建立强力霉素检测的标准曲线。(图7-8)。
实施例6
将40μM的强力霉素标准溶液与BSA-Au NCs检测溶液按体积比1:1混合,用1M的NaOH、HCl调节成混合液的不同pH(3、4、5、6、7、8、9、10、11、12、13、14)。然后在390nm激发下扫描溶液的荧光发射光谱,记录510nm和640nm处的荧光强度并计算比值,观察不同pH下比值的大小,选择比值最大时的pH作为后续检测强力霉素的pH条件。结果表明最佳反应pH为12(图9)。确定好最佳的反应pH后,向荧光比色皿中添加BSA-Au NCs检测溶液、强力霉素后,孵育不同时间(0.5、1、1.5、2、2.5、3、3.5、4、5min),通过使用荧光分光光度计在最佳激发波长的激发下,分别记录不同孵育时间下510nm和640nm处的荧光强度并计算比值,观察不同时间下比值的大小,选择比值最大时的孵育时间作为后续检测强力霉素的反应时间条件。结果表明反应稳定时间在2min(图10)
实施例7
分别配置30μM浓度的各类抗生素和离子溶液,包括强力霉素、土霉素、四环素、金霉素、羧苄青霉素钠、硫酸阿卡米星、阿奇霉素、甲磺酸培氟沙星、硫酸替米考星、马波沙星、硫酸阿霉素、盐酸恩诺沙星、硫酸链霉素、环丙沙星、氯化钙、氯化钾、氯化镁、氯化铁。向荧光比色皿中添加等体积的BSA-Au NCs和上述溶液,在390nm的激发下,记录510nm和640nm处的荧光强度并计算比值,确定BSA-Au NCs对强力霉素的选择性。如图11,与其他抗生素和离子溶液相比,BSA-Au NCs对强力霉素有良好的选择性。
实施例8
分别配置60μM浓度的各类抗生素、离子、氨基酸和蛋白质溶液,包括强力霉素、土霉素、四环素、金霉素、羧苄青霉素钠、硫酸阿卡米星、阿奇霉素、甲磺酸培氟沙星、硫酸替米考星、马波沙星、硫酸阿霉素、盐酸恩诺沙星、硫酸链霉素、环丙沙星、氯化钙、氯化钾、氯化镁、氯化铁、谷氨酰胺、精氨酸、谷氨酸、蛋氨酸、丝氨酸、天冬酰胺、半胱氨酸、赖氨酸、组氨酸、谷胱甘肽、α-胰凝乳蛋白酶、γ-球蛋白、胃蛋白酶、溶菌酶、人血清白蛋白。将强力霉素与上述其他溶液以1:1体积混合,然后在荧光比色皿中添加等体积的BSA-Au NCs和上述混合溶液,在390nm的激发下,记录510nm和640nm处的荧光强度并计算比值,分析在这些干扰物质存在情况下,BSA-Au NCs检测强力霉素的抗干扰力。如图12,与对照相比,在存在其他抗生素、金属离子、氨基酸和蛋白质的情况下,BSA-Au NCs对强力霉素的检测几乎没有影响。
实施例9
为了评估本发明实施例提供的检测强力霉素方法的实用性,对实际食品样品中强力霉素进行了加标回收。本发明实施例在各参数最优条件下,检测了猪肉,鸡肝,牛奶,鸡蛋和蜂蜜中的浓度,步骤为:
(1)用1M NaOH和HCl调节0.01M PBS缓冲液的pH至12。
(2)分别将2mL牛奶、1g鸡蛋和1g蜂蜜放入10mL烧杯中,加入5mL,pH=12,0.01M的PBS缓冲液和1mL的5%三氯乙酸,涡旋10min并以10,000rpm离心10min后,取上清液,用1MNaOH将上清液的pH值调节至12,再以10,000rpm离心10min后,用0.22μm滤膜过滤所得的上清液,分别获得牛奶、鸡蛋、蜂蜜待测样品液。
(3)分别将1g猪肉和1g鸡肝放入10mL烧杯中,加入5mL,pH=12,0.01M的PBS缓冲液,使用细胞粉碎仪搅拌至匀浆,然后加入1mL的5%三氯乙酸,涡旋10min并以10,000rpm离心10min,取上清液,用1M NaOH将上清液的pH值调节至12,再以10,000rpm离心10min后,用0.22μm滤膜过滤所得的上清液,分别获得猪肉、鸡肝待测样品液。
(4)将不同浓度的强力霉素加入到上述上清液中得到待测样品液中;采用本发明检测强力霉素的方法,扫描荧光光谱,根据检测出的510nm和640nm处的荧光强度的比值,并结合强力霉素检测的标准曲线拟合出线性方程,计算出实际食品样品中的强力霉素浓度。检测结果如表1所示。
表1实际样品中强力霉素的测定和回收率
根据表1结果,可以看出本发明实施例提供的检测强力霉素的方法检出量和添加量基本一致,回收率良好,范围在96.0-103.7%之间,由此说明本发明本发明实施例提供的方法能有效应用于实际食品中的强力霉素的检测。
Claims (3)
1.一种双发射金团簇比率荧光探针检测强力霉素的方法,其特征在于,按照下述步骤进行:
(1)分别配制浓度梯度为0、0.2、2、5、10、15、20、30、40、60μM的强力霉素标准溶液;
(2)然后将不同浓度的强力霉素标准溶液与BSA-Au NCs溶液按体积比1:1混合,用1M的NaOH溶液调节混合液的pH调节至12,然后孵育2min,使用390nm作为激发波长,随着强力霉素浓度的增加,记录510nm和640nm处的荧光发射峰,通过510nm和640nm处的荧光强度的比值与加入的强力霉素的浓度进行线性拟合得到线性方程,建立强力霉素检测的标准曲线;
所述双发射金团簇比率荧光探针制备方法包括以下步骤:
(1)在剧烈搅拌下将5 mL,10 mM,37°C的HAuCl4•4H2O溶液添加到5 mL,50 mg/mL,37°C的牛血清白蛋白溶液中;
(2)2min后,逐滴加入1M 的NaOH溶液,使混合液的pH调节至12;
(3)在37℃水浴条件下持续搅拌12h,最终得到产物BSA-AuNCs。
2.一种采用如权利要求1所述的双发射金团簇比率荧光探针检测食品中强力霉素的方法,其特征在于,具体步骤如下:
(1)用1M NaOH和HCl调节0.01MPBS缓冲液的pH至12;
(2)分别将2mL牛奶、1g鸡蛋和1g蜂蜜放入10mL烧杯中,加入5mL,pH = 12,0.01 M的PBS缓冲液和1mL的5%三氯乙酸,涡旋10min并以10,000rpm离心10min后,取上清液,用1M NaOH将上清液的pH值调节至12,再以10,000rpm离心10min后,用0.22μm滤膜过滤所得的上清液,分别获得牛奶、鸡蛋、蜂蜜待测样品液;
(3)分别将1g猪肉和1g鸡肝放入10mL烧杯中,加入5mL,pH = 12,0.01M的PBS缓冲液,使用细胞粉碎仪搅拌至匀浆,然后加入1 mL的5%三氯乙酸,涡旋10min并以10,000 rpm离心10min,取上清液,用1 M NaOH将上清液的pH值调节至12,再以10,000 rpm离心10min后,用0.22μm滤膜过滤所得的上清液,分别获得猪肉、鸡肝待测样品液;
(4)将不同浓度的强力霉素加入到上述上清液中得到待测液待测样品液中;采用检测强力霉素的方法,扫描荧光光谱,根据检测出的510nm和640nm处的荧光强度的比值,并结合强力霉素检测的标准曲线拟合出线性方程,计算出实际食品样品中的强力霉素浓度。
3.双发射金团簇比率荧光探针在检测强力霉素中的应用, 该荧光探针制备方法如下:
(1)在剧烈搅拌下将5 mL,10 mM,37°C的HAuCl4•4H2O溶液添加到5 mL,50 mg/mL,37°C的牛血清白蛋白溶液中;
(2)2min后,逐滴加入1M 的NaOH溶液,使混合液的pH调节至12;
(3)在37℃水浴条件下持续搅拌12h,最终得到产物BSA-AuNCs。
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