CN113617395B - 一种用于食品抗氧化活性检测的纳米酶及其制备方法 - Google Patents
一种用于食品抗氧化活性检测的纳米酶及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于食品抗氧化活性检测的纳米酶及其制备方法,属于纳米生物技术领域。本发明纳米酶的反应原料包括六氰基铁酸钾、柠檬酸、四氯金酸钠(III)和硫酸铜,制备方法为:将六氰基铁酸钾和柠檬酸溶解于水中,然后依次加入四氯金酸钠(III)和硫酸铜,反应得到纳米酶。本发明的纳米酶为金掺杂的纳米酶Au@Cu‑HCF,既能够用于检测氢原子转移类(HAT)抗氧化物质,又能检测单电子转移类(SET)抗氧化物质,可以用于衡量食品中总的抗氧化能力,在植物源性食品中TAC检测方面具有良好的实际应用前景。
Description
技术领域
本发明涉及纳米生物技术领域,特别是涉及一种用于食品抗氧化活性检测的纳米酶及其制备方法。
背景技术
天然抗氧化物质具有降低或清除自由基的能力,它们不仅可以保护天然食物不受氧化损害,而且还能中和人体内的氧化应激,维持人体氧化还原动态平衡。食品的天然抗氧化能力分析和鉴定在营养健康领域受到了极大的关注。
目前,包括气相色谱(GC)和液相色谱(LC)在内的传统仪器分析方法被广泛用作鉴定抗氧化物质的有效手段,但是此类方法耗时长、对操作人员专业要求高、仪器耗材价格昂贵,并且不能够实现实际样品的便携分析。一些快速比色法可以有效克服目前抗氧化能力分析存在的技术限制,其中包括总自由基捕捉法(TRAP)、2,2’-联氮-双(3-乙基苯并噻唑啉-6-磺酸盐)自由基阳离子(ABTS·+)、2,2-二苯基-1-苦基肼自由基(DPPH·)和氧自由基吸收能力测定(ORAC)等方法,可用于比色测定抗氧化物质的抗氧化活性,该方法快速便捷,可被开发为快速检测试剂盒,用于现场的高通量快速分析。
由于食品基质成分复杂,抗氧化能力的检测结果可能因使用的分析方法不同而不同,Miller等人于1993年提出ABTS可在适当氧化剂的作用下被氧化成绿色ABTS·+,而抗氧化物质的存在会抑制ABTS·+的产生。因为抗氧化剂的反应涉及更快的反应动力学,所以ABTS方法被认为是识别抗氧化能力最敏感的技术之一。Floegel等人的一项研究表明,ABTS方法能够更准确地估计饮食中富含抗氧化物质的水果、蔬菜和饮料的抗氧化能力,特别是那些含有亲水性、亲脂性和高色素化合物的食物。
过氧化物酶作为一种天然酶,具有催化效率高、生物相容性好和底物特异性高等特点,在ABTS分析中有着重要的应用。然而,天然过氧化物酶的生产涉及几个缺点,例如成本高、耗时长、纯化方式较繁琐等。此外,天然酶的催化活性易受环境条件的影响,如极端的酸碱度和温度会导致酶显著变性,严重限制了ABTS方法的大规模应用。
发明内容
本发明的目的是提供一种用于食品抗氧化活性检测的纳米酶及其制备方法,以解决上述现有技术存在的问题,使得该酶具有优异的类过氧化物酶活性,从而替代天然过氧化物酶进行食品抗氧化活性检测。
为实现上述目的,本发明提供了如下方案:
本发明的技术方案之一是提供一种纳米酶,所述纳米酶是以六氰基铁酸钾、柠檬酸、四氯金酸钠(III)和硫酸铜为反应原料制备得到;所述六氰基铁酸钾、柠檬酸、四氯金酸钠(III)和硫酸铜的质量比为18.4:150:1:20。
本发明的技术方案之二是提供上述纳米酶的制备方法,包括以下步骤:
将所述六氰基铁酸钾和柠檬酸溶解于去离子水中,然后依次加入所述四氯金酸钠(III)和硫酸铜,反应得到所述纳米酶。
进一步地,所述六氰基铁酸钾和柠檬酸按照固液比为16-17mg:1mL溶解于水中;所述硫酸铜以水溶液的形式加入。
进一步地,所述四氯金酸钠(III)在搅拌状态下加入。
本发明的技术方案之三是提供上述纳米酶在食品抗氧化活性检测中的应用。
进一步地,所述食品抗氧化活性检测的底物为ABTS、TMB或鲁米诺。
进一步地,所述食品抗氧化活性检测中的抗氧化物质为氢原子转移类抗氧化物质和/或单电子转移类抗氧化物质。
进一步地,所述氢原子转移类抗氧化物质为谷胱甘肽或半胱氨酸;所述单电子转移类抗氧化物质为维生素C或维生素E。
所述维生素E为水溶性维生素E,即Trolox。
进一步地,所述食品为植物源食品。
本发明公开了以下技术效果:
本发明制备得到了一种新型的金掺杂纳米酶(Au@Cu-HCF),该纳米酶具有良好的分散性、高稳定性和优异的类过氧化物酶活性,该Au@Cu-HCF纳米酶可以催化氧化还原反应,导致OH·自由基的形成,在抗氧化物质的存在下,底物(ABTS、TMB和鲁米诺)与OH·的相互作用被抑制,导致底物的颜色或化学发光强度随抗氧剂浓度的变化而变化,可以以此来进行抗氧化物质的定量分析。
本发明的Au@Cu-HCF纳米酶既能够用于检测氢原子转移类(HAT)抗氧化物质(例如谷胱甘肽、半胱氨酸),又能检测单电子转移类(SET)抗氧化物质(例如维生素C、维生素E),因此可以用于衡量食品中总的抗氧化能力。通过对莲藕和水果饮料中总抗氧化能力的检测对比研究,证明了该Au@Cu-HCF纳米酶在植物源性食品中TAC检测方面具有良好的实际应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为实施例1制备的Au@Cu-HCF纳米酶的TEM图;
图2为实施例1制备的Au@Cu-HCF纳米酶的元素组成;
图3为实施例1制备的Au@Cu-HCF纳米酶的类过氧化物酶活性检测结果;
图4为以ABTS为显色底物,实施例1中Au@Cu-HCF纳米酶抗氧化活性比色检测结果;其中,图4A为实施例1的Au@Cu-HCF纳米酶用于维生素C检测,图4B为实施例1的Au@Cu-HCF纳米酶用于Trolox检测,图4C为实施例1的Au@Cu-HCF纳米酶用于谷胱甘肽检测,图4D为实施例1的Au@Cu-HCF纳米酶用于半胱氨酸检测;
图5为以TMB为显色底物,实施例1中Au@Cu-HCF纳米酶抗氧化活性比色检测结果;其中,图5A为实施例1的Au@Cu-HCF纳米酶用于维生素C检测,图5B为实施例1的Au@Cu-HCF纳米酶用于Trolox检测,图5C为实施例1的Au@Cu-HCF纳米酶用于谷胱甘肽检测,图5D为实施例1的Au@Cu-HCF纳米酶用于半胱氨酸检测;
图6为以鲁米诺为底物,实施例1中Au@Cu-HCF纳米酶抗氧化活性化学发光检测结果;其中,图6A为实施例1的Au@Cu-HCF纳米酶用于维生素C检测,图6B为实施例1的Au@Cu-HCF纳米酶用于Trolox检测,图6C为实施例1的Au@Cu-HCF纳米酶用于谷胱甘肽检测,图6D为实施例1的Au@Cu-HCF纳米酶用于半胱氨酸检测。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1Au@Cu-HCF纳米酶的合成
将18.4mg六氰基铁酸钾和0.15g柠檬酸溶解于10mL去离子水中,混合均匀。然后,在剧烈搅拌下注入0.2μL的四氯金酸钠(III)(1mg),继续搅拌2min,将无水硫酸铜溶液(4mL,5mg/mL)逐滴滴加到上述混合物中,直至呈酒红色,即得Au@Cu-HCF纳米酶溶液,将该纳米酶溶液以17000转/分钟离心15分钟,分离即得Au@Cu-HCF纳米酶。
采用一步自组装法制备得到了金掺杂纳米酶粒子,透射电子显微镜(TEM)分析表明,Au@Cu-HCF纳米酶粒子形态特征为直径为15nm左右的圆球(图1)。能量色散X射线光谱结果表明,Au@Cu-HCF纳米酶由Cu、Au、Fe、N和C组成(图2)。
实施例2制备PEG-SH修饰的Au@Cu-HCF纳米酶
将实施例1制备得到的酒红色Au@Cu-HCF纳米酶溶液在避光条件下连续搅拌12h后,加入50mg COOH-PEG-SH,继续搅拌12h,将得到的纳米酶溶液以17000转/分钟离心15分钟,分离即得PEG-SH修饰的Au@Cu-HCF纳米酶。
本实施例利用金和硫之间的强作用力,采用COOH-PEG-SH包覆纳米颗粒,对其进行PEG-SH修饰,以进一步提高纳米颗粒的稳定性和生物相容性。
实施例3Au@Cu-HCF纳米酶过氧化物酶活性的测定
取实施例1制备的Au@Cu-HCF纳米酶溶液40μL,与40μL去离子水混合均匀后加入100μL的TMB缓冲液(TMB缓冲液A+B,缓冲液A:取无水醋酸钠8.2g、β-环糊精2.5g和过氧化氢脲0.4g溶于1L水;缓冲液B:取100mg TMB溶于10mL DMSO)进行反应,待显色15min反应结束后加入100μL的H2SO4终止反应,用紫外-可见分光光度计在400nm-800nm范围内记录TMB体系的紫外-可见吸收光谱。
将50μL实施例1制备的Au@Cu-HCF纳米酶溶液与50μL的去离子水混合均匀,再加入50μL的ABTS缓冲液(含有4mL PBS,200μL 3%的H2O2和10mg ABTS)进行显色,反应10min后,用紫外-可见分光光度计在400nm-800nm范围内记录ABTS体系的紫外-可见吸收光谱。
对于TMB底物,在H2O2存在时,通过紫外-可见分光光度计检测到反应生成的氧化产物。在λmax 650nm处测量了体系中TMB从无色溶液到深蓝色(TMB+)的转化,而在H2SO4终止后,体系黄色产物在450nm具有最高吸收峰,见图3;
对于ABTS底物,在H2O2存在下,体系颜色由无色溶液变为深绿色,在414nm处有特征吸收峰,见图3。
上述结果表明,Au@Cu-HCF纳米酶具有过氧化物酶活性,在Au@Cu-HCF纳米酶的存在下,ABTS和TMB有氧化产物的形成,也揭示了Au@Cu-HCF纳米酶对TMB和ABTS具有很好的催化性能,并发生显著的比色反应。
实施例4基于Au@Cu-HCF的抗氧化剂比色检测
选择氢原子转移类(HAT)和单电子转移类(SET)抗氧化物质进行测试。用水配制抗坏血酸(VC)、Trolox(水溶性VE)、谷胱甘肽(GSH)和半胱氨酸(Cys)的标准品。
对于基于TMB的比色生物传感系统,将20μL标品和20μL实施例1制备的Au@Cu-HCF纳米酶探针溶液与50μL TMB缓冲液(TMB缓冲液A+B)混合,在室温下反应15min后,加入50μL的终止缓冲液以终止反应,然后利用酶标仪在450nm处测量吸光度。
在基于ABTS的比色传感系统中,将ABTS(10mg)和200μL的3%H2O2加入到4mL磷酸盐-柠檬酸缓冲液(pH5.0)中,以配置ABTS工作液。在比色检测过程中,将标准品(50μL)与实施例1的Au@Cu-HCF纳米酶探针溶液(50μL)混合,然后加入50μL的ABTS工作液,在室温下反应,反应10min后,使用酶标仪在414nm处测量吸光度。
在基于鲁米诺化学发光传感系统中,将17.7mg鲁米诺和40mg NaOH溶解在100mL去离子水中,在暗室中放置4天,制备鲁米诺储存液。将57μL的30%双氧水稀释到5mL去离子水中,然后稀释10倍,在检测过程中,在含有20μL实施例1Au@Cu-HCF纳米酶探针溶液的96孔板中加入40μL/孔的标准品,然后将等量的鲁米诺储存液和H2O2储存液混合并加入上述96孔板中,立即在暗色盒中成像。
基于Au@Cu-HCF纳米酶的比色检测机理是:TMB或ABTS底物和抗氧化剂(AH)与OH·自由基在纳米酶表面发生竞争反应。它们产生具有颜色的TMB或ABTS氧化产物,这些氧化产物的吸光度信号与AH浓度成反比。
对于ABTS比色法,反应温度、pH值、实施例1制得的Au@Cu-HCF纳米酶溶液稀释倍数和反应时间分别设定为25℃、7、32倍和10min。对于TMB比色法,反应条件设定为25℃、7、16倍和15min。
基于Au@Cu-HCF纳米酶的传感系统测定HAT和SET的抗氧化剂分析,包括Trolox、半胱氨酸、维生素C和谷胱甘肽。图4显示了光密度值随不同浓度的目标物的变化而变化,利用ABTS·+在414nm处相应的光密度值来定量评估信号对目标抗氧化剂浓度的响应。如图4所示,光密度值与Trolox、半胱氨酸、维生素C和谷胱甘肽的浓度呈线性关系。
在基于ABTS的比色策略中,维生素C检测方程为y=1.5303x+0.0004,R2=0.999,范围为0.007~0.25mM(图4A)。Trolox的检测方程为y=1.5262x-0.0002,R2=0.993,检测范围为0.007~0.25mM(图4B)。谷胱甘肽检测的检测方程为y=0.1674x+0.0054,R2=0.997,检测范围为0.031~2mM(图4C)。半胱氨酸的检测方程为y=0.9851x+0.0003,R2=0.998,检测范围为0.007~0.25mM(图4D)。上述分析的维生素C、Trolox、谷胱甘肽和半胱氨酸实际检测限分别为0.007mM、0.007mM、0.031mM和0.007mM。
对于基于TMB的分析,维生素C的方程为y=0.9954-0.9897/(1+(x/0.3652)1.0182),R2=0.999,检测范围为0.008~0.568mM(图5A)。Trolox的检测方程为y=0.6994-0.6892e(-x/0.2134),R2=0.996,检测范围为0.007~0.5mM(图5B)。谷胱甘肽的检测方程为y=0.5432+0.6074e(-x/0.0890),R2=0.995,检测范围为0.012~3.3mM(图5C)。半胱氨酸的检测方程为y=0.6698-0.6723/(1+(x/0.1654)1.0374),R2=0.998,范围为0.012~0.412mM(图5D)。
以上结果表明,Au@Cu-HCF纳米酶具有模拟过氧化物酶的性质,能够用于HAT和SET两种类型的抗氧化物质传感分析,基于Au@Cu-HCF纳米酶的ABTS和TMB分析可用作检测抗氧化剂总抗氧化能力的传感策略。
抗氧化物质可以清除自由基抑制化学发光,鲁米诺-H2O2的氧化化学发光反应会减弱,发光强度会降低。在0.2-0.6mM范围内,化学发光强度的差值与维生素C浓度呈线性关系,线性回归方程为y=-72.4819+72.6528/(1+(x/27.6980)1.672),R2为0.999(图6A)。然后将其进一步用于Trolox的检测,得到了相似的结果,在0.05-0.4mM范围内,化学发光强度与Trolox浓度成正比(R2=0.993)(图6B),线性方程为y=-526.2718+526.4566/(1+(x/26.3453)1.9461)。对于谷胱甘肽的检测,随着谷胱甘肽浓度从0.025mM增加到0.4mM,化学发光强度逐渐增强,这是因为GSH分子中的巯基可以增强鲁米诺-H2O2反应体系的化学发光强度(图6C),化学发光强度与其浓度呈良好的线性关系(y=15.1664-15.1666/(1+(x/3887.5170)0.5005),R2=0.995)。研究并未得到针对半胱氨酸的检测曲线(图6D)。
结果表明,将基于Au@Cu-HCF的化学发光传感策略作为总抗氧化物质检测方法是困难的,这是因为当用于SET和HAT两种类型的抗氧化物质检测时存在相反的线性趋势。
实施例5基于Au@Cu-HCF纳米酶的比色策略用于实际样品分析
莲藕含有丰富的抗氧化物质,如维生素C、维生素E、酚类(包括类黄酮)和类胡萝卜素,被消费者评价为“抗氧化功能性”食品之一。本实施例制备了莲藕提取物,并将其直接稀释,通过上述比色传感方法检测总抗氧化能力。
在目前研制的商用ABTS试剂盒中,大都通过测量ABTS·+的吸光度,计算出样品的总抗氧化能力。Trolox是一种维生素E类似物,具有与维生素E相似的抗氧化能力,一直被用作其他抗氧化剂总抗氧化能力的参考。例如,Trolox的总抗氧化能力为1,其他样品在相同浓度下的总抗氧化能力表示为其抗氧化能力与Trolox相比的倍数。因此,为了与商品化的ABTS试剂盒进行比较,本实施例基于Au@Cu-HCF的比色策略以Trolox为标准物质构建标曲。
如表1所示,以Au@Cu-HCF为基础的ABTS比色法测得莲藕的总抗氧化能力为4.78;基于Au@Cu-HCF的TMB比色策略,莲藕的总抗氧化能力被检测为4.65。基于Au@Cu-HCF的传感策略得到的所有结果与商用ABTS试剂盒获得的结果(4.55)一致。同时,基于Au@Cu-HCF的传感策略的标准偏差(ABTS检测为0.16,TMB检测为0.08)表明,与商业ABTS试剂盒获得的结果相比,所开发的比色策略具有可接受的准确性和重复性。此外,基于智能手机的传感模式具有便携、高通量和快速的明显优势。
本实施例还利用所提出的传感策略对一种商品化柑橘汁和一种功能性柠檬饮料的总抗氧化能力进行了测定,并与商品化ABTS试剂盒进行了比较。对柑橘汁,ABTS法和TMB法测得的总抗氧化能力分别为6.11和6.02。对于柠檬饮料,ABTS法、TMB法测得的总抗氧化能力分别为1.04、1.08。这些结果都与商业ABTS试剂盒得到的结果一致(柑橘汁6.27,柠檬功能饮料1.11)。说明所建立的方法在测定这两种植物源性饮料的总抗氧化能力时,与常规试剂盒相比,具有可接受的准确度和再现性。
表1
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (6)
1.一种纳米酶,其特征在于,所述纳米酶以六氰基铁酸钾、柠檬酸、四氯金酸钠(III)和硫酸铜为反应原料制备得到;所述六氰基铁酸钾、柠檬酸、四氯金酸钠(III)和硫酸铜的质量比为18.4:150:1:20;
所述纳米酶的制备方法包括以下步骤:
将所述六氰基铁酸钾和柠檬酸溶解于去离子水中,然后依次加入所述四氯金酸钠(III)和硫酸铜,反应得到所述纳米酶;
所述六氰基铁酸钾和柠檬酸按照固液比为16-17mg:1mL溶解于水中。
2.根据权利要求1所述的纳米酶,其特征在于,所述硫酸铜以水溶液的形式加入。
3.根据权利要求1所述的纳米酶,其特征在于,所述四氯金酸钠(III)在搅拌状态下加入。
4.如权利要求1所述的纳米酶在食品抗氧化活性检测中的应用,其特征在于,所述食品抗氧化活性检测的底物为ABTS、TMB或鲁米诺;
所述食品抗氧化活性检测中的抗氧化物质为氢原子转移类抗氧化物质和/或单电子转移类抗氧化物质。
5.根据权利要求4所述的应用,其特征在于,所述氢原子转移类抗氧化物质和单电子转移类抗氧化物质为谷胱甘肽、半胱氨酸、维生素C、维生素E。
6.根据权利要求4所述的应用,其特征在于,所述食品为植物源食品。
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