CN113916872A - 一种检测黄曲霉毒素b1的比色法 - Google Patents
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Abstract
本发明公开了一种检测黄曲霉毒素B1的比色法,利用AFB1核酸适体与金纳米棒的自组装调控金纳米棒的蚀刻,产生丰富的颜色变化。适配体通过静电作用与金纳米棒自组装,促进了TMB2+对金纳米棒的蚀刻,使得金纳米棒的吸收峰发生蓝移,溶液产生从青色到粉色的多色变化。当黄曲霉毒素B1存在时,其特异性结合核酸适配体导致其构象发生变化,金纳米棒与核酸适体无法进行自组装,其蚀刻作用减弱,吸收峰和溶液颜色无明显变化。本发明方法简便易行,对黄曲霉毒素B1的检测限达8pg/mL,30min内可完成样品检测,可用于对目标物的高灵敏快速检测。
Description
技术领域
本发明涉及一种检测黄曲霉毒素B1的比色法,属于有害物质检测领域。
背景技术
黄曲霉毒素B1(AFB1)主要是由黄曲霉和寄生曲霉产生的次级代谢产物,具有强烈的致癌,致畸和致突变性。其毒素分布广泛,能通过污染农产品,食品和饲料或间接污染动物产品进入食物链,严重影响食品安全,对人类和动物健康构成主要威胁。国内外政府机构为保护消费者健康,制定了一系列严格的AFB1限量标准。
AFB1快速检测技术是真菌毒素污染食品的重要筛查和检验手段,目前的研究和应用主要集中于基于免疫学的的酶联免疫法、侧流层析法以及荧光免疫层析法等技术。但这些技术因其所依赖的抗体存在制备周期长、成本高、易失活等缺陷而受到限制。核酸适体作为一种新型的识别元件,具有可人工合成,与靶标特异性结合,稳定性好,易修饰等独特优点,将其结合多种纳米材料制备传感器用于AFB1的检测研究已逐渐成为研究热点。
比色传感器方法因为操作简便、结果可视化、检测设备简单低廉等优点,在现场检测中具有良好应用前景。
但常规的比色法因显色单一而存在可视化的分辨度低,方法检测灵敏度低等缺陷;金纳米棒因存在强的局域表面等离子体共振(LSPR)效应,在不同长径比下呈现出多种丰富的颜色。
发明内容
本发明的目的在于提供一种检测黄曲霉毒素B1的比色法,利用核酸适体作为识别元件,通过核酸适体与金纳米棒自组装调控金棒的蚀刻,实现对黄曲霉毒素B1高灵敏的快速检测。该方法操作简便,响应速度快,并显著提高检测的灵敏度和可视化的分辨度。
为了解决上述技术问题,本发明是通过以下技术方案实现的:步骤如下:
步骤一,配制金纳米棒和十六烷基三甲基溴化铵(CTAB)的混合液;
步骤二,将一定量的黄曲霉毒素B1适体加入到步骤一中制备的金纳米棒混合液中,在室温下孵育,形成AFB1和金纳米棒的自组装体;
步骤三,将一定量的辣根过氧化物酶HRP与TMB显色液混合孵育,然后加入一定量的HCl溶液终止反应,制备出蚀刻试剂;
步骤四,分别将不同浓度的黄曲霉毒素B1标准品加入到步骤二制备的自组装体中进行孵育,然后加入一定量步骤三制备的蚀刻试剂,得到显色溶液;蚀刻一定时间后观察所述显色溶液的颜色变化,并利用微量紫外分光光度计测量金纳米棒的等离子体共振吸收峰λ。
进一步地:步骤一所述的金纳米棒的粒径为50-60nm、表面等离子共振峰(SPR)在673-675nm、溶液颜色为青色。
进一步地:步骤一所述十六烷基三甲基溴化铵(CTAB)的使用浓度为0.1M,金纳米棒与十六烷基三甲基溴化铵(CTAB)的混合比例优选1:4,混合温度优选40-60℃。
进一步地:步骤二中黄曲霉毒素B1适体为5'-GTT GGG CAC GTG TTG TCT CTC TGTGTC TCG TGC CCT TCG CTA GGC CCA CA-3',适体浓度优选0.5μM,孵育时间优选5-10min。
进一步地:步骤三中所述的HRP的浓度为0.01~0.05μg/mL,显色液采用单组分TMB显色液,反应时间优选5min,HCl浓度优选2mol/L。
进一步地:步骤四中所述的蚀刻反应时间优选5min。
进一步地:步骤四中所述的溶液颜色变化为粉色-紫色-蓝色-青色等多种颜色。
进一步地:所述的黄曲霉毒素B1的可视化定性分析根据溶液颜色的变化判定。
进一步地:所述的黄曲霉毒素B1含量的测定是通过建立黄曲霉毒素B1的浓度梯度与金纳米棒的等离子体共振吸收峰的位移变化之间的线性关系获得的。
与现有技术相比,本发明的优点是:
本发明利用核酸适体与金纳米棒自组装作用调控金纳米棒的蚀刻,使检测体系呈现多颜色变化,对检测溶液中黄曲霉毒素B1的最低检测限为8pg/mL,明显提高检测的灵敏度和可视化的分辨度。同时该方法操作简便,试剂耗量少,信号响应速度快,30min内即可完成样品的检测,满足现场检测的需求。
附图说明
为了易于说明,本发明由下述的具体实施及附图作以详细描述。
图1为本发明为比色法的原理图;
图2为本发明黄曲霉毒素B1含量的定性和定量分析图;
图3为本发明不同浓度AFB1下金纳米棒蚀刻后的透射电镜图;
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
在本发明的描述中,需要理解的是,术语“中心”、“纵向”、“横向”、“长度”、“宽度”、“厚度”、“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系,仅是为了便于描述本发明和简化描述,而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作,因此不能理解为对本发明的限制。
此外,术语“第一”、“第二”仅用于描述目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此,限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中,“多个”的含义是至少两个,例如两个,三个等,除非另有明确具体的限定。
在本发明中,除非另有明确的规定和限定,术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解,例如,可以是固定连接,也可以是可拆卸连接,或成一体;可以是机械连接,也可以是电连接或彼此可通讯;可以是直接相连,也可以通过中间媒介间接相连,可以是两个元件内部的连通或两个元件的相互作用关系,除非另有明确的限定。对于本领域的普通技术人员而言,可以根据具体情况理解上述术语在本发明中的具体含义。
参阅图1-3为本发明一种检测黄曲霉毒素B1的比色法的一种实施例:
步骤一,配制金纳米棒和十六烷基三甲基溴化铵(CTAB)的混合液;
步骤二,将一定量的黄曲霉毒素B1适体加入到步骤一中制备的金纳米棒混合液中,在室温下孵育,形成AFB1和金纳米棒的自组装体;
步骤三,将一定量的辣根过氧化物酶HRP与TMB显色液混合孵育,然后加入一定量的HCl溶液终止反应,制备出蚀刻试剂;
步骤四,分别将不同浓度的黄曲霉毒素B1标准品加入到步骤二制备的自组装体中进行孵育,然后加入一定量步骤三制备的蚀刻试剂,得到显色溶液;蚀刻一定时间后观察所述显色溶液的颜色变化,并利用微量紫外分光光度计测量金纳米棒的等离子体共振吸收峰λ。
具体操作步骤分为四个阶段
首先第一阶段为配制金纳米棒混合液;
采用金种子液生长法制备金纳米棒(AuNRs);
分为以下四步:
(1)、配置金种子液:将300μL HAuCl4·3H2O(0.01M)添加到9.8mL的CTAB(0.1M)溶液中。在30s剧烈搅拌下,加入600μL新鲜制备的冰冷NaBH4(0.01M),再混合搅拌2min。最终溶液在28℃水浴中保持2h后使用。
(2)配置生长溶液:在磁力搅拌下,将600μL的HAuCl4·3H2O(0.05M)水溶液和50mL的CTAB(0.1M)在锥形瓶中混合;将150μL AgNO3(0.01M)加入,400rpm搅拌下15min,加入500μL AA(0.1M),剧烈搅拌30s直到溶液变成无色;制备后立即使用。
(3)取步骤一中制备的金种子液200μL,在室温下加入步骤二制备的生长溶液中,混合剧烈搅拌20s,然后将锥形瓶在28℃下静置过夜,将获得的金纳米棒在8500rpm下离心15min去除过量的CTAB后重新分散在无菌超纯水中,置于4℃下避光保存备用。
(4)将所制备的金纳米棒经透射电镜和紫外-可见光谱分析进行表征,得到的粒径为50-60nm,其特征表面等离子共振峰(SPR)在673-675nm;溶液颜色呈青色。
配制金纳米棒和十六烷基三甲基溴化铵(CTAB)混合液的具体过程:
将步骤三中制备的AuNRs与CTAB按AuNRs:CTAB=1:4的比例现配现用混合;
CTAB在50-60℃下加热溶解,与金纳米棒混合时的温度控制在40-60℃,其中浓度优选0.1M。
第二阶段为黄曲霉毒素B1比色检测方法的构建
由以下三步完成
(1)对于金纳米棒与核酸适体的自组装体的制备过程
将30μL的黄曲霉毒素B1适体加入到100μL步骤二中制备的金纳米棒混合液中,室温下孵育5-10min,形成AFB1和金纳米棒的自组装体。
并且采用的黄曲霉毒素B1适体序列为:5'-GTT GGG CAC GTG TTG TCT CTC TGTGTC TCG TGC CCT TCG CTA GGC CCA CA-3';黄曲霉毒素B1适体浓度优选0.5μM,孵育时间优选5min。
(2)对于步骤三中金纳米棒蚀刻试剂的制备
将50μL的HRP与10μL的TMB显色液混合孵育,然后加入一10μL HCl溶液终止反应,制得TMB2+蚀刻试剂。
这里的HRP的浓度为0.01~0.05μg/mL,显色液为单组分TMB显色液,HCl浓度为2M;混合孵育时间控制在5min之内;反应过程中无需额外加入H2O2。
(3)黄曲霉毒素B1比色检测方法的构建
分别将30μL不同浓度的黄曲霉毒素B1标准品加入到步骤一制备的自组装体中,孵育5-10min,然后加入50μL步骤二中制得的试剂进行蚀刻反应,观察溶液的颜色变化,并利用紫外分光光度计测量金纳米棒的等离子体共振吸收峰λ。
黄曲霉毒素B1标准品的浓度依次为0、0.01、0.05、0.1、0.2、0.5、1、5、10、100ng/mL;蚀刻反应时间优选5min。
黄曲霉毒素B1含量的定性分析是以观察到不同黄曲霉毒素B1浓度下反应溶液颜色变化为粉色-紫色-蓝色-青色来确定的。
黄曲霉毒素B1的定量分析以黄曲霉毒素B1的浓度为横坐标,反应前后金纳米棒的共振峰位移差△λ为纵坐标作图,得到在0.01ng/mL~0.5ng/mL的浓度范围内,△λ与黄曲霉毒素B1浓度之间呈现线性相关(R2=0.9903),并得到黄曲霉毒素B1的最低检测限为8pg/mL。
第三阶段为检测方法的特异性研究
配制10ng/mL的赭曲霉毒素、伏马菌素、呕吐毒素、玉米赤酶烯酮单一溶液及其混合液作为干扰物质,同时配制0.5ng/mL的黄曲霉毒素B1作为对照,分别采用实施例2方法进行检测。
结果显示,高浓度的干扰物质的加入并没有引起金纳米棒蚀刻颜色和共振峰位置的明显变化,表明方法对黄曲霉毒素B1的检测具有良好的特异性。
第四阶段为样品检测
在阴性玉米油和花生碎中分别添加不同浓度的黄曲霉毒素B1,使其终浓度分别为0、0.05、0.1、0.5ng/mL,样品提取后采用黄曲霉毒素B1比色检测方法进行检测,获得样品中黄曲霉毒素B1的添加回收率在93%~117%之间,标准偏差为1.3%-8.4%,说明此方法可以用于实际样本中黄曲霉毒素B1的检测。
以上所述仅为本发明的具体实施例,但本发明的技术特征并不局限于此,任何本领域的技术人员在本发明的领域内,所作的变化或修饰皆涵盖在本发明的专利范围之中。
Claims (8)
1.一种检测黄曲霉毒素B1的比色法,其特征在于:步骤如下:
步骤一,配制金纳米棒和十六烷基三甲基溴化铵CTAB的混合液;
步骤二,将黄曲霉毒素B1适体加入到步骤一中制备的金纳米棒混合液中,在室温下孵育,形成黄曲霉毒素B1和金纳米棒的自组装体;
步骤三,将辣根过氧化物酶HRP与TMB显色液混合孵育,然后加入一定量的氯化氢HCl溶液终止反应,制备出蚀刻试剂;
步骤四,分别将不同浓度的黄曲霉毒素B1标准品加入到步骤二制备的自组装体中进行孵育,然后加入步骤三制备的蚀刻试剂,得到显色溶液;蚀刻后观察所述显色溶液的颜色变化,并利用微量紫外分光光度计测量金纳米棒的等离子体共振吸收峰λ。
2.根据权利要求1所述的检测黄曲霉毒素B1的比色法,其特征在于:步骤一中所述的金纳米棒的粒径为50-60nm、表面等离子共振峰SPR在673-675nm、溶液颜色为青色。
3.根据权利要求2所述的检测黄曲霉毒素B1的比色法,其特征在于:步骤一中所述的十六烷基三甲基溴化铵(CTAB)使用浓度为0.1M,金纳米棒与十六烷基三甲基溴化铵CTAB的混合比例1:4,混合温度优选40-60℃。
4.根据权利要求3所述的检测黄曲霉毒素B1的比色法,其特征在于:步骤二中黄曲霉毒素B1适体为5'-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCACA-3',适体浓度优选0.5μM,孵育时间优选5-10min。
5.根据权利要求4所述的检测黄曲霉毒素B1的比色法,其特征在于:步骤三中所述的辣根过氧化物酶HRP的浓度为0.01~0.05μg/mL,TMB显色液采用单组分TMB显色液,反应时间优选5min,氯化氢HCl浓度优选2mol/L。
6.根据权利要求5所述的检测黄曲霉毒素B1的比色法,其特征在于:步骤四中所述的蚀刻反应时间为5min。
7.根据权利要求6所述的检测黄曲霉毒素B1的比色法,其特征在于:根据观察到所述显色溶液颜色的变化对所述黄曲霉毒素B1进行定性分析。
8.根据权利要求7所述的检测黄曲霉毒素B1的比色法,其特征在于:建立黄曲霉毒素B1的浓度梯度与金纳米棒的等离子体共振吸收峰λ的位移变化之间的线性图。
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