CN114136944B - 一种检测牛奶中卡那霉素残留的荧光适配体传感器的制备方法 - Google Patents
一种检测牛奶中卡那霉素残留的荧光适配体传感器的制备方法 Download PDFInfo
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Abstract
本发明提供了一种检测牛奶中卡那霉素(KAN)残留的荧光适配体传感器制备方法，属于食品安全检测领域。本发明包括特定荧光波长DNA‑银纳米簇(DNA‑AgNCs)合成和荧光传感器构建。特定的DNA模板可以产生红色荧光的DNA‑AgNCs，减少牛奶固有成分产生的相对短的波长荧光信号干扰。利用红色荧光较强穿透能力进一步减少干扰信号的影响。利用DNA‑AgNCs和金纳米颗粒(AuNPs)的荧光共振能量转移(FRET)构建荧光传感方法。由于静电相互作用，DNA被吸附在AuNPs上。DNA‑AgNCs与AuNPs距离的减小会产生FRET，从而导致DNA‑AgNCs荧光被猝灭。当加入KAN时，适配体和KAN可以特异性结合，导致DNA‑AgNCs和AuNPs之间的静电相互作用减弱。DNA‑AgNCs和AuNPs之间的FRET将降低，致使DNA‑AgNCs的荧光信号得到恢复。该传感器对KAN的检测限低至22.6nM，具有令人满意的特异性和灵敏度。
Description
技术领域
本发明提供一种检测牛奶中卡那霉素残留的适配体传感器的制备方法，属于食品安全检测技术领域。
背景技术
卡那霉素(KAN)是用于治疗由革兰氏阳性菌和革兰氏阴性菌引起感染的重要氨基糖苷类抗生素，会引起严重的副作用，会毒害肾脏、损害听力以及神经等，还会引起对药物的过敏反应；
因此，开发灵敏和选择性技术来检测动物源性食品如牛奶中的KAN是至关重要的；目前，毛细管电泳、比色检测、高效液相色谱、免疫分析和酶联免疫等灵敏检测方法已被开发用于检测牛奶残留物中的KAN，但由于周转时间长，步骤复杂，设备昂贵和技术要求高等缺点限制了它们的应用。
发明内容
发明的目的在于需求一种简单、方便、高选择性和灵敏的检测技术检测牛奶中的KAN。
其技术方案为：适配体是人工筛选的单链DNA或RNA序列，结合各种靶标，从小分子到蛋白质甚至细胞，由于它们价格低廉，化学稳定性和特异性优良，合成简单且免疫原性低，适配体对生物传感器的应用具有很大的吸引力。
所述的一种检测牛奶中KAN残留的适配体传感器的制备方法(图1)，其特征在于：通过控制DNA-AgNCs合成的DNA模板和AuNPs的粒径，DNA-AgNCs的荧光发射光谱可以有效地重叠AuNPs的吸收光谱；通过静电相互作用，带负电荷的AuNPs表面可以吸附ssDNA适配体的带正电荷的碱基，从而减少DNA-AgNCs与AuNPs之间的距离；荧光共振能量转移(FRET)是在DNA-AgNCs和AuNPs之间产生；AuNPs可以导致DNA-AgNCs的荧光猝灭，这是由于能量从DNA-AgNCs转移到AuNPs的原因；当加入KAN时，适配体和KAN可以具有高亲和力的特异性结合，导致DNA-AgNCs和AuNPs之间的静电相互作用减弱；这导致了DNA-AgNCs和AuNPs的FRET的减少，以及DNA-AgNCs荧光信号的恢复；最后，荧光强度的变化可以用于检测KAN。
所述的一种检测牛奶中KAN残留的适配体传感器的制备方法，其特征在于：荧光传感器检测牛奶中的兽药残留物，这通常受到复杂的牛奶基质的干扰；特定的DNA模板可以产生可见红色波长范围内的DNA-银纳米簇(DNA-AgNCs)；这将通过避免在相对较短的波长范围内牛奶固有成分产生的荧光信号来减少牛奶的荧光干扰；此外，红色荧光具有相对较高的穿透性，这也可以减少干扰物质的影响。
为达到以上目的，采取以下技术方案实现：银纳米簇的制备，首先，将DNA5、DNA6、DNA7、DNA8和DNA9溶液(100μM)在1XTE缓冲液(pH＝7.8-8.2)中加热至95℃10min，然后冷却至正常温度1小时；然后分别将1XTE缓冲液(pH＝7.8-8.2)溶解的DNA1、DNA2、DNA3、DNA4、DNA5、DNA6、DNA7、DNA8和DNA9溶液(100μL)加入1XTEBuffer(pH＝7.8-8.2)中；然后，分别在这些混合物中加入60μL的硝酸银溶液(1mM)；将混合溶液剧烈摇晃1min，然后在黑暗中与冰水浴孵育30min；随后，分别将60μL新鲜制备的硼氢化钠溶液(1mM)加入到上述混合溶液中；然后将混合溶液剧烈摇晃1min；最后，将得到的溶液在黑暗中存储在4℃处。
为达到以上目的，采取以下技术方案实现：在检测KAN之前，在整个实验过程中，需要将合成的10μM DNA-AgNCs溶液(以DNA浓度为参考)稀释至1μM作为最终浓度；对于KAN检测，将400μL的AuNPs溶液加入到制备的DNA-AgNCs溶液的等体积(400μL)溶液中，反应在正常温度下持续30min；然后，将一系列不同浓度的KAN溶液(每一份为100nM～2500nM)分别加入到DNA-AgNCs/AuNPs溶液中；混合溶液在常温下孵育90min；在激发波长为575nm，狭缝宽度为5nm时，得到了所得混合物的荧光发射光谱。
所述适配体传感器的制备工艺如下：预处理前，牛奶样品分别添加200nM、500nM和800nM；在那之后，预处理是去除蛋白质和脂肪的必要步骤；方法如下：首先，将4mL3个添加的牛奶样品分别加入16mL冰冷甲醇中20min；然后，将混合溶液以12000rpm离心15min，去除沉淀；上清液通过0.22μm的滤膜；滤液在60℃下干燥，然后用氮气干燥剩余溶液；随后，用超纯水重组残基，并再次通过0.22μm的滤膜；最后，将超纯水溶液的体积设置为4mL。
附图说明
图1为适配体传感器的构建过程。
图2为纳米材料的电镜表征。
图3为牛奶的3D荧光光谱。
图4为构建DNA-AgNCs的DNA序列。
图5为DNA序列的表征。
图6试验优化。
图7为试验标准曲线的建立。
图8为特异性的测试。
图9为牛奶实际样品中的KAN测试。
具体实施方式
实施例1：DNA4-AgNCs的透射电镜图像如图2A、B所示，结果显示DNA-AgNCs呈圆形分散；粒径分布如图2C所示，显示AgNCs的平均粒径为3.16nm；为了测量DNA-AgNCs的荧光稳定性，我们研究了100小时内在655nm处的荧光发射强度变化(I/I0)；I0表示DNA-AgNCs荧光强度的初始测量值，我指给定时间的荧光强度；如图2D所示，荧光发射强度在10h～24h达到平台，24h～100h下降，因此，我们决定在实验期间在10h-24h内测量荧光强度；采用TEM和SEM方法对AuNPs的大小和形状进行了表征，图像如图2E、F所示；从图像来看，AuNPs的形状接近球形，平均直径为96nm(图2G)；AuNPs的最大紫外-可见吸收峰在563nm处(图2H)。
实施例2：对传感器的DAN-AgNCs进行荧光表征(图5)，不同DNA模板(A、DNA1；B、DNA2；C、DNA3；D、DNA4；E、DNA5；F、DNA6；G、DNA7；H、DNA8；I、DNA9、)合成的DNA-AgNCs的荧光激发和发射光谱；其最大激发波长分别为609nm、563nm、549nm、575nm、580nm、573nm、577nm、579nm、565nm；最大发射波长分别为681nm、640nm、622nm、655nm、644nm、649nm、646nm、652nm、634nm；插图是DNA-AgNCs在365nm紫外灯下和日光下的照片；以DNA4合成的银纳米簇在3d光谱区域干扰较少，因此选择DNA4为模板较为合适。
实施例3：对试验中的条件进行优化(图6)，基于DNA-AgNCs/AuNPs荧光传感器的卡那霉素检测优化；(A)DNA-AgNCs合成中pH的优化；(B)在浓度为0-16.5pm的DNA-AgNCs在655nm处存在AuNPs，最佳浓度为2.28pM；(C)荧光猝灭的孵育时间(0-50min)，最佳淬灭时间为30min；(D)荧光恢复的孵育时间(0-180min)，最佳恢复时间为90min。
实施例4：通过测量不同浓度的KAN，评估该传感器测定分析物的性能，如图7所示，在0～2500nM范围内，荧光强度随着KAN浓度的增加而逐渐增加，在100nM～1100nM范围内，KAN浓度呈线性相关；线性回归方程如下：I＝0.04625CKAN+143.89332；相关系数(R2)为0.99449；根据3σ/K公式计算出KAN的检测限(LOD)为22.6nM；其中σ为空白解10次重复测量的标准差，k为线性回归线的斜率。
实施例5：为了评价该传感器的特异性，通过与其他抗生素如红霉素(ERY)、氯霉素(CHL)、土霉素(OXY)、四环素(TET)和妥布霉素(TOB)进行比较，确定了基于DNA-AgNCs的荧光适应体的特异性；其他抗生素的浓度为KAN的10倍，如图8所示，I和I0分别表示不同类型抗生素存在和无抗生素存在下的荧光强度；且KAN对荧光适增剂的反应高于其他抗生素；这些结果表明，荧光适应器对检测KAN具有很强的特异性，表明所开发的荧光适应器可用于检测KAN。
实施例6：为了牛奶的荧光分布，三维荧光光谱在图3A、B中显示出4个明显的峰；根据之前的研究，可以推断激发/发射280nm/300-380nm的峰是氨基酸的混合峰；激发/发射峰分别为370nm/530nm/530nm和445nm/535nm的激发/发射峰与核黄素峰有关；激发/发射峰为320nm/410nm，这归因于荧光美拉德产物的峰；激发波长为500nm～600nm，发射波长为600～700nm的荧光强度相对较弱，因此在此区域构建传感器是合适的。
实施例7：为了研究荧光适应剂在牛奶中的适用性，我们将三种不同浓度的卡那霉素加入到实际牛奶中，回收率见图9；可以看出，卡那霉素检测的峰值回收率在107.50％～117.69％之间，相对标准差(RSD)在1.21％～4.77％之间；这些优良的性能表明了我们提出的卡那霉素检测方法在该应用中的可行性。
Claims (3)
1.一种检测牛奶中卡那霉素的荧光适配体传感器的制备方法，其特征在于：构建了一种基于AuNPs和DNA-AgNCs的荧光适配体传感器，用于检测卡那霉素(KAN)；
所述DNA-AgNCs的制备过程如下：
首先，将DNA5、DNA6、DNA7、DNA8和DNA9溶液(100μM)在1XTE缓冲液(pH＝7.8-8.2)中加热至95℃10min，然后冷却至正常温度1小时；然后分别将1XTE缓冲液(pH＝7.8-8.2)溶解的DNA1、DNA2、DNA3、DNA4、DNA5、DNA6、DNA7、DNA8和DNA9溶液(100μL)加入1XTEBuffer(pH＝7.8-8.2)中；然后，分别在这些混合物中加入60μL的硝酸银溶液(1mM)；将混合溶液剧烈摇晃1min，然后在黑暗中与冰水浴孵育30min；随后，分别将60μL新鲜制备的硼氢化钠溶液(1mM)加入到上述混合溶液中；然后将混合溶液剧烈摇晃1min；最后，将得到的溶液在黑暗中存储在4℃处；
其中，DNA1、DNA2、DNA3、DNA4、DNA5、DNA6、DNA7、DNA8和DNA9的DNA序列如下：
DNA1：TGGGGGTTGAGGCTAAGCCGACCCCCCCCCCCC；
DNA2：CCCCCCCCCCCCTGGGGGTTGAGGCTAAGCCGA；
DNA3：TGGGGGTTGAGGCTAAGCCGAAAAAACCCCCCCCCCCC；
DNA4：TGGGGGTTGAGGCTAAGCCGATTTTTCCCCCCCCCCCC；
DNA5：TTTTTCCCCCCCCCCCCAAAAATGGGGGTTGAGGCTAAGCCGA；
DNA6：TGGGGGTTGAGGCTAAGCCGATTTTTCCCCCCCCCCCCAAAAA；
DNA7：CCCCCCCCCCCCAAAAATGGGGGTTGAGGCTAAGCCGATTTTT；
DNA8：AAAAATGGGGGTTGAGGCTAAGCCGATTTTTCCCCCCCCCCCC；
DNA9：CCCCCCCCCCCCAAAAATGGGGGTTGAGGCTAAGCCGATTTTTCCCCCCCCC CCC；
所述AuNPs的平均粒径为96nm；AgNCs的平均粒径为3.16nm。
2.如权利要求1所述的一种检测牛奶中卡那霉素的荧光适配体传感器的制备方法，其特征在于：使用特定的DNA模板可以产生可见红色波长范围内的DNA-银纳米簇(DNA-AgNCs)，避免在相对较短的波长范围内牛奶固有成分产生的荧光信号干扰。
3.如权利要求1所述的一种检测牛奶中卡那霉素的荧光适配体传感器的制备方法，其特征在于：利用AuNPs和DNA-AgNCs进行荧光共振能量转移，在相应的激发波长下通过AuNPs和DNA-AgNCs荧光信号发生变化实现对卡那霉素的检测。
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