CN113984728A - 一种用于单增李斯特菌快速检测的荧光生物传感器构建方法 - Google Patents
一种用于单增李斯特菌快速检测的荧光生物传感器构建方法 Download PDFInfo
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Abstract
本发明涉及一种基于双位点识别策略和荧光内滤效应构建的荧光生物传感器。通过构建一种上转换纳米粒子(UCNPs)介导的荧光生物传感器,并用于单增李斯特菌的快速、灵敏检测。将万古霉素(Van)作为第一识别分子,制备MNPs‑Van磁纳米探针;以生物素化的核酸适配体(aptamer)作为第二识别分子,可与单增李斯特菌细胞壁上的内化蛋白A结合。利用两种识别分子分别靶标目标菌上的不同位点,从而特异性捕获目标菌,形成三明治型复合物(MNPs‑Van/单增李斯特菌/aptamer)。通过辣根过氧化物酶标记物(HRP‑SA),引入HRP‑TMB酶催化系统而产生蓝色物质,结合UCNPs的荧光内滤效应,导致UCNPs荧光强度的降低,从而实现对单增李斯特菌的定量检测。
Description
技术领域
本发明属于食品安全检测分析技术领域。具体涉及一种基于双位点识别策略和荧光内滤 效应介导的荧光生物传感器的构建,进而实现对食品中单增李斯特菌的快速检测新方法。
背景技术
“民以食为天,食以安为先”,食品安全是老百姓最关心的民生问题之一。而食源性致病微 生物引起的食源性疾病是重要的公共卫生问题,也是各国食品安全的重点监测对象。每年都 出现食物中毒、食品卫生事件的报道,尤以致病微生物为主,极不利于国家健康有序的发展。 从各国食源性疾病爆发的数据分析来看,不论美国、欧盟、还是中国,引起疾病爆发事件的 致病因素主要是微生物性因素。常见的食源性致病微生物有沙门氏菌、大肠杆菌、葡萄球菌、 副溶血性弧菌、单增李斯特菌、黄曲霉菌及病毒、禽流感病毒、口蹄疫病毒等。根据GB29921- 2013所规定,一般除了金黄色葡萄球菌和副溶血性弧菌在允许范围内可以检出,食品中不得 检出致病菌是非常重要的食品卫生质量指标。而食品产业链长,易污染环节多,食品营养丰 富,适合致病菌生长,因此需要多种不同的方法对食品中致病菌进行检测,这对食品安全非 常重要。
单增李斯特菌,作为世界四大食源性致病菌之一,也是李斯特菌中唯一一种人畜共患病原 菌,其可以使人类患上类流感疾病和严重的并发症,如脑膜炎、败血症,甚至孕妇的自然流 产等,致死率高达30%。此外,单增李斯特菌分布广泛,能在高盐、低温、酸碱性等多种环 境中生存和繁殖,俗称“冰箱杀手”;常污染的食物有肉制品、奶制品等全产业链和即时食品、 冷冻冷藏食品,因此,建立一种快速、灵敏、可靠地检测单增李斯特菌的方法是非常重要的。
目前,单增李斯特菌的常见检测方法主要有:传统的微生物培养法、基于抗原-抗体特异性 识别的免疫检测方法和基于碱基互补配对原则的核酸扩增检测方法、以及生物传感器等。微 生物培养法不需要使用特殊的仪器设备,检测灵敏度高,一直是细菌检测的金标准;但是该 过程繁琐复杂,周期较长(4-7天),易出现错检漏检,且需要专门的实验人员完成检测过程, 这限制了该法的实际应用,无法满足食品生产和现场分析的快速检测。免疫分析主要包括酶 联免疫吸附测定法(ELISA)和胶体金免疫层析试纸条方法。ELISA具有操作相对简单、高通量 等优势;胶体金免疫层析试纸条具有操作简单、反应速度快、适合现场快速检测等优点。但 是两者灵敏度一般,且高特异性有赖于高质量的抗体,而抗体制备复杂,保存条件严苛,货 架期较短,成本较高,不利于该检测技术进一步推广使用。利用核酸扩增技术将致病菌的检 测提升到分子水平,可实现低浓度目标菌的有效检测。常见的核酸扩增方法比如qPCR、mPCR、 环介导等温扩增法等,但是其操作复杂,需要专业人员和专门仪器,而且引物、探针设计要 求高、难度大,易出现假阳性结果。
近年来,国内外研究者们一直致力于探索适合现场分析的食源性致病菌检测方法,其中生 物传感器成为研究热点。生物传感器具有分析速度快、成本低、便携性好等优点,容易实现
发明内容
本发明的目的是提供一种基于双位点识别策略和荧光内滤效应的荧光生物传感器的制备 方法及其应用,可以实现单增李斯特菌的快速非免疫检测。该传感器具有较宽的线性范围, 且灵敏度较高、检测速度较快。
为了实现上述目的,本发明的技术方案包括:以磁纳米颗粒作为载体(MNPs)制备MNPs- Van纳米探针,结合特异性适配体(aptamer),实现双位点非免疫识别单增李斯特菌。利用 UCNPs的荧光内滤效应,即UCNPs的发射波长与有色产物的吸收波长出现重叠,诱导荧光 强度发生变化,将传统的紫外吸光值信号转换为上转换荧光信号,建立单增李斯特菌定量检 测的标准曲线。
对于单增李斯特菌的检测,与现有的技术相比,本发明的优点在于:
1.本发明使用万古霉素和核酸适配体作为生物识别分子,规避了传统免疫分析中抗体的 使用,降低了成本。
2.本发明将双位点识别策略和UCNPs的荧光内滤效应相结合,实现了信号转换和信号 放大,克服了传统比色传感方法灵敏度低,线性范围窄等缺点,可满足一定浓度范围的单增 李斯特菌检测。
3.本发明建立的荧光生物传感器制作方便,需样品量少,抗外界干扰能力强,且易于实 现食源性致病菌的现场即时检测。
附图说明
图1是本发明制备的荧光生物传感器检测单增李斯特菌的原理示意图。
图2是UCNPs的表征图:(A)OA-UCNPs的TEM图像;(B)UCNPs的 X-衍射结果;(C)ADA-UCNPs的TEM图像;(D)UCNPs的傅里叶变换红外 光谱;(E)UCNPs的荧光光谱;(F)ADA-UCNPs,MNPs and MNPs-Van的Zeta 电位。
图3是MNPs和MNPs-Van的表征图:(A)和(B)MNPs和MNPs-Van的 TEM图像;(C)MNPs和MNPs-Van的水合粒径;(D)MNPs和MNPs-Van的 傅里叶变换红外光谱;(E)单增李斯特菌的电镜图像;(F)MNPs-Van偶联物捕 获单增李斯特菌的电镜图像。
图4是优化实验参数:(A)MNPs-Van偶联物的浓度;(B)适配体的浓度(C) HRP-SA的浓度。
图5是构建的荧光传感器检测单增李斯特菌的灵敏度分析:(A)荧光强度 变化量;(B)标准曲线。
图6是构建的荧光传感器特异性检测单增李斯特菌结果。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应 该明了,所述实施例仅是帮助理解本发明,不应视为对本发明的具体限制。
实施例1
1.荧光生物传感器的制备
(1)UCNPs的制备
1)分别称取0.1164g YCl3·6H2O、0.0892g GdCl3·6H2O、0.062g YbCl3·6H2O 和0.006g ErCl3·6H2O溶于4mL甲醇溶液中,超声至完全溶解,然后倒入用少 许甲醇润洗过的三口烧瓶中(三口烧瓶必须保持洁净,若有需要可用王水浸泡过 夜);然后再倒入6mL油酸和14mL 1-十八烯。
2)在连续的磁力搅拌下,将上述混合物置于氮气保护条件下,加热至160℃, 保持30min,然后冷却至50℃左右。
3)分别称取0.2964g NH4F和0.2g NaOH溶于20mL甲醇溶液中,并超声 约15min至完全溶解;然后在连续搅拌下,将该溶液逐滴加入到上述烧瓶溶液 中。随后将混合物置于50℃水浴条件下保持40min,然后在70℃水浴条件下 保持60min,以便于甲醇的挥发。
4)随后,在连续搅拌下,将上述混合物加热至300℃并保持60min。待冷 却至室温,10000rmp离心3min,弃上清;并用环己烷和乙醇洗涤沉淀3次(10000 rmp离心3min),最后将收集到的固体在60℃下过夜真空干燥,并密封保存。
上述合成的上转换纳米颗粒是油酸包裹着的(OA-UCNPs),所以是疏水性 的,这不利于后续的进一步使用。为了将疏水性的UCNPs转变成亲水性的 UCNPs,本研究采用配体交换的方式对UCNPs进行表面修饰,而阿伦膦酸(ADA) 被用作配体去置换UCNPs表面的原始疏水性配体。具体方法如下:称取50mg ADA和200mg OA-UCNPs固体混合,并分散于10mL三氯甲烷、4mL乙醇和 6M1超纯水中,超声约5min;用1M HCl调节pH至2-3,并在连续搅拌下反应30min。反应结束后,分别用乙醇和纯水洗涤3-4次(10000rmp离心3min)。 最后将得到的产品(ADA-UCNPs)重新分散在10mL超纯水中,置于4℃条件 下,备用。
(2)MNPs-Van偶联物的制备
首先,将1mg羧基包覆的磁纳米颗粒(MNPs)悬液转移到1.5mL离心管 中,用MEST(10mM MES,pH=6.0,0.05%Tween-20)洗涤2次并转移至新的 1.5mL离心管中。磁分离后,在室温条件下,用50μL EDC和NHS(10mg/mL) 活化羧基化的磁纳米颗粒20min。将试管置于磁分离架上分离,用MEST洗涤2 次,然后分散于含有1.0mg万古霉素的磷酸盐缓冲溶液(10mM PBS,pH=7.4) 中,在室温条件下,连续轻柔振荡、孵育6h。磁分离并去除上清液后,在试管 中加入1mL PBST和1%BSA,孵育30min,封闭残留位点。最后,将MNPs-Van 偶联物用PBST洗涤3次,然后将其分散于1mL PBST和0.5%BSA中,于4℃ 下保存备用。
2.样品检测
(1)检测方法
1)将已知浓度的单增李斯特菌菌液梯度稀释成102,2×102,2×103,2×104, 2×105,2×106,2×107和2×108CFU/mL。
2)将100μL MNPs-Van偶联物分别与400μL不同浓度(102-2×108CFU/mL) 的目标菌菌液于1.5mL无菌离心管中混合,在37℃下持续振荡、孵育30min。
3)磁分离后用PBST缓冲液洗涤3次,加入生物素化的适配体100mL,在 37℃下振荡、孵育30min。
4)磁分离、洗涤3次,加入100μL HRP-SA溶液,在37℃下振荡、孵育 30min。磁分离、洗涤3次,用100μL去离子水重新分散复合物,再与200μL TMB 溶液在黑暗条件下反应10min。
5)磁分离后,从上清液中吸取200μL溶液,与100μL UCNPs溶液(1mg/mL) 混匀。随后置于具有980nm激光源的荧光分光光度计上,测定上述混合物的上 转换荧光发射光谱。
每个点分析3次(n=3),而且对于每个间隔,荧光强度变化量(ΔFL intensity)计算公式为:ΔFL intensity=|FL intensitysample-FL intensityblank|。
(2)条件优化
在(1)检测方法的基础上,对MNPs-Van偶联物浓度、适配体浓度和HRP-SA 稀释比进行优化,如图4所示,得到MNPs-Van偶联物和适配体的浓度分别为 0.1mg/mL和0.1mM,HRP-SA稀释比1∶2000。
(3)标准曲线建立
在(1)检测方法的基础上,以样品浓度(CFU/mL)的对数为横坐标,以 ΔFLintensity值为纵坐标作图,如图5所示,建立荧光强度变化量和单增李斯特 菌浓度间的标准曲线。
(4)特异性验证
以革兰氏阳性菌(金黄色葡萄球菌和嗜热脂肪芽孢杆菌)和革兰氏阴性菌(大 肠杆菌和肠道沙门氏菌),这四种病原菌为阴性对照组,验证方法的特异性。如 图6所示,检测单增李斯特菌时的ΔFL intensity明显高于阴性对照。
(5)实际样品检测
回收率采用标准加入法进行了研究,即在空白火腿样品中加入不同浓度的单 增李斯特菌。如下表所示,火腿样品中检测单增李斯特菌的平均回收率为88.0% -108.5%,表明了该方法具有一定的可行性和准确性。
申请人声明,本发明通过上述实施例来说明本发明的工艺方法,但本发明并不局限于上 述工艺步骤,即不意味着本发明必须依赖上述工艺步骤才能实施。所属技术领域的技术人员 应该明了,对本发明的任何改进,对本发明所选用原料的等效替换及辅助成分的添加、具体 方式的选择等,均落在本发明的保护范围和公开范围之内。
Claims (1)
1.基于双位点识别策略和荧光内滤效应的荧光生物传感器的构建及应用,其特征是步骤如下:
步骤一,传感器的制备
(1)UCNPs的制备
利用热分解法合成镧系金属掺杂的上转换纳米颗粒UCNPs,具体制备方法如下:
1)分别称取0.1164g YCl3·6H2O、0.0892g GdCl3·6H2O、0.062g YbCl3·6H2O和0.006gErCl3·6H2O溶于4mL甲醇溶液中,超声至完全溶解,然后倒入用少许甲醇润洗过的三口烧瓶中,然后再倒入6mL油酸和14mL 1-十八烯;
2)在连续的磁力搅拌下,将上述混合物置于氮气保护条件下,加热至160℃,保持30min,然后冷却至50℃左右。
3)分别称取0.2964g NH4F和0.2g NaOH溶于20mL甲醇溶液中,并超声约15min至完全溶解,然后在连续搅拌下,将该溶液逐滴加入到上述烧瓶溶液中,随后将混合物置于50℃水浴条件下保持40min,然后在70℃水浴条件下保持60min,以便于甲醇的挥发;
4)随后,在连续搅拌下,将上述混合物加热至300℃并保持60min,待冷却至室温,10000rmp离心3min,弃上清,并用环己烷和乙醇洗涤沉淀3次,最后将收集到的固体在60℃下过夜真空干燥,并密封保存;
上述合成的上转换纳米颗粒是油酸包裹着的OA-UCNPs,采用配体交换的方式对UCNPs进行表面修饰,即称取50mg ADA和200mg OA-UCNPs固体混合,并分散于10mL三氯甲烷、4mL乙醇和6Ml超纯水中,超声约5min;用1M HCl调节pH至2-3,并在连续搅拌下反应30min。反应结束后,分别用乙醇和纯水洗涤3-4次,10000rmp离心3min,最后将得到的产品ADA-UCNPs重新分散在10mL超纯水中,置于4℃条件下,备用;
(2)MNPs-Van偶联物的制备
首先,将1mg羧基包覆的磁纳米颗粒MNPs悬液转移到1.5mL离心管中,用10mM MES,pH=6.0,0.05%Tween-20制成的MEST洗涤2次并转移至新的1.5mL离心管中,磁分离后,在室温条件下,用50μL EDC和10mg/mL NHS活化羧基化的磁纳米颗粒20min,将试管置于磁分离架上分离,用MEST洗涤2次,然后分散于含有1.0mg万古霉素的含有10mM PBS和pH=7.4的磷酸盐缓冲溶液中,在室温条件下,连续轻柔振荡、孵育6h;磁分离并去除上清液后,在试管中加入1mL PBST和1%BSA,孵育30min,封闭残留位点,将MNPs-Van偶联物用PBST洗涤3次,然后将其分散于1mL PBST和0.5%BSA中,于4℃下保存备用;
步骤二,单增李斯特菌的检测方法
首先,将已知浓度的单增李斯特菌菌液梯度稀释成102,2×102,2×103,2×104,2×105,2×106,2×107和2×108CFU/mL;然后将100μL MNPs-Van偶联物分别与400μL的102-2×108CFU/mL不同浓度的目标菌菌液于1.5mL无菌离心管中混合,在37℃下持续振荡、孵育30min;磁分离后用PBST缓冲液洗涤3次,加入生物素化的适配体100mL,在37℃下振荡、孵育30min;磁分离、洗涤3次,加入100μL HRP-SA溶液,在37℃下振荡、孵育30min;磁分离、洗涤3次,用100μL去离子水重新分散复合物,再与200μL TMB溶液在黑暗条件下反应10min;磁分离后,从上清液中吸取200μL溶液,与100μL的1mg/mL UCNPs溶液混匀;随后置于具有980nm激光源的荧光分光光度计上,测定上述混合物的上转换荧光发射光谱;每个点分析3次(n=3),而且对于每个间隔,荧光强度变化量(ΔFL intensity)计算公式为:ΔFL intensity=|FL intensitysample-FL intensityblank|;
步骤三,数据监测与记录
通过一台自主搭建的荧光检测系统对其进行荧光强度(FL intensity)的测定。
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CN114686611A (zh) * | 2022-04-24 | 2022-07-01 | 常州先趋医疗科技有限公司 | 用于检测单增李斯特菌的引物组及其应用 |
WO2023225904A1 (zh) * | 2022-05-25 | 2023-11-30 | 深圳先进技术研究院 | 一种用于检测颅内葡萄球菌感染的试剂底物以及试剂盒的应用 |
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