CN116953219A - 一种用于多重细菌鉴别的荧光阵列传感器及其检测方法 - Google Patents

一种用于多重细菌鉴别的荧光阵列传感器及其检测方法 Download PDF

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CN116953219A
CN116953219A CN202310163242.1A CN202310163242A CN116953219A CN 116953219 A CN116953219 A CN 116953219A CN 202310163242 A CN202310163242 A CN 202310163242A CN 116953219 A CN116953219 A CN 116953219A
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吴丽
杨笑笑
冀海伟
王�琦
秦玉岭
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Abstract

本发明公开了一种多重细菌鉴别的检测方法，构建了由4种不同的荧光抗菌肽组成的荧光阵列传感器。这一传感器具有高灵敏度和高选择特性，由于各种细菌表面结构差异，探针分子响应不同细菌能产生不同的荧光信号，对所获得的不同荧光强度进行线性判别分析，实现了对不同种类的细菌进行快速区分和判断。

Description

一种用于多重细菌鉴别的荧光阵列传感器及其检测方法
技术领域
本发明属于生物分析检测领域，具体涉及一种用于多重细菌鉴别的荧光阵列传感器，为生物科学、医疗卫生等领域中的多重细菌检测提供一种方法。
背景技术
无处不在的致病菌几乎危害到了人类生活的方方面面，尤其是人类健康方面。致病细菌及其有毒副产品对食品、空气和水的污染可能引发各种疾病，造成严重的公共卫生危机和事故，还可能会导致全球气候变化和环境污染。每年有3亿多疾病和500多万人死于致病性细菌感染，造成不可估量的财产损失。耐药性细菌的出现更是加剧了这种情况，并增加了死亡率。因此及时有效的消除细菌将大大有利于环境保护和临床治疗，提高存活率，而这很大程度上依赖于细菌的快速识别和检测。此外，还考虑到一种疾病可能与多种细菌物种有关,因此迫切需要开发一个平台，可以同时快速、可靠地鉴定多种细菌。
迄今为止，已经提出了许多用于细菌检测的方法。传统的病原微生物检测方法几乎完全依赖于特定的微生物培养基来分离和计数活细菌细胞。尽管传统检测方法是有效的，并且已经用于分离和鉴定细菌病原体很多年了，被认为是微生物检测的金标准，但是该过程属于劳动密集型的检测方法，耗时的富集步骤已将分析时间延长至几天，并且增加了微生物吸收或传播的风险。因此，传统的基于微生物培养的方法不足以对病原微生物进行及时评估。聚合酶链反应(PCR)、酶联免疫吸附试验(ELISA)、表面增强拉曼散射(SERS)和质谱等一系列细菌特异性检测方法虽克服了耗时的问题，但通常受到需要昂贵的机器、复杂的操作和专业的检测人员等限制。
近几十年来，研究重点是开发生物传感器，实现快速灵敏的细菌检测方法.现如今，许多生物传感器已经提供了优于现有技术的优点，例如，便捷的操作步骤、高灵敏度、易微型化、成本低等。然而，目前大多数的生物传感器通常只能识别一种细菌，难以实现同时分析多种细菌的目标。因此，开发一种快速简单的同时鉴别多种细菌的平台成为当前的挑战。
发明内容
发明目的：为解决上述技术问题，本发明的目的在于提供一种用于多重细菌鉴别的荧光阵列传感器及其检测方法，这一方法利用4种偶连上荧光分子的抗菌肽与不同细菌表面作用方式的不同，产生不同程度的荧光增强作为信号，并使用线性判别分析荧光数据，创建不同的响应模式，实现对不同细菌的区分和鉴别。此方法具有操作简单，检测灵敏快速，普适性强等特点。
技术方案：一种用于快速鉴别多重细菌的荧光阵列传感器，所述的荧光阵列传感器由不同抗菌肽修饰同一种荧光分子响应不同类型细菌后能产生差异的荧光信号，通过对测量后的荧光强度进行线性判别分析，即可快速区分多重细菌。
作为优化：所述的荧光阵列传感器包括4种偶连上荧光分子的抗菌肽，其中，
抗菌肽Tritrpticin，其序列为：
H-Val-Arg-Arg-Phe-Pro-Trp-Trp-Trp-Pro-Phe-Leu-Arg-Arg-OH；
抗菌肽SPFK，其序列为：
H-Pro-Lys-Leu-Leu-Lys-Thr-Phe-Leu-Ser-Lys-Trp-Ile-Gly-OH；
抗菌肽Indolicidin，其序列为：
H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2；
抗菌肽CM15，其序列为：
H-Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-NH2；
荧光探针，分子结构式如下：
一种快速区分不同细菌的荧光阵列传感器检测方法，具体按照如下步骤进行：
步骤(1)细菌培养：将不同细菌置于LB培养基中，在37℃下震荡培养8-12h；
步骤(2)细菌收集：在3000-5000rpm条件下离心5-10分钟，弃上清，用10mM、pH＝7.4的PBS缓冲液清洗细菌2-3次，最后用PBS重悬细菌；
步骤(3)荧光检测：在待检测细菌中加入各个荧光探针孵育一定时间后，用荧光光谱仪检测，在480nm激发波长下测量荧光得到荧光光谱图，以荧光增强和荧光比率变化(I-I0/I0)作为探针响应信号，用于区分不同种类细菌；
步骤(4)数据处理：每种细菌样本并行测量5次，因此所有细菌得到了4×9×5的数据阵列，对获得的数据进行线性判别分析。
作为优化：步骤(2)中用PBS重悬的细菌OD600＝0.1。
作为优化：步骤(3)中加入的荧光探针Tritrpticin浓度为：30μg/mL，SPFK浓度为：30μg/mL，Indolicidin浓度为：30μg/mL，CM15浓度为：50μg/mL。
作为优化：步骤(3)中不同细菌与各个探针的孵育时间为60-120min。
作为优化：所述的荧光探针在溶液中可自由旋转几乎不发出任何荧光，通过抗菌肽结合在细菌表面后，限制了该探针的空间旋转，在530nm处发出强烈的绿色荧光。
作为优化：检测细菌的浓度范围为大于109CFU/mL，探针的浓度大于等于30μg/mL。
作为优化：该方法用于同时快速准确鉴别各种不同的细菌。
作为优化：所述的细菌为：金黄色葡萄球菌、表皮葡萄球菌、粪肠球菌、枯草芽孢杆菌、屎肠球菌、大肠杆菌、铜绿假单胞菌、鲍曼不动杆菌、肺炎克雷伯菌。
有益效果：本发明的优点和有益效果为：
1.由于传感器阵列的交叉反应性，它能集合不同细菌的检测结果形成指纹，这可以用作分析复杂基质的高通量分化的参考，为细菌成像和鉴别提供了一种替代方法，并应在多种微生物的鉴别中具有潜在应用。
2.与以往基于模式的微生物鉴定相比，直接选择完整的活微生物而不是微生物裂解物作为分析目标，缩短预处理时间，实现快速检测和实时监测。
3.在不同抗菌肽的作用下，仅使用一种荧光探针来区分多种细菌，简化了荧光传感器阵列的构造。
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，下面将对实施例或现有技术描述中所需要使用的附图做简单地介绍：
图1为四种抗菌肽连上荧光分子的紫外吸收光谱，其中(A)为Tritrpticin；(B)为Indolicidin；(C)为SPFK；(D)为CM15；
图2为四种荧光抗菌肽与九种细菌结合后荧光响应光谱图，其中(A)为Tritrpticin；(B)为Indolicidin；(C)为SPFK；(D)为CM15；
图3为四种荧光抗菌肽与金黄色葡萄球菌结合后荧光成像图，其中(A)为Tritrpticin；(B)为Indolicidin；(C)为SPFK；(D)为CM15；
图4为根据四种荧光抗菌肽与九种细菌结合后的荧光强度分析所得的线性判别图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，以使本领域的技术人员能够更好的理解本发明的优点和特征，从而对本发明的保护范围做出更为清楚的界定。本发明所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例，基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：荧光阵列传感器的构建
根据固相合成法合成4种抗菌肽(Tritrpticin、Indolicidin、SPFK、CEA-MET)后，通过氨基与羧基的缩合反应，抗菌肽与荧光分子相连。图1所示为4种抗菌肽偶连上荧光分子后的紫外吸收光谱，在500nm处各个抗菌肽均出现荧光分子的特征峰，验证抗菌肽与荧光分子的偶连成功。
实施例2：采用荧光光谱仪验证细菌与探针响应情况
步骤一：将不同细菌置于LB培养基中，在37℃下震荡培养8-12h；
步骤二：在3000-5000rpm条件下离心5-10分钟，弃上清，用10mM、pH＝7.4的PBS缓冲液清洗细菌2-3次，最后用PBS重悬细菌至OD600＝0.1；
步骤三：：在细菌中加入探针(Tritrpticin：30μg/mL、SPFK：30μg/mL、Indolicidin：30μg/mL、CM15：50μg/mL)后孵育60-120min，用荧光光谱仪检测，在480nm激发光波长下，检测不同细菌与探针结合后的荧光响应程度。
如图2所示，9种不同的细菌与4种连上荧光分子的抗菌肽结合后，分别有不同程度的荧光增强。
实施例3：采用荧光显微镜验证细菌与探针结合情况
步骤一：将金黄色葡萄球菌置于LB培养基中，在37℃下震荡培养8-12h；
步骤二：在3000-5000rpm条件下离心5-10分钟，弃上清，用10mM、pH＝7.4的PBS缓冲液清洗细菌2-3次，最后用PBS重悬细菌至OD600＝0.1；
步骤三：在细菌中加入探针(Tritrpticin：30μg/mL、SPFK：30μg/mL、Indolicidin：30μg/mL、CM15：50μg/mL)后孵育60-120min；
步骤四：将孵育后的细菌在3000-5000rpm条件下离心5-10分钟，弃上清，用10mM、pH＝7.4的PBS缓冲液清洗细菌2-3次以洗去多余的荧光探针，最后用PBS重悬细菌，在荧光显微镜下观察细菌与探针的结合情况。
如图3所示，金黄色葡萄球菌与4种连上荧光分子的抗菌肽结合后，在荧光显微镜下，部分金黄色葡萄球菌发出明显的绿色荧光。
实施例4：采用荧光阵列传感器检测法快速判断9种常见细菌
细菌表面是有多种组分组成的，细菌基因的任何改变都可能使细胞表面产生微小的变化，因此通过多个连有不同抗菌肽的荧光探针与各种细菌表面的非特异性相互作用产生的不同响应，可对多种细菌进行快速检测和判断。基于实例2，已证实不同细菌和不同探针作用可产生不同程度的荧光增强，为荧光阵列传感器快速区分多重细菌提供基础。
步骤一：将9种不同细菌置于LB培养基中，在37℃下震荡培养8-12h；
步骤二：在3000-5000rpm条件下离心5-10分钟，弃上清，用10mM、pH＝7.4的PBS缓冲液清洗细菌2-3次，最后用PBS重悬细菌至OD600＝0.1；
步骤三：在细菌中加入探针(Tritrpticin：30μg/mL、SPFK：30μg/mL、Indolicidin：30μg/mL、CM15：50μg/mL)后孵育60-120min，用荧光光谱仪检测，在480nm激发光波长下，获得不同细菌与探针结合后的荧光强度。
步骤四：用4种探针对9种细菌样本并行测量5次，因此所有细菌得到了4×9×5的数据阵列，对获得的数据进行线性判别分析
如图4所示，根据线性判别分析结果，这9种细菌被很好地区分开了，没有任何重叠。

Claims (10)

1.一种用于快速鉴别多重细菌的荧光阵列传感器，其特征在于：所述的荧光阵列传感器由不同抗菌肽修饰同一种荧光分子响应不同类型细菌后能产生差异的荧光信号，通过对测量后的荧光强度进行线性判别分析，即可快速区分多重细菌。
2.根据权利要求1所述的用于快速鉴别多重细菌的荧光阵列传感器，其特征在于：所述的荧光阵列传感器包括4种偶连上荧光分子的抗菌肽，其中，
抗菌肽Tritrpticin，其序列为：
H-Val-Arg-Arg-Phe-Pro-Trp-Trp-Trp-Pro-Phe-Leu-Arg-Arg-OH；
抗菌肽SPFK，其序列为：
H-Pro-Lys-Leu-Leu-Lys-Thr-Phe-Leu-Ser-Lys-Trp-Ile-Gly-OH；
抗菌肽Indolicidin，其序列为：
H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2；
抗菌肽CM15，其序列为：
H-Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-NH2；
荧光探针，分子结构式如下：
3.一种快速区分不同细菌的荧光阵列传感器检测方法，其特征在于：具体按照如下步骤进行：
步骤(1)细菌培养：将不同细菌置于LB培养基中，在37℃下震荡培养8-12h；
步骤(2)细菌收集：在3000-5000rpm条件下离心5-10分钟，弃上清，用10mM、pH＝7.4的PBS缓冲液清洗细菌2-3次，最后用PBS重悬细菌；
步骤(3)荧光检测：在待检测细菌中加入各个荧光探针孵育一定时间后，用荧光光谱仪检测，在480nm激发波长下测量荧光得到荧光光谱图，以荧光增强和荧光比率变化(I-I0/I0)作为探针响应信号，用于区分不同种类细菌；
步骤(4)数据处理：每种细菌样本并行测量5次，因此所有细菌得到了4×9×5的数据阵列，对获得的数据进行线性判别分析。
4.根据权利要求3所述的快速区分不同细菌的荧光阵列传感器检测方法，其特征在于：步骤(2)中用PBS重悬的细菌OD600＝0.1。
5.根据权利要求3所述的快速区分不同细菌的荧光阵列传感器检测方法，其特征在于：步骤(3)中加入的荧光探针Tritrpticin浓度为：30μg/mL，SPFK浓度为：30μg/mL，Indolicidin浓度为：30μg/mL，CM15浓度为：50μg/mL。
6.根据权利要求3所述的快速区分不同细菌的荧光阵列传感器检测方法，其特征在于：步骤(3)中不同细菌与各个探针的孵育时间为60-120min。
7.根据权利要求3所述的快速区分不同细菌的荧光阵列传感器检测方法，其特征在于：所述的荧光探针在溶液中可自由旋转几乎不发出任何荧光，通过抗菌肽结合在细菌表面后，限制了该探针的空间旋转，在530nm处发出强烈的绿色荧光。
8.根据权利要求3所述的快速区分不同细菌的荧光阵列传感器检测方法，其特征在于：检测细菌的浓度范围为大于109CFU/mL，探针的浓度大于等于30μg/mL。
9.根据权利要求2-8中任一权利要求项所述的鉴别多重细菌的荧光阵列传感器检测方法，其特征在于：该方法用于同时快速准确鉴别各种不同的细菌。
10.根据权利要求9所述的鉴别多重细菌的荧光阵列传感器检测方法，其特征在于：所述的细菌为：金黄色葡萄球菌、表皮葡萄球菌、粪肠球菌、枯草芽孢杆菌、屎肠球菌、大肠杆菌、铜绿假单胞菌、鲍曼不动杆菌、肺炎克雷伯菌。
CN202310163242.1A 2023-02-24 2023-02-24 一种用于多重细菌鉴别的荧光阵列传感器及其检测方法 Pending CN116953219A (zh)

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