CN106950212B - 一种快速检测微生物的基于星状环糊精的荧光传感器 - Google Patents
一种快速检测微生物的基于星状环糊精的荧光传感器 Download PDFInfo
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Abstract
本工作拟研发了一种星状环糊精的荧光传感器快速检测微生物，并对微生物膜的抗生素抗菌机制进行筛选，这种复合体结合统计分析工具可以特异性识别病原微生物膜。该复合体结合特征微生物膜，特征微生物竞争作用使其表面的荧光蛋白游离，每种微生物膜结合强度和相互作用力不一样导致可以得到特征的响应信号。基于上述的实验，进一步把这种复合体用到抗生素机制评估和筛选。本工作创新性体现在：借助于星状季铵盐化环糊精与荧光蛋白形成复合体来检测微生物膜，微生物膜体系无需任何前处理，只需要简单的步骤，为解决涉及微生物检测方法中“在线”、“便携式”和“全自动”的机电工程与生物学复合问题提供参考和借鉴。
Description
技术领域
一种基于星状季铵盐化环糊精与荧光蛋白形成复合体阵列检测和微生物膜并响应微生物膜对抗生素的抗菌机制。
背景技术
微生物生物膜是由微生物群体及其包被的细胞外多聚物和基质网组成，它们彼此黏附或者黏附到组织或物体的表面。最近的研究发现高达60%手术后导致的疾病都是手术器材微生物感染导致。微生物生物膜与微生物的耐药性形成、基因的转移以及引起机体的持续性感染等都密切相关。当前常用的对微生物膜进行检测的方法包括基于培养方法、血液分析、显微镜观察、组织切片观察、探针成像、免疫诊断分析和分子检测。培养分析方法能够准确阐明临床相关疾病确实是微生物膜感染导致，这种方法存在的不足之处是需要时间长，一般3-5天才能获得分析结果（Rogers, G.B., Carroll, M.P., Bruce, K.D., 2009.Journal of medical microbiology 58(Pt 11), 1401-1418.）。血液分析通常是分析血液中验证因子，但是这种方法没有特异性（Mathur, T., Singhal, S., Khan, S.,Upadhyay, D.J., Fatma, T., Rattan, A., 2006. Indian journal of medicalmicrobiology 24(1), 25-29）。最近相关成像技术也被用于微生物活体诊断，比如核磁共振成像、超声波、内窥镜、计算机断层扫描等（Temple, C.L., Ross, D.C., Bennett,J.D., Garvin, G.J., King, G.J., Faber, K.J., 2005. The Journal of handsurgery 30(3), 534-542.）,但是这些方法的成本非常高。酶链免疫反应可以快速诊断微生物膜，但是其抗体稳定性不好，很容易失活（Hall-Stoodley, L., Stoodley, P.,Kathju, S., Høiby, N., Moser, C., William Costerton, J., Moter, A.,Bjarnsholt, T., 2012. FEMS Immunology & Medical Microbiology 65(2), 127-145.）。
基于纳米材料阵列响应来快速诊断微生物得到了响应的关注。比如利用纳米金与带负电荧光聚合物可以选择性检测微生物（Phillips, R.L., Miranda, O.R., You, C.-C., Rotello, V.M., Bunz, U.H.F., 2008. Angewandte Chemie InternationalEdition 47(14), 2590-2594.）。我们也前期设计了磁性颗粒与带负电荧光聚合物来选择性检测微生物（Wan, Y., Sun, Y., Qi, P., Wang, P., Zhang, D., 2014. Biosensorsand Bioelectronics 55, 289-293）。本方案则采用基于星状环糊精与荧光蛋白的阵列响应来检测微生物膜，并使其来筛选微生物的抗生素的抗菌机制。
发明内容
为实现上述目的，本发明采用技术方案为：
一种快速检测微生物的基于星状环糊精的荧光传感器，其特征包括季铵盐化的环糊精与绿色荧光蛋白、红色荧光蛋白、蓝色荧光蛋白形成的复合体。
作为优选所述的一种快速检测微生物的基于星状环糊精的荧光传感器，所述的季铵盐化的环糊精，包括：苯基官能团的季铵盐化环糊精，正己烷官能团的季铵盐化环糊精，二茂铁基官能团的季铵盐化环糊精，辛烷基官能团的季铵盐化环糊精，和甲基官能团的季铵盐化环糊精。
作为优选所述的环糊精，包括：-环糊精，-环糊精和-环糊精。
附图说明
图1 星状季铵盐化环糊精与荧光蛋白形成检测微生物的复合体阵列示意图。
图2星状季铵盐化环糊精与荧光蛋白形成复合体阵列检测八种不同微生物。
图3星状季铵盐化环糊精与荧光蛋白形成复合体阵列检测抗生素机制（利用爱德华弧菌生物膜作为来源）。
具体实施方式
下面通过实施例对本发明做进一步说明。
实施例1：
纳米传感器构建：浓度分别为（100 n M）的绿色荧光蛋白、红色荧光蛋白和蓝色荧光蛋白与星状季铵盐的环糊精进行滴定反应，直到三种荧光强度减少到最低值。既可以获得星状季铵盐化环糊精与荧光蛋白形成复合体。
实施例2：
大肠杆菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M 硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。往每孔中加入200 μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定大肠杆菌的种类。
实施例3：
沙门杆菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M 硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。往每孔中加入200 μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定沙门杆菌的种类。
实施例4：
金黄色葡萄球菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M 硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。往每孔中加入200 μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定金黄色葡萄球菌的种类。
实施例5：
铜绿假单胞菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。往每孔中加入200 μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定铜绿假单胞菌的种类。
实施例6：
爱德华弧菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。往每孔中加入200 μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定爱德华弧菌的种类。
实施例7：
链球菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M 硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。往每孔中加入200μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定链球菌的种类。
实施例8：
大肠杆菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M 硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。再加入微生物膜半死亡浓度的青霉素，培育24小时，微孔板清洗三次。加入往每孔中加入200 μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定抗生素的种类。
实施例9：
大肠杆菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M 硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。再加入微生物膜半死亡浓度的头孢菌素，培育24小时，微孔板清洗三次。加入往每孔中加入200 μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定抗生素的种类。
实施例10：
大肠杆菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M 硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。再加入微生物膜半死亡浓度的四环素，培育24小时，微孔板清洗三次。加入往每孔中加入200 μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定抗生素的种类。
实施例11：
金黄色葡萄球菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M 硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。再加入微生物膜半死亡浓度的万古霉素，培育24小时，微孔板清洗三次。加入往每孔中加入200μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定抗生素的种类。
实施例12：
大肠杆菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M 硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。再加入微生物膜半死亡浓度的多粘菌素B，培育24小时，微孔板清洗三次。加入往每孔中加入200 μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定抗生素的种类。
实施例13：
金黄色葡萄球菌在LB培养基中37度培养12小时，再用离心机清洗三次。在600nm处测量悬浮的微生物浓度，加入培养基进行培养。培养中加入0.1%葡萄糖、1m M 硫酸镁和0.15M 硫酸铵及34 m M柠檬酸，并调整到pH=7，加入微孔板。培养板在室温下培养3天，其中第二天去除悬浮的菌液，加入新鲜的培养基。等到第三次去除菌液并用PBS冲洗三次。再加入微生物膜半死亡浓度的替考拉宁，培育24小时，微孔板清洗三次。加入往每孔中加入200μL的纳米传感器复合体，培育15分钟，用酶标仪测量三种荧光阵列响应信号。结合线性判别分析既可以确定抗生素的种类。
Claims (3)
1.一种快速检测微生物的基于星状环糊精的荧光传感器，其特征在于 包括季铵盐化的环糊精与绿色荧光蛋白、红色荧光蛋白、蓝色荧光蛋白形成的复合体。
2.如权利要求1所述的一种快速检测微生物的基于星状环糊精的荧光传感器，其特征在于所述的季铵盐化的环糊精，包括：苯基官能团的季铵盐化环糊精，正己烷官能团的季铵盐化环糊精，二茂铁基官能团的季铵盐化环糊精，辛烷基官能团的季铵盐化环糊精，和甲基官能团的季铵盐化环糊精。
3.如权利要求2所述的一种快速检测微生物的基于星状环糊精的荧光传感器，其特征在于，所述的环糊精，包括：-环糊精， -环糊精和 -环糊精。
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