Stopped-flow microarray immunoassay for detection of viable E. coli by use of chemiluminescence flow-through microarrays
Stopped-flow microarray immunoassay for detection of viable E. coli by use of chemiluminescence flow-through microarrays
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DOI：
10.1007/s00216-010-4414-0
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发表时间：
2011-01-01
影响因子：
4.3
通讯作者：
Seidel, Michael
中科院分区：
文献类型：
--
作者：
Langer, Veronika;Niessner, Reinhard;Seidel, Michael
在水质分析中，微生物的快速多路分析对控制细菌污染具有重要的健康和安全意义。通过流式微阵列免疫分析直接定量细菌需要新的分析策略来优化灵敏度和分析时间。对于细菌和颗粒来说，流体动力和沉降作用是在流动系统中结合在表面上的主要作用，而扩散作用则微不足道。因此，我们实施了一种停止和流动技术来定量活的大肠杆菌细胞。该方法采用静置容积元件交替并向前泵送元件的方法，比连续流方法更有效地分析细菌。为了定量活的大肠杆菌细胞，采用抗体微阵列和基于流动注射的微阵列分析进行了化学发光三明治免疫分析测试格式。抗体作为选择性捕获分子，被固定在聚合物修饰的玻璃表面作为微阵列底物。对于细菌识别步骤，使用第二种检测抗体，在微阵列的每个点形成三明治免疫分析。采用辣根过氧化物酶催化化学发光反应进行检测。所有的分析步骤都是通过自动流动的化学发光微阵列读出系统进行的。67 min可检出活的大肠杆菌细胞，检出限为4 × 10(5)个细胞mL（-1）。通过引入停流技术并优化相互作用时间和步骤，在微孔板上对大肠杆菌的检测速度更快，灵敏度提高了两个数量级。
Rapid multiplexed analysis of microorganisms is important in water analysis to control bacterial contamination for health and safety reasons. Direct quantification of bacteria by means of flow-through microarray immunoassays requires new analysis strategies for optimising sensitivity and the analysis time. For bacteria and for particles, hydrodynamic forces and sedimentation are the dominating effects for binding on surfaces in a flow-through system, whereas diffusion is insignificant. Therefore, we have implemented a stop and flow technique for quantification of viable E. coli cells. The method, with alternation of resting volume elements and pumping the elements forward, was more effective than continuous-flow approaches for analysis of bacteria. For quantification of viable E. coli cells, a chemiluminescence sandwich immunoassay test format was performed by means of antibody microarrays and flow-injection-based microarray analysis. Antibodies, which served as selective capture molecules, were immobilised on polymer-modified glass surfaces serving as microarray substrate. For the bacteria recognition step, a second detection antibody was used, forming a sandwich immunoassay at each spot of the microarray. Detection was carried out with a horseradish peroxidase catalysed chemiluminescence reaction. All assay steps were conducted with an automated flow-through chemiluminescence microarray readout system. Living E. coli cells could be detected in 67 min with a detection limit of 4x10(5) cells mL(-1). By introduction of the stopped-flow technique and optimisation of interaction time and interaction steps the achieved detection of E. coli was faster and two orders of magnitude more sensitive than with a conventional ELISA technique in microplates.