Development of an advanced electrochemical DNA biosensor for bacterial pathogen detection
Development of an advanced electrochemical DNA biosensor for bacterial pathogen detection
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DOI：
10.2353/jmoldx.2007.060052
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发表时间：
2007-04-01
影响因子：
4.1
通讯作者：
Haake, David A.
中科院分区：
文献类型：
--
作者：
Liao, Joseph C.;Mastali, Mitra;Haake, David A.
电化学传感器能够快速、准确地检测生物液中的各种目标分子。我们开发了一种电化学传感器检测方法，包括将细菌16S rRNA与荧光素修饰的检测器探针和固定在传感器表面的生物素修饰的捕获探针杂交。信号由与抗荧光素单抗Fab结合的辣根过氧化物酶作用产生的氧化还原电流产生。先前的一项研究发现，这种电化学传感器策略可以识别临床尿液标本中的尿路病原体。为了提高检测灵敏度，我们研究了影响电化学信号电流幅度的关键步骤。首先用Triton X-100和溶菌酶处理，然后用碱性裂解，可以有效地裂解和释放革兰氏阴性和阳性细菌的16S rRNA，使电化学信号比单独碱性裂解增加12倍。探测器和捕获探针的目标杂交位点之间的核苷酸距离以及探测器探针上荧光素修饰的位置导致信号强度变化23倍。这些结果证明了目标-探针和探针-探针相互作用在使用电化学DNA传感器方法检测细菌16S rRNA中的重要性。
Electrochemical sensors have the capacity for rapid and accurate detection of a wide variety of target molecules in biological fluids. We have developed an electrochemical sensor assay involving hybridization of bacterial 16S rRNA to fluorescein-modified detector probes and to biotin-modified capture probes anchored to the sensor surface. Signal is generated by an oxidation-reduction current produced by the action of horseradish peroxidase conjugated to an anti-fluorescein monoclonal Fab. A previous study found that this electrochemical sensor strategy could identify uropathogens in clinical urine specimens. To improve assay sensitivity, we examined the key steps that affect the current amplitude of the electrochemical signal. Efficient lysis and release of 16S rRNA from both gram-negative and -positive bacteria was achieved with an initial treatment with Triton X-100 and lysozyme followed by alkaline lysis, resulting in a 12-fold increase in electrochemical signal compared with alkaline lysis alone. The distance in nucleotides between the target hybridization sites of the detector and capture probes and the location of fluorescein modification on the detector probe contributed to a 23-fold change in signal intensity. These results demonstrate the importance of target-probe and probe-probe interactions in the detection of bacterial 16S rRNA using an electrochemical DNA sensor approach.