Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens
Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens
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DOI：
10.1128/jcm.44.2.561-570.2006
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发表时间：
2006-02-01
影响因子：
9.4
通讯作者：
Haake, DA
中科院分区：
文献类型：
--
作者：
Liao, JC;Mastali, M;Haake, DA
我们描述了使用微型制造的电化学传感器阵列首次对人类临床液体样本中的细菌病原体进行物种特异性检测。阵列中的16个传感器中的每个都由三个单层金电极组成-工作电极、参考电极和辅助电极。每个工作电极都包含一个捕获探针库中的一个代表，每个探针库都针对临床上相关的细菌尿路病原体。文库包括大肠杆菌、奇异变形杆菌、铜绿假单胞菌、肠球菌属和克雷伯氏菌-肠杆菌群的探针。来自一步细菌裂解的细菌16S rRNA靶标与传感器表面的生物素修饰的捕获探针和第二个荧光素修饰的检测器探针杂交。通过将辣根过氧化物酶(HRP)偶联的抗荧光素抗体结合到检测器探针上，实现了对目标探针杂交物的检测。在工作电极和参比电极之间的固定电位为-200 mV时，获得了催化HRP反应的安培法测量结果。在样品处理开始后的45天内，实现了对培养、接种的尿液和临床尿液样本中少至2,600个泌尿系致病菌的物种特异性检测。在用临床盲法尿样进行的电流法检测系统的可行性研究中，该传感器阵列对直接检测革兰氏阴性菌具有100%的灵敏度，而不需要进行核酸纯化或扩增。98%的革兰氏阴性细菌可用物种特异性探针进行鉴定。当与基于微流体的样品制备模块相结合时，该集成系统可以作为快速诊断尿路感染的护理设备。
We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes-working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Entero-cococcits spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of -200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 ruin from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.