Nanocomposites Research Group, Department of Chemical Engineering, Anna University, Chennai, TN 600 025, India.
Anal Biochem. 2011 Oct 1;417(1):73-9. doi: 10.1016/j.ab.2011.05.034. Epub 2011 May 27.
The present study was aimed at the development and evaluation of a DNA electrochemical biosensor for Mycobacterium sp. genomic DNA detection in a clinical specimen using a signal amplifier as dual-labeled AuNPs. The DNA electrochemical biosensors were fabricated using a sandwich detection strategy involving two kinds of DNA probes specific to Mycobacterium sp. genomic DNA. The probes of enzyme ALP and the detector probe both conjugated on the AuNPs and subsequently hybridized with target DNA immobilized in a SAM/ITO electrode followed by characterization with CV, EIS, and DPV analysis using the electroactive species para-nitrophenol generated by ALP through hydrolysis of para-nitrophenol phosphate. The effect of enhanced sensitivity was obtained due to the AuNPs carrying numerous ALPs per hybridization and a detection limit of 1.25 ng/ml genomic DNA was determined under optimized conditions. The dual-labeled AuNP-facilitated electrochemical sensor was also evaluated by clinical sputum samples, showing a higher sensitivity and specificity and the outcome was in agreement with the PCR analysis. In conclusion, the developed electrochemical sensor demonstrated unique sensitivity and specificity for both genomic DNA and sputum samples and can be employed as a regular diagnostics tool for Mycobacterium sp. monitoring in clinical samples.
本研究旨在开发和评估一种基于 DNA 的电化学生物传感器，用于临床标本中分枝杆菌属基因组 DNA 的检测，该传感器使用信号放大器作为双标记 AuNPs。该 DNA 电化学生物传感器采用三明治检测策略构建，涉及两种针对分枝杆菌属基因组 DNA 的特异性 DNA 探针。探针酶 ALP 和探测器探针都连接在 AuNPs 上，随后与固定在 SAM/ITO 电极上的靶 DNA 杂交，然后通过 CV、EIS 和 DPV 分析用 ALP 通过对硝基苯酚磷酸盐的水解生成的电活性物质对硝基苯酚进行表征。由于每个杂交都携带大量的 ALP，因此获得了增强灵敏度的效果，并在优化条件下确定了检测限为 1.25ng/ml 基因组 DNA。该双标记 AuNP 促进的电化学生物传感器还通过临床痰样本进行了评估，显示出更高的灵敏度和特异性，结果与 PCR 分析一致。总之，开发的电化学生物传感器对基因组 DNA 和痰样本均具有独特的灵敏度和特异性，可作为临床样本中分枝杆菌属监测的常规诊断工具。
