Automated detection and quantitation of bacterial RNA by using electrical microarrays
Automated detection and quantitation of bacterial RNA by using electrical microarrays
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DOI：
10.1021/ac0600914
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发表时间：
2006-07-15
影响因子：
7.4
通讯作者：
Hintsche, Rainer
中科院分区：
文献类型：
--
作者：
Elsholz, B.;Woerl, R.;Hintsche, Rainer
低密度电16 S rRNA特异性寡核苷酸微阵列和自动分析系统已被开发用于病原体的鉴定和定量。病原体是大肠杆菌、铜绿假单胞菌、粪肠球菌、金黄色葡萄球菌和表皮葡萄球菌，它们通常涉及尿路感染。具有纳米级结构的叉指型金阵列电极（IDA电极）已被用于非常灵敏的分析。巯基修饰的寡核苷酸作为捕获探针固定在金IDA上。它们通过杂交介导靶16 S rRNA的特异性识别。另外，三个未标记的寡核苷酸在捕获位点附近杂交。它们是支持分子，因为它们改善了捕获位点的RNA杂交。还允许生物素标记的检测寡核苷酸与捕获的RNA序列杂交。生物素标记能够结合抗生物素蛋白碱性磷酸酶缀合物。磷酸酶将电化学介体对氨基苯酚从其电惰性磷酸盐衍生物中释放出来。通过安培氧化还原循环产生电信号，并通过独特的多电位仪检测。微阵列的读出信号是位置特异性电流，并且随时间与分析物浓度成比例地变化。如果通过支持寡核苷酸将两种额外的生物素引入亲和结合复合物中，则测定的灵敏度增加超过60%。大肠杆菌总RNA的检测限已确定为0.5 ng/μ L。用于可变分析格式的射流控制以及多通道电读出和数据处理都已完全自动化。快速和简单的程序不需要通过PCR对靶核酸进行任何扩增。
Low-density electrical 16S rRNA specific oligonucleotide microarrays and an automated analysis system have been developed for the identification and quantitation of pathogens. The pathogens are Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis, which are typically involved in urinary tract infections. Interdigitated gold array electrodes (IDA-electrodes), which have structures in the nanometer range, have been used for very sensitive analysis. Thiol-modified oligonucleotides are immobilized on the gold IDA as capture probes. They mediate the specific recognition of the target 16S rRNA by hybridization. Additionally three unlabeled oligonucleotides are hybridized in close proximity to the capturing site. They are supporting molecules, because they improve the RNA hybridization at the capturing site. A biotin labeled detector oligonucleotide is also allowed to hybridize to the captured RNA sequence. The biotin labels enable the binding of avidin alkaline phophatase conjugates. The phosphatase liberates the electrochemical mediator p-aminophenol from its electrically inactive phosphate derivative. The electrical signals were generated by amperometric redox cycling and detected by a unique multipotentiostat. The read out signals of the microarray are position specific current and change over time in proportion to the analyte concentration. If two additional biotins are introduced into the affinity binding complex via the supporting oligonucleotides, the sensitivity of the assays increase more than 60%. The limit of detection of Escherichia coli total RNA has been determined to be 0.5 ng/mu L. The control of fluidics for variable assay formats as well as the multichannel electrical read out and data handling have all been fully automated. The fast and easy procedure does not require any amplification of the targeted nucleic acids by PCR.