CombiMatrix oligonucleotide arrays:: Genotyping and gene expression assays employing electrochemical detection
CombiMatrix oligonucleotide arrays:: Genotyping and gene expression assays employing electrochemical detection
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DOI：
10.1016/j.bios.2006.06.024
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发表时间：
2007-04-15
影响因子：
12.6
通讯作者：
McShea, Andy
中科院分区：
文献类型：
--
作者：
Ghindilis, Andrey L.;Smith, Maria W.;McShea, Andy
CombiMatrix 寡核苷酸微阵列平台的电化学检测和分析性能已得到开发，该平台在半导体矩阵中包含 12,544 个可单独寻址的微电极（特征）。该方法基于检测靠近特定微阵列电极的氧化还原活性化学物质（例如辣根过氧化物酶 (HRP) 和相关底物 TMB）。首先，微阵列探针与生物素标记的靶标杂交，其次，HRP-链霉亲和素缀合物与生物素结合，然后发生电子供体底物的酶促氧化。检测电流是由于 HRP 反应产物的电还原而产生的，并使用 CombiMatrix ElectraSense (TM) Reader 进行测量。 ElectraSense (TM) 平台的性能已使用基因表达和基因分型测定进行表征，以分析：(i) 信号与浓度依赖性、(ii) 测定分辨率、(iii) 变异系数、(CV) 和 (iv) 阵列间重现性和数据相关性。 ElectraSense (TM) 平台还与标准荧光检测进行了比较，发现这两种不同的检测技术之间具有良好的一致性。与荧光检测的检测限 1.5 pM 相比，ElectraSense (TM) 的检测限较低为 0.75 pM。因此，ElectraSense (TM) 平台已用于开发核酸检测，对多种病原体进行高精度基因分型，包括生物威胁因子（如炭疽杆菌、鼠疫耶尔森氏菌和其他微生物，包括大肠杆菌、枯草芽孢杆菌等）和呼吸道常见病原体（如甲型流感病毒）。 (c) 2006 Elsevier B.V. 保留所有权利。
Electrochemical detection has been developed and assay performances studied for the CombiMatrix oligonucleotide microarray platform that contains 12,544 individually addressable microelectrodes (features) in a semiconductor matrix. The approach is based on the detection of redox active chemistries (such as horseradish peroxidase (HRP) and the associated substrate TMB) proximal to specific microarray electrodes. First, microarray probes are hybridized to biotin-labeled targets, second, the HRP-streptavidin conjugate binds to biotin, and enzymatic oxidation of the electron donor substrate then occurs. The detection current is generated due to electro-reduction of the HRP reaction product, and it is measured with the CombiMatrix ElectraSense (TM) Reader. Performance of the ElectraSense (TM) platform has been characterized using gene expression and genotyping assays to analyze: (i) signal to concentration dependence, (ii) assay resolution, (iii) coefficients of variation, (CV) and (iv) array-to-array reproducibility and data correlation. The ElectraSense (TM) platform was also compared to the standard fluorescent detection, and good consistency was observed between these two different detection techniques. A lower detection limit of 0.75 pM was obtained for ElectraSense (TM) as compared to the detection limit of 1.5 pM obtained for fluorescent detection. Thus, the ElectraSense (TM) platform has been used to develop nucleic acid assays for highly accurate genotyping of a variety of pathogens including bio-threat agents (such as Bacillus anthracis, Yersinia pestis, and other microorganisms including Escherichia coli, Bacillus subtilis, etc.) and common pathogens of the respiratory tract (e.g. influenza A virus). (c) 2006 Elsevier B.V. All rights reserved.