Ghindilis Andrey L, Smith Maria W, Schwarzkopf Kevin R, Roth Kristian M, Peyvan Kia, Munro Sandra B, Lodes Michael J, Stöver Axel G, Bernards Karen, Dill Kilian, McShea Andy
CombiMatrix Corporation, 6500 Harbour Heights Pkwy, 301, Mukilteo, WA 98275, USA.
Biosens Bioelectron. 2007 Apr 15;22(9-10):1853-60. doi: 10.1016/j.bios.2006.06.024. Epub 2006 Aug 7.
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 Reader. Performance of the ElectraSense 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 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 as compared to the detection limit of 1.5 pM obtained for fluorescent detection. Thus, the ElectraSense 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).
针对CombiMatrix寡核苷酸微阵列平台，已开发出电化学检测方法并研究了其检测性能。该平台在半导体基质中包含12,544个可单独寻址的微电极（特征点）。该方法基于对特定微阵列电极附近的氧化还原活性化学物质（如辣根过氧化物酶（HRP）和相关底物TMB）的检测。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。由于HRP反应产物的电还原产生检测电流，并用CombiMatrix ElectraSense Reader进行测量。已使用基因表达和基因分型分析对ElectraSense平台的性能进行了表征，以分析：（i）信号与浓度的依赖性，（ii）检测分辨率，（iii）变异系数（CV）和（iv）阵列间的重现性和数据相关性。还将ElectraSense平台与标准荧光检测进行了比较，观察到这两种不同检测技术之间具有良好的一致性。与荧光检测获得的1.5 pM检测限相比，ElectraSense的检测下限为0.75 pM。因此，ElectraSense平台已用于开发核酸检测方法，用于对多种病原体进行高精度基因分型，包括生物威胁因子（如炭疽芽孢杆菌、鼠疫耶尔森菌等）以及呼吸道常见病原体（如甲型流感病毒）。
