Disposable Magnetic DNA Sensors for the Determination at the Attomolar Level of a Specific Enterobacteriaceae Family Gene
Disposable Magnetic DNA Sensors for the Determination at the Attomolar Level of a Specific Enterobacteriaceae Family Gene
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DOI：
10.1021/ac801319b
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发表时间：
2008-11-01
影响因子：
7.4
通讯作者：
Pingarron, Jose M.
中科院分区：
文献类型：
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作者：
Loaiza, Oscar A.;Campuzano, Susana;Pingarron, Jose M.
一次性磁性DNA传感器使用酶扩增策略的肠杆菌科细菌家族相关的基因的特异性检测，基于链霉亲和素-过氧化物酶的耦合生物素化的lacZ基因的靶序列，已经开发。将生物素化的25-mer捕获探针连接至链霉亲和素修饰的磁珠，并允许与生物素化的靶进行杂交。然后，将链霉亲和素-过氧化物酶聚合物附着到生物素化的靶标上，并通过四硫富瓦烯（TTF）修饰的金丝网印刷电极（Au/SPE）表面上的磁场捕获所得修饰的磁珠。在加入过氧化氢后在-0.15 V下获得的安培响应用于检测杂交过程。为了提高测定的灵敏度和缩短测定时间，对测定方案的不同变量进行了优化。方法的检出限为5.7fmol，稳定性好（RSD = 7.1%，n = 10）。在磁珠上的DNA非特异性吸附可以忽略不计，因此所获得的结果证明了以高特异性和灵敏度检测杂交事件的可能性。所开发的方法用于分析大肠杆菌DNA片段（326个碱基）的聚合酶链反应（PCR）扩增从细胞培养物中提取的。低至2.5 aM的不对称PCR产物可以检测与开发的方法。
Disposable magnetic DNA sensors using an enzyme-amplified strategy for the specific detection of a gene related to the Enterobacteriaceae bacterial family, based on the coupling of streptavidin-peroxidase to biotinylated lacZ gene target sequences, has been developed. A biotinylated 25-mer capture probe was attached to streptavidin-modified magnetic beads and hybridization with the biotinylated target was allowed to proceed. Then, a streptavidin-peroxidase polymer was attached to the biotinylated target, and the resulting modified magnetic beads were captured by a magnetic field on the surface of tetrathiafulvalene (TTF) modified gold screen-printed electrodes (Au/SPEs). The amperometric response obtained at -0.15 V after the addition of hydrogen peroxide was used to detect the hybridization process. In order to improve the sensitivity of the determination and reduce the assay time, different variables of the assay protocol were optimized. A low detection limit (5.7 fmol) with good stability (RSD = 7.1%, n = 10) was obtained. The DNA nonspecific adsorption at the magnetic beads was negligible, the obtained results thus demonstrating the possibility to detect the hybridization event with great specificity and sensitivity. The developed method was used for the analysis of Escherichia coli DNA fragments (326 bases) in polymerase chain reaction (PCR) amplicons extracted from a cell culture. As low as 2.5 aM asymmetric PCR product could be detected with the developed methodology.