Enzyme-based impedimetric detection of PCR products using oligonucleotide-modified screen-printed gold electrodes
Enzyme-based impedimetric detection of PCR products using oligonucleotide-modified screen-printed gold electrodes
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DOI：
10.1016/j.bios.2004.08.025
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发表时间：
2005-04-15
影响因子：
12.6
通讯作者：
Mascini, M
中科院分区：
文献类型：
--
作者：
Lucarelli, F;Marrazza, G;Mascini, M
本文介绍了一种基于酶的电化学基因传感器的优化和分析性能，开发使用一次性的修饰的丝网印刷金电极。固定的硫醇系探针定性研究通过法拉第阻抗谱。阻抗谱证实，硫醇部分明确地驱动寡核苷酸探针的固定化。此外，探针表面密度和杂交效率进行了定量，通过计时库仑measurement.Electrochemical转导的杂交过程中也进行了法拉第阻抗谱的装置，后耦合的链霉亲和素-碱性磷酸酶共轭和生物催化沉淀的不溶性和绝缘产品上的传感接口。计时库仑结果允许讨论杂交信号的大小方面的探针表面密度和它们相应的杂交效率。在12 pmol/L和12 nmol/L之间，基因传感器响应随寡核苷酸靶浓度在三个数量级上线性变化（r（2）= 0.9998）。估计的检测限为1.2 pmol/L（即，7.2通过分析从pBI 121质粒和含有1%和5%遗传修饰产物的大豆和玉米粉末获得的扩增样品，最终证明了阻抗测定基因传感器的分析有用性。这种未修饰的扩增子的感测通过与生物素化的信号传导探针的夹心杂交来实现。电化学酶放大试验允许明确鉴定所有转基因样品，而在所有阴性对照的情况下均未检测到显著的非特异性信号。(c)2004 Elsevier B. V.保留所有权利。
This paper describes the optimisation and the analytical performances of an enzyme-based electrochemical genosensor, developed using disposable oligonucleotide-modified screen-printed gold electrodes.The immobilisation of a thiol-tethered probe was qualitatively investigated by means of faradic impedance spectroscopy. Impedance spectra confirmed that the thiol moiety unambiguously drives the immobilisation of the oligonucleotide probe. Furthermore, both probe surface densities and hybridisation efficiencies were quantified through chronocoulometric measurements.Electrochemical transduction of the hybridisation process was also performed by means of faradic impedance spectroscopy, after coupling of a streptavidin-alkaline phosphatase conjugate and bio-catalysed precipitation of an insoluble and insulating product onto the sensing interface. Chronocoulometric results allowed discussion of the magnitude of hybridisation signals in terms of probe surface densities and their corresponding hybridisation efficiency. The genosensor response varied linearly (r(2) = 0.9998) with the oligonucleotide target concentration over three orders of magnitude, between 12 pmol/L and 12 nmol/L. The estimated detection limit was 1.2 pmol/L (i.e., 7.2 x 10(6) target molecules in 10 mu L of sample solution).The analytical usefulness of the impedimetric genosensor was finally demonstrated analysing amplified samples obtained from the pBI121 plasmid and soy and maize powders containing 1 and 5% of genetically modified product. Sensing of such unmodified amplicons was achieved via sandwich hybridisation with a biotinylated signaling probe. The electrochemical enzyme-amplified assay allowed unambiguous identification of all genetically modified samples, while no significant non-specific signal was detected in the case of all negative controls. (c) 2004 Elsevier B.V. All rights reserved.