Institute of Analytical Sciences, University of Lyon, UMR CNRS 5280, 5 rue de la Doua, Villeurbanne 69100, France; Laboratory of Environmental Engineering, Badji Mokhtar University of Annaba, BP 12, Annaba 23000, Algeria.
Institute of Analytical Sciences, University of Lyon, UMR CNRS 5280, 5 rue de la Doua, Villeurbanne 69100, France.
Mater Sci Eng C Mater Biol Appl. 2014 May 1;38:286-91. doi: 10.1016/j.msec.2014.02.013. Epub 2014 Feb 16.
In this work, using electrochemical impedance spectroscopy (EIS), we have, for the first time, label-free monitored protein immobilization on a gold surface through a strategy of electroaddressing, compatible with the production of microarrays for multi-detection. This functionalization is achieved via the alkyne/azide cycloaddition, better known as the "click" reaction. The electroaddressing was applied to a polythiol hexynyl derivative previously grafted onto the gold surface. This compound consists of two dithiol phosphate groups and a hexynyl function and was synthesized through a supported synthesis approach, from a dithiol reagent, phosphoramidite (DTPA), and a hexynyl phosphoramidite. Next, an azide-PEG3-biotin derivative was grafted onto the modified gold surface by electro-chronocoulometry. The "click" reaction was controlled by electrochemical impedance spectroscopy, showing the change in impedance only when the electroaddressing was performed at -300 mV. No effect on the EIS signal was observed when a positive potential was applied, confirming the specificity of the electroactivation. Biotin-modified electrodes were used to fix streptavidin and the immobilization was monitored using EIS. Fluorescent streptavidin-functionalized silica nanoparticles were also specifically grafted onto the biotinylated gold surface in order to confirm the "click" reaction using fluorescence microscopy. The obtained streptavidin platform was used to detect the surface coverage by biotinylated human serum albumin (HSA). The lowest detectable concentration is 10 pg/mL, and surface saturation is obtained with concentrations higher than 100 ng/mL.
在这项工作中，我们首次通过电寻址策略，无标记地监测金表面上蛋白质的固定，该策略与用于多检测的微阵列生产兼容。这种功能化是通过炔烃/叠氮化物环加成反应实现的，该反应通常称为“点击”反应。电寻址应用于先前接枝在金表面上的聚硫醇炔烃衍生物。该化合物由两个二硫醇磷酸基团和一个炔烃官能团组成，通过从二硫醇试剂、亚磷酰胺（DTPA）和炔基亚磷酰胺合成的支撑合成方法合成。接下来，通过电 chronocoulometry 将叠氮化物-PEG3-生物素衍生物接枝到修饰的金表面上。通过电化学阻抗谱控制“点击”反应，仅当在-300 mV 时进行电寻址时才会观察到阻抗的变化。当施加正电势时，对 EIS 信号没有影响，这证实了电激活的特异性。生物素修饰的电极用于固定链霉亲和素，并用 EIS 监测固定化。还使用荧光链霉亲和素功能化的二氧化硅纳米粒子特异性地接枝到生物素化的金表面上，以使用荧光显微镜确认“点击”反应。获得的链霉亲和素平台用于检测生物素化人血清白蛋白（HSA）的表面覆盖率。最低可检测浓度为 10 pg/mL，高于 100 ng/mL 的浓度即可达到表面饱和。
