Mirsky V M, Riepl M, Wolfbeis O S
University of Regensburg, Institute of Analytical Chemistry, Chemo- and Biosensors, Regensburg, Germany.
Biosens Bioelectron. 1997;12(9-10):977-89. doi: 10.1016/s0956-5663(97)00053-5.
Self-assembled monolayers of omega-mercaptohexadecanoic acid and omega-mercaptohexadecylamine on gold electrodes are stable at neutral pH and display pure capacitive behavior at frequencies around 20 Hz. Different methods of covalent immobilization of proteins on these monolayers are compared. Various reagents including succinimides, thionylchloride, p-nitrophenol and carbodiimides were used to activate the carboxy groups of the adsorbed monolayer of omega-mercaptohexadecanoic acid. Glutaraldehyde, cyanuric chloride and phenylene diisocyanate were used to activate the amino groups of the monolayer of omega-mercaptohexadecylamine. The immobilization of albumin on the activated surface was studied by capacitive measurements. The N-hydroxysuccinimide and carbodiimide methods were identified as most suitable for protein immobilization in that they did not compromise the insulating properties of the alkylthiol layer and led to maximal increase of its dielectric thickness. These approaches were used for a layer-by-layer preparation of a capacitive immunosensor. Specifically, antibodies to human serum albumin were immobilized on the alkylthiol mono-layer. Binding of the antigen led to a decrease of the electrode capacitance. The detection limit of the immunosensor is as low as 15 nM (1 mg/l).
ω-巯基十六烷酸和ω-巯基十六胺在金电极上的自组装单分子层在中性pH值下稳定，在20Hz左右的频率下表现出纯电容行为。比较了在这些单分子层上共价固定蛋白质的不同方法。使用包括琥珀酰亚胺、亚硫酰氯、对硝基苯酚和碳二亚胺在内的各种试剂来活化吸附的ω-巯基十六烷酸单分子层的羧基。使用戊二醛、三聚氯氰和亚苯基二异氰酸酯来活化ω-巯基十六胺单分子层的氨基。通过电容测量研究了白蛋白在活化表面上的固定化。N-羟基琥珀酰亚胺和碳二亚胺方法被确定为最适合蛋白质固定化，因为它们不会损害烷基硫醇层的绝缘性能，并导致其介电厚度最大增加。这些方法用于电容免疫传感器的逐层制备。具体而言，将人血清白蛋白抗体固定在烷基硫醇单分子层上。抗原的结合导致电极电容降低。免疫传感器的检测限低至15 nM（1 mg/l）。
