We applied the acoustic transverse shear mode (TSM) method for study of the surface properties of a DNA aptasensor that specifically binds human immunoglobulin E (IgE). The biotinylated 45-mer DNA aptamers were immobilized on the surface of a self-assembled layer composed of a mixture of polyamidoamine dendrimers of the fourth generation with 1-hexadecanetiol covered by neutravidin. Using the TSM method, we studied the kinetics of changes of the series resonant frequency, f(s), and the motional resistance, R(m), of a quartz crystal transducer, used as a support for formation of the sensing layer. We have shown that attachment of the biotinylated DNA aptamers onto the surface covered by neutravidin results in a decrease of f(s), but in an increase of R(m). Similar changes of f(s) and R(m) were observed following addition of IgE. This suggests the contribution of friction forces to the crystal oscillation, which was taken into account in the calculation of the mass changes at the sensor surface following binding processes.
The poly(amidoamine)(PAMAM) dendrimers of fourth generation (G4) and 1-hexadecanethiol (HDT) has been used for formation of self assembled monolayers on a gold surface for preparation of quartz crystal microbalance (QCM) immunosensor for detection of human IgG. The anti-IgG antibodies were immobilized on a surface of G4-HDT layers or those covered by Protein A. In later case the oriented immobilization of anti-IgG takes place due to preferred interaction of F c portion of antibodies with the Protein A. The detection limit in a flow mode was 7 nM both for biosensor with and without Protein A. However, in the case of Protein A the number of bounded Ab was 1.4 times lower in comparison with those without Protein A. Thus, the sensitivity of the Protein A based sensor was higher due to orientation effect in comparison with randomly oriented Ab at a dendrimer surface without Protein A. Increased concentration of IgG and formation of Ab-Ag complexes resulted in increase of motional resistance, that suggest increased contribution of viscoelasticity, as revealed from impedance analysis. The most important property of the immunosensor was its ability for regeneration using 0.1 M glycine-HCl.
We applied atomic force microscopy (AFM) to study the surface topography of the supported thin films of various composition: hexadecanethiol (HDT), poly(amidoamine) dendrimers (PAMAM) of first generation (G1), mixture of HDT þ G1, glucose oxidase (GOX) and HDT þ G1 þ GOX layers. The AFM image of dendrimers in air revealed dome-shaped, randomly distributed aggregates of the size from 100 to 200 nm. GOX adsorbed on the gold support also formed aggregates of the diameter between 700 -900 nm and height up to 36 nm. The AFM images of the HDT þ G1 þ GOX layer in air revealed smooth surfaces with irregular pinholes, although these were corrugated in the buffer. Application of the voltage of þ 670 mV to the layers during one hour resulted in significant increase of the surface root means square roughness (R rms ). The electrical properties of G1 layers studied by cyclic voltammetry do not differ substantially from bare gold electrode, while HDT þ G1 mixed layers revealed intermediate electrical properties between HDT and G1. Immobilization of GOX onto the HDT þ G1 resulted further decrease of the layer permeability.
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