Effect of the local environment and state of the immobilized ligand on its reaction with a macromolecular receptor

Abstract

We consider a simple statistical model for interaction between a macromolecular receptor and a surface functionalized with low molecular weight ligands of the same type. For the example of the reaction between immobilized sulfamethoxazole and a selective antibody, we have used surface plasmon resonance to demonstrate that the proposed approach is appropriate for describing the dependence of the adsorption capacity on the surface density of the immobilized ligand and its charge state.Processes occurring at the interface between the surface of a sensor element and the external medium are a major source of information about the qualitative and quantitative composition of the analyte substance. Despite the fact that the result of such a reaction, as for any chemical reaction, depends on the reaction conditions and the ratio of reagents, there are a number of fundamental differences for surface processes which put this field into the separate category of the chemistry of grafted surface compounds [1]. From the standpoint of modern aspects of development of biosensor technologies, there is special interest in the interaction between macromolecular receptors (proteins, viruses, cells, etc.) and surface-modified solids when the area of projection of the receptor onto the surface is many times greater than the characteristic dimension of the grafted ligand (Table 1). A classic example of this type of problem is development of sensitive elements for immunospecific sensors for low molecular weight xenobiotics (toxins, hormones, pesticides, antibiotics etc.) based on competitive assay methods [2].In contrast to most homogeneous reactions in solution, occurrence of heterogeneous reactions at the interface between phases is due to such characteristic features as the decreased dimensionality of the grafted layer (two-dimensionality), interaction of molecules in the layer and/or with the support, polyfunctionality, the presence of vibrational and rotational degrees of freedom in the absence of lateral mobility, and ultralow absolute content in the sample [1]. From the standpoint of biosensor applications, this suggests that we take into account the composition (the type and state of the selective surface center, the chemical functionality of the auxiliary matrix), the structure (surface architecture and grafting density), immobilization procedures (one-center/multicenter, with/without an additional spacer), stability of the structure during analysis/reconstruction, and also technological compatibility with existing and future production processes. Thus the fixed 0040-5760/06/4204-0217

Interface functional architecture using mixed thiol monolayers: Effect of the composition and spatial organization on the immobilization of low-molecular-weight ligands

“…The result obtained, on one hand, supports the description of the interface processes proposed above and, on the other, confirms a significant level of nonspecific adsorption (~250¢¢-300¢¢) characteristic of the surface architecture studied. The latter effect is probably due to the hydrophobic nature of the base matrix, whose high nonspecific adsorption of proteins has been noted previously [3,12].…”

“…The immobilization of SMX was carried out similarly to our previous method [3] by binding the surface hydroxyl groups and terminal SMX amino groups by the trichlorotriazine heterocycle. In the same work, we carried out a detailed examination of the interaction for medium and low densities of the surface sites and showed that the system under such conditions operates satisfactorily.…”

“…In the same work, we carried out a detailed examination of the interaction for medium and low densities of the surface sites and showed that the system under such conditions operates satisfactorily. The studies were carried out using the PLAZMON/BioSuplar surface plasmon resonance spectrometer developed at the Institute of Semiconductor Physics of the National Academy of Sciences of Ukraine, which has been described in detail in our previous work [3,4,9]. A typical procedure for an adsorption experiment in a stationary cell (without flow of the probe) involved the following steps: 1) stabilization of the baseline in the working phosphate buffer (200 µL PBS, pH 7.3), 2) addition of a solution of the anti-SMX antibodies (200 µL in PBS, dilution in the cell 1 : 2000) to the buffer with rapid stirring, 3) monitoring of the reaction up to the establishment of a stationary response, and 4) reverse replacement of the probe in the PBS buffer (Fig.…”

“…In this case, TCT becomes incapable of binding with the surface at the second position and the subsequent immobilization of SMX proceeds in accord with the classical mechanism [10]. The increase in the adsorption capacity of the surface with a further decrease in z results from more favorable conditions for the anti-SMX-SMX reaction due to leveling out of the steric limitations caused by excess immobilized SMX molecules in the contact region [3].…”

“…Approximation of the SPR responses in the framework of various dynamic models indicated that all the kinetic curves can be described with high precision by a nonexponential functional equation DSPR » DSPR sat (1 -exp (-(t/t) b )) [3,13]. It is assumed in this model that the process occurs under conditions of space-time heterogeneity.…”