Specific Binding of Large Aggregates of Amphiphilic Molecules to the Respective Antibodies

Abstract

The Binding of nonylphenol to respective antibodies immobilized on solid substrates was studied with the methods of total internal reflection ellipsometry (TIRE) and QCM (quartz crystal microbalance) impedance spectroscopy. The binding reaction was proved to be highly specific having an association constant of KA=1.6x10(6) mol(-1) L and resulted in an increase in both the adsorbed layer thickness of 23 nm and the added mass of 18.3 microg/cm2 at saturation. The obtained responses of both TIRE and QCM methods are substantially higher than anticipated for the immune binding of single molecules of nonylphenol. The mechanism of binding of large aggregates of nonylphenol was suggested instead. Modeling of the micelle of amphiphilic nonylphenol molecules in aqueous solutions yielded a micelle size of about 38 nm. The mechanism of binding of large molecular aggregates to respective antibodies can be extended to other hydrophobic low-molecular-weight toxins such as T-2 mycotoxin. The formation of large molecular aggregates of nonylphenol and T-2 mycotoxin molecules on the surface was proved by the AFM study.

Total internal reflection ellipsometry as a label-free assessment method for optimization of the reactive surface of bioassay devices based on a functionalized cycloolefin polymer

“…With this design, we were able to incorporate five reaction microwells onto a sensing substrate which is a half of a gold-coated standard glass slide (26 mm ´ 76 mm). Furthermore, the volume of the microwells were substantially reduced to approximately 4.7 µl compared to a typical few millilitres in previous works [16][17][18][19][20][21][22]. This feature is very important for experiments requiring expensive analytes like proteins or oligonucleotides.…”

“…This feature is very important for experiments requiring expensive analytes like proteins or oligonucleotides. Total internal reflection ellipsometry (TIRE) or surface plasmon enhanced ellipsometry (SPEE) is a spectroscopic ellipsometry measurement method operated under the total internal reflection (TIR) condition [16][17][18][19][20][21][22][23][24]. As in a conventional spectroscopic ellipsometry method, TIRE also measure two ellipsometric angles Y and D versus wavelengths.…”

“…A four-layer ellipsometric model summarized in Table 1 was used in the fitting of the measured Y and D spectra by using the PsiDelta 2 software (Jobin Yvon Horiba, France) to deduce the thickness of each add layer to the Au surface [19][20][21][22]. The refractive indices of all four layers in the model were fixed in the fitting model.…”

“…! various analytes to a metal surface, typically gold [16][17][18][19][20][21][22][23][24]. Here, we propose to apply TIRE to monitor the binding of the biomolecules to a ZR surface functionalized with film containing carboxyl groups.…”

Evaluation of Different Nonspecific Binding Blocking Agents Deposited Inside Poly(methyl methacrylate) Microfluidic Flow-Cells

“…Fluorescent intensity was measured inside the microwells of the PMMA flow-cells to evaluate the effectiveness of each coatings against the non-specific adsorption of Cy5-labeled anti-IgG. Finally, total internal reflection ellipsometry (TIRE) [14][15][16][17][18][19][20][21] with spectra and kinetic data of adsorption of anti-a-human chorionic gonadotropin (anti-a-hCG) was used to evaluate the stability of the coatings under buffer rinsing. Our results could be useful when choosing a blocking agent, its deposition and characteristics, for suppression of non-specific binding of biomolecules on a PMMA surface.…”