A nanoparticle-based immobilization assay for prion-kinetics study

Abstract

Magnetic and gold coated magnetic nanoparticles were synthesized by co-precipitation of ferrous and ferric chlorides, and by the micromicelles method, respectively. Synthesized nanoparticles were functionalized to bear carboxyl and amino acid moieties and used as prion protein carriers after carbodiimide activation in the presence of N-hydroxysuccinimide. The binding of human recombinant prion protein (huPrPrec) to the surface of these nanoparticles was confirmed by FTIR and the size and structures of the particles were characterized by transmission electron microscopy. Findings indicate that the rate of prion binding increased only slightly when the concentration of prion in the reaction medium was increased. Rate constants of binding were very similar on Fe3O4@Au and Fe3O4-LAA when the concentrations of protein were 1, 2, 1.5, 2.25 and 3.57 microg/ml. For a 5 microg/ml concentration of huPrPrec the binding rate constant was higher for the Fe3O4-LAA particles. This study paves the way towards the formation of prion protein complexes onto a 3-dimensional structure that could reveal obscure physiological and pathological structure and prion protein kinetics.

Figure 1

TEM images and particle sizes distribution of gold coated magnetic nanoparticles- Fe₃O₄@Au (a) and huPrPrec functionalized gold coated magnetic nanoparticles- Fe₃O₄@Au-huPrPrec (b).

Figure 2

TEM images and particle sizes distribution of magnetic nanoparticles- Fe₃O₄-LAA (a) and huPrPrec functionalized magnetic nanoparticles Fe₃O₄-LAA- huPrPrec (b).

Figure 3

Optical properties of pure gold solution and gold coated magnetic nanoparticles. The absorption maxima were found at 520 and 558 nm for Au and Fe3O4@Au, respectively.

Figure 4

Functionalization of magnetic and gold coated magnetic nanoparticles: the chemisorption of LAA onto Fe₃O₄ (a), the carboxylation of Fe₃O₄@Au using mercaptopropionic acid (b), the activation of the carboxyl groups on the particle, the formation of N-hydroxysuccinimidyl ester in the presence of EDC (c), and the immobilization of huPrPrec onto particles (d). R₁ denotes nanoparticles.

Figure 5

FTIR spectra of Fe₃O₄@Au (a), Fe₃O₄-LAA (b) huPrPrec (c), Fe₃O₄@Au-huPrPrec (d) Fe₃O₄-LAA-huPrPrec(e).

Figure 6

Plots of huPrPrec binding onto Fe₃O₄@Au and Fe₃O₄-LAA (a) as a function of time for various concentrations of huPrPrec.

Figure 7

Dependence of the rate constants of huPrPrec binding on the concentrations of reacting huPrPrec.