The adsorption of monolayers and biofunctionalization of nanoparticles were qualitatively assessed by FTIR spectroscopy. Figure 5 shows the FTIR spectra of Fe₃O₄@Au, Fe₃O₄-LAA, huPrPrec, Fe₃O₄@Au-huPrPrec, and Fe₃O₄-LAA-huPrPrec. The coating of Fe₃O₄@Au and Fe₃O₄ with carboxyl groups and LAA was noted by the appearance of various peaks in the regions from 1400 to 1650 cm^-1 in the spectra of Figure 5a and 5b. The multitude of small peaks crowding the spectra with features associated with various functional groups hinder identification of peaks specific to carbonyl and amine groups in the chemisorbed regions making the differentiation between Fe₃O₄@AuCOOH (Fe₃O₄@Au bearing carboxyl groups) and Fe₃O₄-LAA difficult. Spectra in Figure 5c depicts the functional groups related to pure huPrPrec via characteristic bands of proteins at 1490, 1541, and 1645 cm^-1 wave numbers assignable to the symmetric stretching of the dissociated carboxylic group originating from the amino acid, amide I and amide II as shown in the spectra of Figure 5d and 5e. In the 1415-1300 cm^-1 region of the spectra in Figure 5c, d and 5e a weak band typical to the spectra of the carboxylate group attributable to proteins was also noted. The presence of protein characteristics on the spectra of pure huPrPrec and on the spectra of Fe₃O₄@AuCOOH-huPrPrec, Fe₃O₄-LAA-huPrPrec confirmed that huPrPrec was effectively bound to the nanoparticles.

huPrPrec was immobilized onto functionalized Fe₃O₄@Au and Fe₃O₄-LAA particles after activation by EDC in the presence of NHS. The amount of huPrPrec was determined spectrophotometrically using different concentrations of huPrPrec. The rate of binding expressed as the change in the concentration of unbound protein in the reaction medium was measured using a linear regression analysis of the plots of huPrPrec concentration as a function of time. The coefficient of variation between replicates was less than 4%. Figure 6a, b, c, d, e and 6f show plots of the decrease in huPrPrec when the initial concentrations were 1, 1.5, 2, 2.25, 3.75, and 5 μg/ml, respectively. Rate constants of huPrPrec presented in table 1 shows an increase in the initial concentration of prion protein from approximately 0.06 to 0.194 h^-1, and 0.058 to 0.212 h^-1 for Fe₃O₄@Au and Fe₃O₄-LAA, respectively, in the range of huPrPrec concentrations tested. For each concentration of protein, the rate constant was about the same, irrespective of the type of nanoparticles, except when a concentration of 5 μg/ml of huPrPrec was used. The binding occurred gradually and reached approximately 94%, 87%, 65%, 73%, 79%, and 74% for Fe₃O₄@Au and 94%, 87%, 67%, 73%, 81%, and 71% for Fe₃O₄-LAA, when the initial concentrations of huPrPrec were 1.5, 2, 2.25, 3.75, and 5 μg/ml, respectively, after 20 h of reaction. Figure 7 show plots of the rate constant as a function of the initial concentration of huPrPrec. The trend observed shows that the rate constant increased exponentially with the initial concentration of huPrPrec. Fischer et al. 25 immobilized the disease associated prion protein using 0.1 mg/ml of reacting prion protein for immobilizing the disease associated prion protein solution onto magnetic beads. Here, we were able to quantify down to 0.6 % of 1 μg/ml of reacting huPrPrec. The low concentration of huPrPrec coupled to the increase in the binding rate constants observed with increased protein concentration suggests that the detection methodology is sensitive. Table 2 shows the percentage of huPrPrec binding to the nanoparticles. The trend was quite similar for the immobilization carried out using both carriers although the overall binding rate constants were slightly higher in Fe₃O₄-LAA than the Fe₃O₄@Au conjugates. This indicated that binding was slightly more effective when Fe₃O₄-LAA complex was used. The bigger sizes of Fe₃O₄@Au should have favored the rate of binding since it offers a greater surface area, but this is not the case here where Fe₃O₄-LAA exhibited a better binding affinity. The difference in the affinity of binding could be attributed to the fact that LAA possess amino acids that originated from the protein that may exhibit higher affinity to the surface of the particles. The difference in the rate constants observed at this concentration may be associated with the difference in functional groups on the surface of the nanoparticles used. Prion protein is a complex entity, and although numerous binding partners have been found for the protein, its function still remains unclear, since each portion of the protein has its own functional properties 26 . Although significant effort has been made in elucidating prion related diseases, numerous questions on the structure and conformation of the protein are still to be answered. Huang et al 16 demonstrated that immobilizing specific proteins onto magnetic nanoparticles allows the molecules to expand and acquire a better conformation that could lead to an improved activity. As the possibility to move magnetic nanoparticles using an external magnetic field is an essential asset for molecular manipulation for diagnosis purposes, the immobilization of prion protein onto magnetic and gold coated magnetic nanoparticles and the kinetics data obtained in this study may contribute to an improved biological assay in which the protein could be directed toward target locations such as monoclonal prion protein antibodies 8 by appropriate directed magnetic fields. Furthermore, data on binding kinetics can be useful in the design and evaluation of molecular carriers and in the measurement of the efficacy of the surface chemistry developed. Developing methodologies for attachment of prion protein to magnetic nanoparticles can also contribute to enhancing in vivo and in vitro manipulation of the molecules which is essential in elucidating the structure of the protein and the resulting activity.

Iron (II) chloride tetrahydrate 97 %, iron (III) chloride hexahydrate 99%, sodium hydroxide, acetic anhydride, nitric acid, 1-butanol, octane, toluene, methanol, L-aspartic acid (LAA), cetyltrimethylammonium bromide (CTAB), 3-mercaptopropionic acid (3-MPA), sodium tetrahydridoborate (NaBH₄), and Phosphate Buffer Saline (PBS), pH 7.4 were obtained from Sigma-Aldrich Inc. (St Louis, USA). Hydrogen tetrachloroaurate (III) hydrate (HAuCl₄) was obtained from Sigma-Aldrich Inc. (Milwaukee, WI). Streptavidin, a strain of Streptomyces avidini was purchased from Sigma-Aldrich Inc. (MO, USA) and 1-ethyl 3-(3-dimethylaminepropyl) carbodiimide hydrochloride (EDC) from Pierce (Rockford, IL, USA) was used to complete the streptavidin-biotin reaction in the presence of N-hydroxysuccinimide (NHS) (Sigma-Aldrich, Allentown, PA). Human recombinant prion protein histidine-tagged (23–231), huPrPrec was obtained from Abcam Inc (Cambridge, MA).

Magnetic nanoparticles Fe₃O₄ were prepared by hydrothermal co-prepcipitation of ferric and ferrous using NaOH as a base as described by Kouassi et al 21 . Typically iron (II) chloride and iron (III) chloride (1:2) were dissolved in nanopure water at a concentration of 0.25 M iron ions and chemically precipitated at room temperature (25°C) by repeatedly adding 1 M NaOH to maintain a constant pH of 10. The precipitates were heated at 80°C for 35 min under continuous mixing and washed 4 times in water and several times in ethanol. During washing, the magnetic nanoparticles were separated from the washing liquid using a magnetic separator of strength greater than 20 megaoersted (MOe). The particles were finally dried in a vacuum oven at 70°C.

Fe₃O₄@Au were prepared using reverse micelle of CTAB using 1-butanol as a cosurfactant and octane as the oil phase by modification of the procedure developed by Jun et al. 22 . The size of the reverse micelle is dependent on the molar ratio of water to surfactant. In this work, particles were prepared by choosing a molar ratio of water to CTAB, w as [H20]/[CTAB] = 8. The procedure and components of the experiment were described by Jun et al. 19 . To a 2.5 ml of solution A containing 1 M FeCl₃, 0.5 M FeCl₂, 0.17 mole of CTAB, 0.7 mole of butanol, and 0.17 mole of octane, was added a 2.5 ml of solution B containing 1 M NaBH₄ and the same composition of CTAB, butanol, and octane as in solution A. The blend was heated at 60°C while vigorously mixing for 20 min to form magnetic nanoparticles. A 2 ml amount of a solution C containing 0.44 mole of HAuCl₄, 0.8 mole of CTAB, 0.25 mole butanol, and 0.011 mole octane and an equivalent volume of a solution D containing 1.6 M NaBH₄, 0.8 mole of CTAB, 0.25 mole of butanol, and 0.11 mole of octane were successively added. The pH was kept at 11 by adding minute amounts of 0.5 M NaOH. The mixture was continuously mixed for 15 minutes. The gold coated magnetic nanoparticles formed were washed four times with water, several times with methanol and dried in a vacuum oven for 6 h. To demonstrate that a gold layer was formed around the magnetic nanoparticles, gold colloidal and Fe₃O₄@Au solutions were prepared by dissolving 1.5 mg of HAuCl₄ and Fe₃O₄@Au, respectively, in 4 ml of water and the absorbance was measured using a UV-Vis Beckman Du spectrophotometer.

1.5 g of magnetic nanoparticles was immersed in 50 ml of 0.1 M LAA solution prepared in nitric acid of pH ≈ 2. The mixture was sonicated for 15 min and vigorously stirred for 10 h at room temperature. An external magnetic field was applied to recover the particles and washed two times with nanopure water. The process is expected to ensure the chemisorption of aspartic acid, bearing carboxyl and amino groups onto the surface of the particles.

The carboxylation of gold coated magnetic nanoparticles was done to allow the formation of amide bond between carboxyl groups on the surfaces of the nanoparticles with amino groups from the protein molecules. Magnetic nanoparticles (1.5 g) were added to 15 ml ethanolic solution of 3-MPA 20 mM, sonicated for 48 h and rinsed in nanopure water and dried in a vacuum oven for 6 h.

One mg of EDC, 1.2 mg of NHS and 8 mg of carboxyl or LAA functionalized particles were added to 3 ml phosphate buffer solution (pH 7.4) containing 3–15 μg of huPrPrec. The mixture was then sonicated at 4°C for 10 min and continuously shaken for 18 h at room temperature. At 4 h time intervals a magnetic separator was used to separate the particles from the reaction medium and 30 μl of the reacting solution was taken and used for the determination of protein content using the Bio-Rad Protein Assay using human IGg as the protein standard. Portion of the particles were taken and washed with PBS to separate unbound protein from the particle surfaces and used for characterization.

The concentration of protein in the supernatant was monitored every four hours for a period of twenty hours to evaluate the concentration of bound huPrPrec and the rate constants were determined using linear regression analysis from the plot of protein concentration versus time. Each point is the average of two measurements. The sensitivity of the assay was demonstrated by examining the dose response of the rate constant versus huPrPrec concentration in the concentration range between 2 and 5 μg/ml.

The sizes of magnetic and gold coated magnetic nanoparticles were characterized by transmission electron microscopy (TEM, JEM 1200 EXII, JEOL) and the attachment of biomolecules qualitatively by FTIR spectroscopy (Biorad FTS 6000, Cambridge, MA). The samples for TEM analysis were prepared as follows: a drop of magnetic nanoparticles was dispersed in nanopure water and the resulting solution was sonicated for 5 min to obtain better particle dispersion characteristics. A drop of the dispersed solution was then deposited onto a copper grid and dried overnight at room temperature. Confirmation of the binding of huPrPrec onto the magnetic nanoparticles was done using FTIR spectroscopy. Nanoparticles bearing huPrPrec obtained after the immobilization procedure were separated using a magnetic separator and washed with PBS to remove unbound huPrPrec. A small amount of the remaining huPrPrec-magnetic nanoparticle conjugates were mixed in 5 ml of PBS and a drop of the mixture was deposited on the FTIR micro-ATR sample holder for analysis.