Nanotech 2010

Functionalized Magnetite Nanoparticles with a Polymeric Coating and single chain Fv antibody fragments (scFvs) for the 5’- Nucleotidase.

C. Chapa Gonzalez, R. Terrazas Reza, O. Zavala Tapia, A. Martínez Martínez, C. Martínez Pérez, P. Garcia Casillas
Universidad Autonoma de Ciudad Juarez, MX

Keywords: nanoparticles, magnetite, chemical co-precipitation, protein separation


5-‘Nucleatidase is an enzyme that can be used as a bioindicator for disease diagnosis such as cholestasis, hepatic ischemia, liver necrosis, liver tumor, hepatitis and hepatotoxic drugs, for this reason a new technique that could be used for early diseases detection has been developed. This work describes the synthesis of a new composite magnetite-polymeric single chain-Fv antibody for separation of this enzyme. Magnetite nanoparticles with main diameter of 16 nm, 27 nm and 200 nm were obtained thought the control of the aging conditions in chemical co-precipitation methods. The nanoparticles have spherical shape and superparamagnetic behavior and were coated with silica, aminosilane, and silica-aminosilane shell. The influence of the particle size, saturation magnetization, and coating on protein conjugation was determinate by the use of bovine serum albumin (BSA). The magnetic saturation for nanoparticles oscillates between 48 to 78 emu/g depending of the coating. The best combination to obtain the highest BSA protein adsorption (10.87 mg BSA/mg nanoparticles) is when an average particle size of 16 nm coated with a silica-aminosilane shell. Those particles with the major protein affinity of BSA were selected to conjugate with scFvs (obtained by the phage display method). Using external magnetic field, separation assays were carried out and the amount of isolated protein was determinate by the difference of A280 before and after assays. The scFvs were obtained by recombinant DNA technology; foreign peptide domains were fuse to coat protein pIII in phage display vector pComb3X. The nanoparticles were characterized by Field Emission Scanning Electron Microscopy (FE-SEM), Energy Dispersive X-Ray Spectroscopy (EDS), Vibrating Sample Magnetometry (VSM) and X-Ray Diffraction (XRD). Protein adsorption was determined using a UV-Vis spectrometer.
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