Functionalization and characterization of magnetic nanonanoparticles for bioseparation

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1 Functionalization and characterization of magnetic nanonanoparticles for bioseparation Chi-Hsien Liu, 1 Yi-Fan Hsien, 1 1 Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan Abstract Novel magnetic nanonanoparticles are prepared by covalently binding cationic lysine and arginine onto the surface of nanonanoparticles via a carbodiimide coupling method. The adsorption of plasmid onto surface-modified magnetic nanonanoparticles from solution containing polyethylene glycol (PEG) was studied herein. Nanonanoparticles modified by cationic ligands were characterized using Fourier-transformed infrared spectra, transmission electron micrography, base titration, and ninhydrin assay. Effects of PEG molecular weight and its concentration on plasmid purification by the magnetic nanoparticles were evaluated. PEG with average molecular weight of 8000 could efficiently assist the plasmid purification. Effects of chloride salts on the adsorption of plasmid were also investigated. The adsorption kinetics of cationic nanonanoparticles indicated that arginine-modified nanoaprticles had better capability for plasmid purification. Overall, the results demonstrated that the arginine-modified magnetic adsorbent increased the efficiency and speed of purification of plasmid. Introduction Magnetic separation has received considerable attention in biomedical research because of its important properties of large surface/volume ratios, biocompatibility, and low toxicity [1]. Functional magnetic particles can bind to selected targets in the presence of other suspended solids and can be easily isolated from solution by a magnetic field. Pretreatment of complex mixtures such as with sedimentation, centrifugation, and filtration processes can be eliminated using magnetic technologies. Magnetic particles with specific modifications are being widely studied for bio-separation applications including separation of bio-products [2]. New magnetic particles with functionalized surfaces were developed for the complicated separation processes in biotechnology fields. Magnetic separation of nucleic acids has several advantages compared to other techniques used for the same purpose. For example, nucleic acids can be isolated directly from crude sample materials such as tissue homogenates, cultivation media, microbial lysis, etc. Due to the possibility of

2 adjusting the magnetic and surface properties of the particles, they can be removed relatively easily and selectively even from viscous sample suspensions. In fact, magnetic separation is the only feasible method for the recovery of small particles (diameter approx μm) in the presence of biological debris and other fouling material of similar size [3]. Furthermore, the efficiency of magnetic separation is especially suited for large-scale purifications. These upcoming separation techniques also serve as a basis of various automated low- to high-throughput procedures that allow to save time and money[4]. Centrifugation steps can be avoided and the risk of cross-contamination when using traditional methods is no longer encountered. Although various types of magnetic particles are commercially available for nucleic acid purification, functionalized magnetic nanoparticles can offer new chances for efficient separation and magnetic transfection. Often, surface modifications of magnetic particles are necessary to improve their functionality and specificity. Carbodiimide is frequently used to functionalize magnetic particles by introducing specific ligands onto their surfaces. Ligand-modified magnetic particles are applied to bind targeted molecules so that they can be separated from the complex heterogeneous solution by a magnetic field gradient. Various ligands were investigated for purification of RNA or DNA including carboxyethyl phosphonic acid[5], arginine[6], bis-(2-hydroxyethyl methacrylate) phosphate[7], tetraethoxysilane[8], histidine[9], and polyethylenimine [10]. In this study, novel magnetic particles with lysine or arginine modification were developed to adsorb plasmid DNA. Our aim was to assess the possibility of developing a selective adsorbent for plasmid adsorption by incorporating lysine and arginine via a carbodiimide coupling method on particle surfaces. Functionalization of particles was characterized using Fourier-transformed infrared spectra, transmission electron micrography, base titration, and ninhydrin assay. Effects of PEG molecular weight and chloride salts on plasmid purification by the magnetic nanoparticles were also evaluated. The adsorption kinetics of cationic nanonanoparticles was studied by using three models. Materials and methods Chemicals N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), arginine, and lysine were purchased from Sigma (St. Louis, MO, USA). Paramagnetic iron oxide nanoparticles (USPIO-101, NH2-surface modified) were purchased from Taiwan Advance Nanotech (Taoyuan, Taiwan). Plasmid EGFP-C3 was obtained from Takara Bio (Shiga, Japan). The plasmid was amplified in E coli DH-5 alpha and purified by

3 using a plasmid maxiprep kit (GeneMark, Taiwan). EGFP encodes a red-shifted variant of wild-type green fluorescence protein which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.). All other chemicals were of analytic grade and were used without further purification. The water used in this study was freshly purified by the Milli-Q Gradient A10 system (Millipore, Molsheim, France). Preparation of ligand-coated magnetic particles Lysine modification of paramagnetic iron oxide nanoparticles was achieved by a coupling reaction between the carboxylic groups of ligand and the amino groups on the nanoparticles using EDC as a coupling reagent. One milligrams of magnetic nanoparticles was mixed with 0.2 ml of a solution containing 0.1 M amino acid and 0.64 mg of EDC, and reacted with a constant vortex rate (of magnitude 4) for 4 h at 37 C. The particles were then washed three times with phosphate-buffered saline (PBS) and recovered using a magnetic separation system. Lysine immobilization on the magnetic nanoparticles was determined according to the amount of lysine consumed after the conjugation reaction. In brief, the reaction solution (0.2mL) and ninhydrin solution (0.2 ml) were put in a test tube and heated in a boiling-water bath for 10 min. After heating, the tubes were immediately cooled in an ice-bath. The absorbance (570 nm) of the reaction mixture was measured after 1 ml of 50% alcohol was added to each tube and thoroughly mixed. Purification of plasmid DNA by modified magnetic nanoparticles The supernatant of DNA solution( 62 μg/ml, 20μL) in a 1.5 ml microcentrifuge tube was gently mixed with a (20 μg) suspension of Lysine-modified magnetic nanobeads in binding buffer (20 μl, 2.5M NaCl, 10% PEG) for 10 min at room temperature. The beads were recovered using a permanent magnet and the supernatant removed and the plasmid DNA desorbed by addition of elution buffer ( 50 μl H2O). The concentration of nucleic acids in solution can be readily calculated from absorbance at 260 nm by NanoDrop Spectrophotometer (Thermo, Wilmington, DE). Agarose gel electrophoresis A 1 % agarose gel containing 0.15 (v/v) safe-view dye (GeneMark).was run in a horizontal gel electrophoresis unit (Yuan Tuo tech. Ltd.) and 1 kb molecular mass standards(gen-kb) were used. The running buffer was TAE (40 mm Tris, 20 mm acetic acid, 1 mm EDTA, ph 8.0). Electrophoresis was carried out at 100 V for 40 min. Gel electrophoresis experiments were carried out for plasmid DNA in four different samples, feed solution, supernatants after adsorption, wash, and desorption

4 experiments. Effects of salt concentration and on desorption of plasmid DNA Effects of salt concentration and ph of elution buffer on desorption of plasmid DNA from Lysine-modified magnetic nanobeads was evaluated in the range of LiCl, NaCl, KCl concentration between M and ph , respectively. To determine the desorption of plasmid from magnetic nanoparticles, plasmid was adsorbed onto the adsorbent under the above-described conditions. Effects of various PEG and salt concentrations on plasmid desorption were evaluated using the following expression: adsorption rate = (DNA adsorbed / total DNA) 100%. Characterization of the nanoparticles FTIR spectra of lysine-modified nanospheres were analyzed using an FTIR spectrophotometer (Bruker Alpha, German). The dry nanoparticles were mixed with KBr (IR grade, Sigma) and pressed into a tablet form for the IR analysis. The morphology of nanospheres was observed using TEM (JEOL JEM 2000 EXII, Tokyo, Japan). Samples were diluted 1:25 with Milli-Q water and dried on carbon film (CF200-Cu, Electron Microscopy Science, Washington, DC) for 12 h and then analyzed by TEM Results and discussion In this study, cationic ligands were used to modify the magnetic particles to adsorb plasmid DNA. Water-soluble carbodiimide, a carboxyl- and amine-reactive cross-linker, conjugates the amino groups on magnetic particles and the carboxyl groups of ligands. First, ligands including lysine, arginine, ethylenediamine, cadaverine, and hexamethylenediamine were used to functionalize the magnetic nanoparticles for plasmid purification. Lysine and arginine have better DNA adsorption capacity as compared to ethylenediamine, cadaverine, and hexamethylenediamine (Fig.1). Previously, arginine modified agarose is successfully applied for the recovery of plasmid in a chromatographic system [6]. Arginine base interactions have been recognized at the atomic level as the most prevalent in numerous protein DNA structures[11-13]. Lysine can also interact with nucleotides at second structure [14, 15]. We also found that arginine or lysine modified nanoparticles could be used to efficiently separate the plasmid DNA herein. The surface modification on the magnetic particles was characterized by IR spectroscopy. The FTIR spectrum is widely used to investigate characteristics of stretching vibrations of functional groups in materials. The FTIR spectra of the original (containing amine groups) and arginine-modified magnetic particles are

5 presented in Fig. 2. Arginine-modified particles showed a distinctive absorption peak at 1652 cm -1, which corresponds to the stretching of the C=O of the amide groups. The broad absorption peak around 3100~3400 cm -1 of the modified particles was due to N-H stretching vibrations of amide. The appearance of the characteristic CO-NH peptide bond around 1652 cm 1 indicates that arginine was successfully attached to the amine group on the particle surfaces [16]. The original magnetic particles did not show distinguished peaks. The existence of arginine on the modified magnetic particles was confirmed herein. In addition, the arginine and lysine consumption in the conjugation reaction was used to evaluate the amino acids on the surfaces of nanoparticles using Ninhydrin analysis. The arginine and lysine density on the magnetic nanoparticles were found to be mole /g, mole /g, respectively. Amount of conjugated amino acid on the particles could be increased by manipulating the concentrations of crosslinker, nanoparticles, and amino acids. As demonstrated in the NaOH-titration curve (Fig 3), the isoelectric points were ranked in the following order arginine> lysine> NH2 group. The theoretical acid ionization constant (pka) of ammonium, lysine, and arginine were 9.2, 10.5, and 12.5, respectively. Therefore our results showed similar trends to the theoretical prediction. The adsorption kinetics of two kinds of magnetic nanoparticles were studied (Fig. 4). Arginine modified nanoparticles had higher plasmid adsorption capacity than lysine modified nanoparticles. Three kinetic models were tested to find out the appropriate adsorption rate expression: the pseudo-firstorder equation (1), pseudo-second-order equation (2), and intraparticle diffusion model (3) as expressed below [17]: 1/qt=k1/q1 1/t+1/ q1 (1) t/qt=1/k2 1/(q2) 2 +1/q2 t (2) qt= kp t C (3) where qt is the amount of DNA adsorbed (mg/ g) at time t, q1 is the maximum adsorption capacity (mg/g) for pseudo-first-order adsorption, k1 is the pseudo-first-order rate constant (min), q2 is the maximum adsorption capacity (mg/g) for pseudo-second-order adsorption, k2 is the pseudo-second-order rate constant (g/mg/ min), C is the intercept, and kp is the intraparticle diffusion rate constant (mg/g/ min 0.5 ). The kinetic parameters (k1, k2, kp, q1, q2, C) and correlation coefficients (r2 ) of two kinds of nanoparticles were calculated and summarized in Table 1. The correlation coefficients for pseudo-first kinetic model were close to 1.0 for the arginine- or lysine-modified nanoaprticles. These results revealed that the adsorption process obeyed the pseudo-first-order kinetic model. In addition, the correlation coefficients for the intraparticle diffusion model were much below1, indicating that the adsorption process did not follow the intraparticle diffusion kinetic

6 model. This is reasonable because the adsorption of plasmid occurred only on the surface of modified nanoparticles and the intraparticle diffusion resistance was absent. Also, all the plots of t/qt versus t were shown in Figs 5, 6, 7. Both revealed that the adsorption process obeyed the pseudo-first-order kinetic model. The maximum adsorption capacities q1 were determined to be 76 and 59 mg/g for Arginine- and lysine- modified nanoparticles, respectively, at 25. The effects of increasing temperature will be discussed in the near future. The ingredients of binding buffer such as the polymers and salts had great impacts on the adsorption of plasmid adsorption. In this study, effects of concentration of PEG 8000 on plasmid adsorption in arginine- or lysine- modified nanoparticles were demonstrated in Fig. 8. Ten percent of PEG 8000 had the maximal plasmid adsorption in the tested magnetic nanoparticles with arginine. Effects of PEG MW on plasmid adsorption in arginine- or lysine- modified nanoparticles were shown in Fig.9. PEG with MW of 8000 could enhance the separation of plasmid by using the magnetic nanoparticles. Finally, Effects of chloride salts on plasmid adsorption in arginine modified nanoparticles were studied in Fig. 10. Sodium chloride had the best adsorption results. In summary, the results showed nano magnetic nanoparticles can be effectively adsorbed plasmid DNA. Figures and Table Table 1. Adsorption Kinetics for the nanoparticles and plasmid at 25 q 1 k 1 R 2 q 2 k 2 R 2 K p C R 2 Arg Lys

7 Fig.1. Effects of various ligands on the adsorption of plasmid DNA Fig.2. FTIR spectra for the Nanoparticles with different modification Fig.3. The titration curves for three kinds of nanoparticles

8 Fig. 4. Plasmid DNA adsorption kinetics for arginine- or lysine- modified nanoparticles Fig.5. Plot of data regression for pseudo-firstorder equation

9 Fig.6. Plot of data regression for pseudo-second-order equation Fig. 7. Plot of data regression for intraparticle diffusion model Fig. 8. Effects of concentration of PEG 8000 on plasmid adsorption in arginine- or lysine- modified nanoparticles

Fig.9. Effects of PEG MW on plasmid adsorption in arginine- modified nanoparticles Fig. 10. Effects of chloride salts on plasmid adsorption in arginine-modified nanoparticles References 1. Ito, A.

10 Fig.9. Effects of PEG MW on plasmid adsorption in arginine- modified nanoparticles Fig. 10. Effects of chloride salts on plasmid adsorption in arginine-modified nanoparticles References 1. Ito, A., et al., Medical application of functionalized magnetic nanoparticles. Journal of Bioscience and Bioengineering, (1): p Altinta, E.B., et al., Use of magnetic poly(glycidyl methacrylate) monosize beads for the purification of lysozyme in batch system. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, (1-2): p Berensmeier, S., Magnetic particles for the separation and purification of nucleic acids. Applied Microbiology and Biotechnology, (3): p Safarik, I. and M. Safarikova, Magnetic techniques for the isolation and

11 purification of proteins and peptides. BioMagnetic Research and Technology, Sarkar, T.R. and J. Irudayaraj, Carboxyl-coated magnetic nanoparticles for mrna isolation and extraction of supercoiled plasmid DNA. Analytical Biochemistry, (1): p Sousa, F., et al., Specific recognition of supercoiled plasmid DNA in arginine affinity chromatography. Analytical Biochemistry, (2): p Xie, X., et al., Preparation and application of surface-coated superparamagnetic nanobeads in the isolation of genomic DNA. Journal of Magnetism and Magnetic Materials, (1-2): p Chiang, C.L., C.S. Sung, and C.Y. Chen, Application of silica-magnetite nanocomposites to the isolation of ultrapure plasmid DNA from bacterial cells. Journal of Magnetism and Magnetic Materials, (2): p Sousa, F., et al., Separation of supercoiled and open circular plasmid DNA isoforms by chromatography with a histidine-agarose support. Analytical Biochemistry, (1): p Chiang, C.L., et al., Application of superparamagnetic nanoparticles in purification of plasmid DNA from bacterial cells. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, (1-2): p Luscombe, N.M., R.A. Laskowski, and J.M. Thornton, Amino acid-base interactions: A three-dimensional analysis of protein-dna interactions at an atomic level. Nucleic Acids Research, (13): p Hoffman, M.M., et al., AANT: The Amino Acid-Nucleotide Interaction Database. Nucleic Acids Research, (DATABASE ISS.): p. D174-D Lejeune, D., et al., Protein-nucleic acid recognition: Statistical analysis of atomic interactions and influence of DNA structure. Proteins: Structure, Function and Genetics, (2): p Gromiha, M.M., K. Yokota, and K. Fukui, Understanding the recognition mechanism of protein-rna complexes using energy based approach. Current Protein and Peptide Science. 11(7): p Zhang, T., et al., Analysis and prediction of RNA-binding residues using sequence, evolutionary conservation, and predicted secondary structure and solvent accessibility. Current Protein and Peptide Science. 11(7): p Kwon, E.J. and T.G. Lee, Surface-modified mesoporous silica with ferrocene derivatives and its ultrasound-triggered functionality. Applied Surface Science, (15): p

12 17. Chang, Y.C. and D.H. Chen, Adsorption kinetics and thermodynamics of acid dyes on a carboxymethylated chitosan-conjugated magnetic nano-adsorbent. Macromolecular Bioscience, (3): p

Spherotech, Inc Irma Lee Circle, Unit 101, Lake Forest, Illinois PARTICLE COATING PROCEDURES

Spherotech, Inc Irma Lee Circle, Unit 101, Lake Forest, Illinois PARTICLE COATING PROCEDURES SPHERO TM Technical Note STN-1 Rev C. 041106 Introduction Currently, there are several methods of attaching biological ligands to polystyrene particles. These methods include adsorption to plain polystyrene