Rapid and selective separation for mixed proteins with thiol functionalized magnetic nanoparticles
© Lee et al.; licensee Springer. 2012
Received: 5 January 2012
Accepted: 31 May 2012
Published: 31 May 2012
Thiol group functionalized silica-coated magnetic nanoparticles (Si-MNPs@SH) were synthesized for rapid and selective magnetic field-based separation of mixed proteins. The highest adsorption efficiencies of binary proteins, bovine serum albumin (BSA; 66 kDa; pI = 4.65) and lysozyme (LYZ; 14.3 kDa; pI = 11) were shown at the pH values corresponding to their own pI in the single-component protein. In the mixed protein, however, the adsorption performance of BSA and LYZ by Si-MNPs@SH was governed not only by pH but also by the molecular weight of each protein in the mixed protein.
KeywordsSilica-coated magnetic nanoparticles Thiol Mixed protein separation BSA LYZ
The ability to separate and identify different proteins accurately and efficiently out of a biological sample is of utmost importance for application such as biomedicine, energy resources, environmental protection, and catalysis [1–3]. However, it remains imperative to understand the inherent physical characteristics of proteins, such as the isoelectric point (pI)1 and molecular weight. Because the technique is underlying the implicit assumption, the behavior of an intact biological system consisting of a plurality of proteins is coexistent mixed state .
In recent two decade, magnetic field-based separation using magnetic nanoparticles (MNPs) have received considerable attention because of their easy operation, uniqueness, and nano-scale size [5, 6]. Besides their size-dependent properties, MNPs applications are affected by their surface functionalization. In this context, silica-coated MNPs (Si-MNPs) has several advantages arising from their stability under aqueous conditions (at least if the pH value is sufficiently low) and easy functionalization by hydroxyl group on the exposed silica surface surrounding MNPs [7, 8]. A key consideration during protein separation process is to control the driving force of the sorbent surface behind the adsorption of proteins involves hydrophobic and electrostatic interactions . Therefore, the successful adsorption of proteins onto MNPs depends on the proper surface modification.
Generally, thiol functional group (−SH) is known to play a significant role of cross-linking proteins, because the strongest nucleophile is present in the cystein thiol group of natural proteins [10, 11]. Moreover, the value of the molecular electrostatic potential bond of thiol group may possess electronegative property . Hence, it is presumed that thiol group can provide hydrophobic (non polar) condition to MNPs surface for improving protein adsorption . To date, coupling biomolecules onto thiol-reactive surface has been achieved mainly for protein immobilization on gold-based biosensor surfaces through self-assembled monolayer system [13, 14]. On the other hand, Si-MNPs are directly functionalized through the coupling of organosilanes (R′-Si(OR)3) including thiol group with free silanol groups of the silica surface . Therefore, Si-MNPs having an easily modifiable silica surface as well as a superparamagnetic MNPs core might serve as an ideal platform for magnetic separation of proteins.
Here, we have exploited thiol group functionalized silica-coated magnetic nanoparticles (Si-MNPs@SH) as a magnetic sorbent for effective protein separation from aqueous condition. Furthermore, we discuss protein adsorption effects by Si-MNPs@SH in mixed proteins with various pI value and molecular weight, which are derived from straightforward experiments of two binary proteins: BSA and LYZ.
Silane coupling reagent were purchased from Aldrich Chemical Co. (St. Louis, MO, USA) and used as received. All other chemicals were of analytical reagent grade. BSA and LYZ were obtained from Sigma. Alexa Flour 488 and Texas Red fluorescence dyes were purchased from Molecular Probes Inc (Eugene, OR, USA). Distilled, deionized water was used for the preparation of all aqueous solutions.
Preparation of Si-MNPs and thiol group functionalization (Si-MNPs@SH)
The silica-coated Fe3O4 magnetic nanoparticles were prepared using the method of Kang et al. . The Si-MNPs were then modified successively with silane coupling agent to introduce the -SH. Briefly, 1 g of Si-MNPs was dispersed in 50 mL of anhydrous toluene containing 1 mg of (3-mercaptopropyl) triethoxysilane (MPTS), and the mixture was allowed to react at 110°C for 8 h under dry nitrogen. The resultants were separated by simple magnetic attraction and washed repeatedly with toluene and finally vacuum dried for further use.
Protein separation experiments
where E ad (%) is the efficiency of protein adsorbed by a unit mass of dry particles, C i (μM) and C f (μM) are the protein concentrations of the initial and final solutions, respectively. All the tests were conducted in triplicate.
Analysis of fluorescence-labeled proteins
For the fluorescence labeling of BSA and LYZ, Alexa Flor 488 and Texas Red Protein Labeling Kit (Molecular Probes, UK) were used according to the manufacturer's instructions. Fifty microliters of 1 M sodium bicarbonate solution was added into a 0.5 mL of 100 μM BSA and LYZ solutions. The protein solutions were mixed with 0.5 mL of reactive Alexa Flor 488 and Texas Red dyes, respectively, for 1 h at room temperature. After the reaction, the remaining traces of free dyes were removed by column chromatography. In mixed protein solution, the adsorption of fluorescence-labeled BSA and LYZ to functionalized Si-MNPs was observed by confocal microscopy. Fluorescence spectra were measured using a microplate reader. The lasers provided excitation of Alexa Flour 488 for BSA and Texas Red for LYZ at 494 and 595 nm, respectively, and emitted fluorescent lights were detected at 519 and 615 nm, respectively.
The particle size and morphology of the Si-MNPs were determined by high resolution transmission electronic microscopy (HR-TEM) using a JEM-2000EX TEM (JEOL, Japan). The wide angle X-ray diffraction (XRD) patterns were taken with 40 kV, 160 mA Cu Kα radiation using a Rigaku Denki instrument. The magnetization of Si-MNPs@SH at room temperature up to 10 kOe was measured using a vibrating sample magnetometer (VSM 4179) (Oxford Instruments, UK). Identification and characterization of thiol functional group were carried out using X-ray photoelectron spectroscopy (XPS) (Sigma Probe equipped with monochromatic Al source, 15 kV and 100 W, Thermo Scientific, UK). Confocal microscopy was performed with a MultiProbe 2001 confocal scanning laser microscope, with an argon/krypton laser and ImageSpace Software from Molecular Dynamics, USA. The fluorescence intensity and absorbance of the samples were measured using microplate reader Infinite M200 (Tecan Ltd., Switzerland). The fluorescence was measured five times for each sample with a 20 μs integration time.
Results and discussion
Synthesis and characterization of Si-MNPs@SH
Separation of mixed proteins with si-MNPs@SH
In conclusion, this study reported the fabrication of thiol group functionalized Si-MNPs (Si-MNPs@SH) and its use for effective protein separation in mixed proteins. For the purpose, binary proteins BSA and LYZ, which have different molecular weights and pI values, were used as model proteins for protein separation. Compared with the single-component proteins, the adsorption performance of BSA and LYZ by Si-MNPs@SH was governed not only by pH but also by the molecular weight of each protein in the mixed protein solution. The phenomenon was visualized by confocal microscope analysis of protein-bound Si-MNPs@SH. We expect that the results may have important implications in the binding effect of mixed proteins onto nano-sized particles and should also contribute to the further application of using magnetic nanoparticle-based technologies to separate proteins from intact biological samples such as blood plasma and serum.
SYL carried out the biological and analytical studies. CYA and JL achieved the synthesis and characterization of the magnetic nanoparticles. JHL involved in instrumental characterization. JHC participated in the design of the study and coordination of the work as lead investigator. All authors contributed to the interpretation of the results and the drafting of the manuscript. All authors read and approved the final manuscript.
Thiol functional group
Thiol group functionalized silica-coated magnetic nanoparticles
Bovine serum albumin
(3- mercaptopropyl) triethoxysilane
X-ray photoelectron spectroscopy.
This work was supported by a grant from the Fundament R&D Program for Core Technology of Materials funded by Ministry of Knowledge Economy, Republic of Korea.
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