Continued advancement of protein array, bioelectrode, and biosensor technologies will necessitate development of methods that allow for increased protein immobilization capacity and more control over protein orientation. Toward these ends, we developed a method involving modification of chitosan with nitrilotriacetic acid (NTA) to achieve immobilization of a larger amount of His-tagged protein than is possible with current methods. The immobilization capacity of our method was evaluated using His-tagged GFP (Green Fluorescent Protein) as a model protein. The average immobilization density on modified glass was about 32 ng/mm2. Our method is suitable for use on a variety of solid surfaces, including glassy carbon, silicon wafers, polycarbonate, and beaten gold.
The immobilization of proteins on the surface of solid materials is a key technique in the production of protein arrays, biosensors, and bioelectrodes for use in the analytical and bioelectronics fields. For these applications, it is necessary to arrange the orientation of immobilized protein molecules so that their function will be maintained. A number of different methods have been reported for immobilizing proteins on a variety of surfaces [
A dot blot is a technique used in molecular biology to detect biomolecules on nitrocellulose or PVDF (polyvinylidene difluoride) membranes [2,3]. Dot blotting is based on physical adsorption and allows for rapid immobilization of substrates and protein molecules. Deactivation of immobilized proteins can be suppressed by spotting them onto polymers such as polyacrylamide gels or polyethyleneglycol applied to a solid surface by inhibiting protease attack and minimizing shear, interfacial temperature or solvent denaturation [
MacBeath and Schreiber immobilized proteins on BSA-NHS (bovine serum albumin-N-hydroxysuccinimide)-coated slides fabricated by attaching a molecular layer of BSA to the surface of a glass slide and then activating the BSA with N,N,disuccinimidyl carbonate [
A self-assembled monolayer is an organized layer of amphiphilic molecules in which one end of the molecule, the “head group”, shows a specific, reversible affinity for a given substrate [1,6,11]. The hydrophilic head groups assemble together on the substrate, while the hydrophobic tail (alkyl chain) groups assemble far from the substrate surface. Because immobilization by this method is based on adsorption, it is difficult to control protein orientation.
Affinity methods such as those based on streptavidin/ biotin or His-tag/Ni-NTA interactions are commonly used in biochemical and histomorphology studies [
For intein-mediated immobilization, target genes are inserted in-frame and the mRNA is translated together with that encoding a target protein. This precursor protein (tag-intein-target protein) undergoes autocatalytic protein splicing, resulting in 2 products (tag-target protein and intein). By using this method, the N-terminal cysteine-containing peptides were specific immobilized onto a thioester-functionalized glass slide, which was subsequently used for screening of epitope mapping of kinase/phosphatase assays [
These protein absorption/immobilization techniques were designed for immobilizing large amounts of highly active proteins at a high density. However, there is still a need for techniques that will enable immobilization of greater amounts of protein while preserving protein activity. Immobilizing a sufficient quantity of a protein of interest over a minimal surface area remains challenging for many applications, as does maintaining the orientation of immobilized proteins so as to preserve their activity. To solve the problem of maximizing the amount and density of protein immobilized, we increased the effecttive surface area available for immobilization by modifying the surface of various solid substrates with a polymer (chitosan). To address the problem of maintaining the proper orientation of an immobilized protein, we modified chitosan bound to the surface with NTA to allow for specific binding between NTA and the Histagged protein. The method we describe here is simple with respect to both preparation of the substrate surface and protein immobilization. In addition, the method is suitable for use with a variety of substrates, and thus should have a wide range of applications.
We purchased 3-aminopropyltriethoxysilane from ShinEtsu Chemical (Tokyo, Japan). Shrimp shell chitosan (>75%, deacetylated) was purchased from Sigma-Aldrich (St. Louis, MO, USA). N-(5-amino-1-carboxypentyl) iminodiacetic monohydrate disodium salt (AB-NTA) was obtained from Dojindo (Kumamoto, Japan). Glutaraldehyde, NiCl2, imidazole, and all other reagents were purchased from Wako (Osaka, Japan).
The His-tag modified GFP expression vector (pGGFPH) was a kind gift from Professor H. Nakano (Graduate School of Bioagricultural Science, Nagoya University, Japan) [
Glass slides printed with highly water-repellent mark (Matsunami; Tokyo, Japan) were washed with acetone and milli-Q water. The slides were soaked in 1 M NaOH overnight at room temperature, and then washed with milli-Q water and 99.5% ethanol. Next, 3-aminopropyltriethoxysilane was vacuum-deposited onto the cleaned glass (CG) surface for 2 h at room temperature, washed with milli-Q water, and then the slides were baked for 2 h at 100˚C after blowing off residual water with dry air. This process produced amino silane-treated CG slides (AGs). The Ags were soaked in 1% (v/v) glutaraldehyde overnight at 37˚C to produce glutaraldehyde-treated AGs (GAGs). The GAGs were incubated overnight at 37˚C in chitosan (0.05% (w/v)) dissolved in 0.1 M acetic acid buffer (pH 5.0) supplemented with 0.25 mM sodium azide, producing chitosan immobilized glass slides (CIGs). The CIGs were rinsed twice with 0.1 M acetic acid buffer (pH 5) and then 3 times with milli-Q water. The CIGs were soaked in 1% (v/v) glutaraldehyde overnight at 37˚C to produce glutaraldehyde-treated CIGs (GCIGs). The GCIGs were incubated overnight at 37˚C in a 0.05% (w/v) solution of N-(5-Amino-1-carboxypentyl) iminodiacetic acid (AB-NTA) in 0.1 M HEPES buffer (pH 8.0), then rinsed 3 times with milli-Q water. The slides were then soaked in blocking solution (1% (v/v) glycine) for 1 h at 37˚C, then rinsed 3 times with milli-Q water, 3 times with 0.5 M NiCl2, and 3 times with milli-Q water to produce Ni-NTA immobilized GCIGs (Ni-NTAGCIGs).
Preparation of glassy carbon plates, silicon wafer plates, and polycarbonate plates was exactly same way to the protocol used to prepare the modified glass slides. Beaten gold for use in immobilization was prepared as follows. Cysteine was used instead of 3-aminopropyltriethoxysilane to modify gold surfaces with amino groups. Beaten gold was soaked in 1 M HNO3 for 2 h, rinsed with milliQ water, then pasted to the slide glass with double-faced tape. Cysteine thiol groups were adsorbed onto the cleaned gold surface by soaking the glass in 1 M cysteine solution (pH 9.4) for 2 h with stirring. The amino groups of cysteine residues bound to the surface were then crosslinked with amino groups of chitosan using glutaraldehyde. The subsequent preparation steps for beaten gold were almost the same as those used in the preparation of modified glass from the step of modified CIGs.
Immobilization of His-tagged GFP was examined on various substrates (CG, AG, GAG, CIG, GCIG, NiNTAGCIG, modified glassy carbon, modified silicon wafers, modified polycarbonate, and a modified gold surface). His-tagged GFP was adjusted to 200 μg/ml with TG buffer (50 mM Tris-HCl (pH 8.0), 10% (v/v) glycerol). A total of 100 ng of His-tagged GFP was spotted on each substrate and incubated for 60 min in a moist, dark chamber. Substrates were then washed with TG buffer, and immobilized His-tagged GFP was observed with a UV illuminator (TFML-20E, Funakoshi; Tokyo, Japan) or a fluorescence microscope (Nikon ECLIPSE TE2000-U; Tokyo, Japan). Images were captured with a digital camera (Nikon DIGITAL CAMERA D80; Tokyo, Japan). The amount of His-tagged GFP immobilized on each surface was determined by comparing the images with those for known amounts of His-tagged GFP (0, 0.25, 0.5, and 2 μg/spot) using Image J version 1.44 (http://rsbweb.nih.gov/ij/). Immobilized His-tagged GFP was also observed under a fluorescence microscope equipped with a Nikon CF Plan 10×/0.30 EPI Infinity lens. The scale was determined by comparison with the appropriate scale on the captured images.
The fluorescence associated with immobilized Histagged GFP at each modification step is shown in
taraldehyde-treated CIG (GCIG) and Ni-NTA-immobilized GCIG (Ni-NTAGCIG). Comparing the fluorescence obtained with GAG and GCIG surfaces demonstrated that His-tagged GFP was immobilized more efficiently on GCIG than on GAG. This result indicated that chitosan modification increased the number of sites available for cross-linking His-tagged GFP. More Histagged GFP was immobilized onto GCIG than NiNTAGCIG surfaces, indicating that the cross-linking of AB-NTA and chitosan was more efficient than the crosslinking of His-tagged GFP and AB-NTA.
The molecular weight of AB-NTA (about 263 Da) is much lower than that of His-tagged GFP (about 27 kDa). Therefore, without orientation control through AB-NTA, considerable steric hindrance associated with His-tagged GFP would be expected. Our results demonstrated that large amounts of His-tagged GFP can be immobilized by increasing the number of cross-linking sites with chitosan and providing orientation control with NTA. Based upon comparison with His-tagged GFP controls, we estimated that 0.26, 0.52, 0.41, and 0.43 μg His-tagged GFP/diameter 4 mm spot was immobilized onto the Ni-NTAGCIG surface (with an average of about 0.45 μg/the spot of diameter 4 mm, or about 32 ng/mm2).
To test the specificity of immobilization, His-tagged GFP immobilized on Ni-NTAGCIG surfaces was striped by soaking the slides in 1 M imidazole solution. Histagged GFP was removed from Ni-NTAGCIG slides by treatment with either imidazole (
We were also able to successfully immobilize Histagged GFP onto other solid surfaces, such as glassy carbon, silicon wafers (polished and unpolished), polycarbonate, and beaten gold (
tion efficiency was higher on the glassy carbon than on the other materials.
The immobilization capacity of our method was evaluated using His-tagged GFP as a model protein. The average immobilization density on modified glass was about 32 ng/mm2. We found that incorporation of chitosan increases the amount of His-tagged GFP that can be immobilized onto a variety of solid surfaces, including glassy carbon, silicon wafers, polycarbonate, and beaten gold. Our method could thus be applied to the immobilization of large amounts of highly active protein to surfaces such as enzyme electrodes or sensors. Experiments aimed at improving the method by eliminating the irregularities associated with polymer modification are ongoing.