ARTICLE pubs.acs.org/JPCC
Novel Biologically Active Silver-Avidin Hybrids Gil Mor,† Sefi Vernick,‡ Hila Moscovich-Dagan,‡ Yael Dror,† and Amihay Freeman*,† †
Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, and ‡Department of Physical Electronics, Faculty of Engineering, Tel Aviv University, 69978, Israel
bS Supporting Information ABSTRACT: Coupling of biologically active proteins, for example, enzymes and binding proteins, with metals carries huge potential inherent in the integration of these hybrids with miniaturized electronics, medical devices, and in vivo imaging. Here we propose and demonstrate feasibility of the preparation of novel, biologically active silver-avidin hybrids by electroless silver deposition directed to the surface of single, soluble avidin molecules, with retention of their solubility and highly specific biotin binding capacity. The process is based on conjugation of silver ions reducing polymers to avidin surface, followed by the addition of silver ions under mild physiological conditions. The partially overlapping silver patches thus obtained on the protein’s surface provided soluble, biologically active hybrids, retaining their specific biotin binding capability of both low-molecular-weight and high-molecularweight biotinylated molecules and exhibiting enhanced thermal stability. The hybrids thus obtained were successfully used for molecular imaging of cancer cells prelabeled with biotinylated monoclonal antibody.
’ INTRODUCTION A major hurdle on the way to improved biosensing and in vivo imaging is a highly effective integration of functions, for example, conductivity or X-ray diffraction, enabled by metals with functions provided by biologically active proteins, for example, biocatalysis and biorecognition. Recent efforts to cope with this challenge were focused on the nanoscale integration of metals and biological macromolecules by two main approaches: conjugation of prefabricated metal nanoparticles or metal electroless deposition (ED).1 Metallization of biological templates by ED was mostly used for the construction of nanowires, for example, by using DNA,2 microtubules,3 and peptide nanostructures4 as biotemplates. Metallization attempts were directed to the surface of the substrate by either nonspecific adsorption of nucleating metal ions, for example, palladium or platinum ions, followed by their chemical reduction, with subsequent enlargement by ED aiming at continuous metal deposition.5 9 These approaches resulted, however, in rough, granular, and relatively thick (10 35 nm) metal deposits.10,11 The feasibility of a novel mechanism for directing electroless silver deposition to the surface of single, soluble enzyme molecules, while retaining their enzymatic activity, was recently demonstrated by our lab.12 The feasibility of this approach was established by the conjugation of a new polymeric silver ions reducing agent to the surface of soluble glucose oxidase (GOx) molecules [polyglutaraldehyde (PGA) displaying Schiff bases with β-alanine], followed by the removal of nonbound reducer and the addition of silver ions. The GOx-silver hybrids thus obtained enabled “nanowiring” of the catalytic site of the metallized GOx molecules to a platinum electrode, enabling glucose determination in the absence of oxygen.12 Furthermore, the feasibility of a second novel mechanism for directing deposition of other metals, for example, palladium deposition r 2011 American Chemical Society
to the surface of GOx molecules, was also recently demonstrated by our lab.13 This mechanism was based on surface modification of enzyme molecules providing a uniform surface display of a multipoint array of a chelating agent. Following chelation of Pd2+ ions, removal of unbound ions, and subsequent reduction of the chelated ions, nucleation sites of metallic Pd were obtained on the surface of GOx. The GOx-Pd hybrid thus obtained retained its solubility and enzymatic activity and demonstrated unique molecular “nanowiring” capability of the enzyme catalytic site to platinum electrode.13 Here we propose and demonstrate feasibility of the fabrication of silver-binding protein hybrid retaining its solubility and specific binding capability of either low-molecular-weight or high-molecular-weight ligands, aiming at their use for labeling and imaging of receptors displayed on cancer cells or tumor surfaces. The fabrication of this envisaged hybrid was illustrated by controlled electroless deposition of silver directed to the surface of single, soluble molecules of avidin, well-known for its high affinity of binding the water-soluble vitamin d-biotin (Kd 10 15) and its numerous applications in biochemistry and biotechnology.14 16 To ensure free access of ligands to silver-Avidin’s binding sites, we attempted protection of these sites by the reversible ligand, HABA (2-(4-Hydroxyphenylazo) benzoic acid, readily displaced by biotin displaying ligands.17,18 Silver deposition was directed to Avidin’s surface by the conjugation of silver reducing polymeric chains to lysine residues. Controlled enlargement of the silver nucleation sites thus obtained into metallic patches without blocking ligand’s access to the binding sites was subsequently affected by the addition of soluble reducing polymeric chains. The whole Received: April 12, 2011 Revised: October 6, 2011 Published: October 06, 2011 22695
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Figure 1. Schematic description of avidin metallization with silver by directed electroless deposition. (A) Conjugation of HABA to the biotin binding site and silver ions reducer (PGA-β alanine) to amine residues (indicated in blue). (B) Magnification of an area from A, presenting the formation of silver ions reducing agent [polyglutaraldehyde (PGA) displaying Schiff bases with β-alanine] conjugated to the surface of the protein. (C) Formation of silver nucleation sites on the protein surface. (D F) Horizontal expansion of silver patches from the nucleation sites thus obtain by additional silver reducer (PGA-β-alanine).
process was designed to be carried out under mild physiological conditions.
’ EXPERIMENTAL SECTION Preparation of Silver-Avidin (Ag-Av) Hybrids. Ag-Av hybrid was prepared by modification and optimization of the method previously applied for enzyme metallization by Dagan-Moscovich et al.12 as follows: A solution (3 mL) of 6 μM (tetramer) of egg white avidin (Sigma, cat. no. A9275) dissolved in 50 mM HEPES buffer, pH 8, containing 1.8 mM of PGA (molecular weight ∼1000, prepared as previously described),19 was incubated overnight at 4 °C. Nonbound PGA was removed by centrifugation (4000 rpm for 10 min) in ultrafiltration tubes (Millipore, Amicon Ultra-0.5, Ultracel-10 Membrane, 10 kDa, cat. no. UFC501024). The PGA-activated avidin (6 μM, 3 mL), resuspended in HEPES buffer, was incubated with 22 mM β-alanine for 2 h at 4 °C to display Schiff-base silver reducing groups on Avidin’s surface. Nonbound β-alanine was removed by centrifugation in ultrafiltration tubes as described above. To protect the binding sites during silver reduction, we incubated the avidin-PGA-β-alanine complex (∼12 μM, 1.5 mL) with 120 μM HABA (2-(4-hydroxyphenylazo) benzoic acid, Sigma, cat. no. H5126) for 5 min. The complex thus obtained was subsequently treated with 2.4, 3.6, or 4.8 mM AgNO3 (Frutarom, cat. no. 5553260) dissolved in DDW, corresponding to avidin/ AgNO3 molar ratios of 1:200, 1:300, and 1:400 (abbreviated as Ag-Av 200, Ag-Av 300, and Ag-Av 400, respectively) providing different numbers of nucleation sites of silver on the protein surface. Following 2 h of incubation, we added 750 μL of PGA-βalanine solution (6 mM PGA, 100 mM β-alanine, incubated for 2 h in room temperature) to the modified avidin to enlarge and thicken the silver nuclei. Metallization was allowed to proceed overnight at room temperature. Excess amounts of silver ions were subsequently removed by ultrafiltration as described above.
Ag-Av Purification. The Ag-Av hybrids were purified by cation exchange chromatography (HiTrap SP HP 1 mL, GE € KTA system (GE Healthcare, cat. no. 17-1151-01) using an A Healthcare Life Sciences) controlled by Unicorn 5.10 software, equilibrated with buffer A (50 mM HEPES buffer, pH 8). Following washing with buffer A to remove freely soluble PGAβ-alanine and the protecting HABA molecules, hybrids were eluted from the matrix with a 0 100% gradient of buffer B (50 mM HEPES buffer and 1 M NaCl, pH 8). Fractions were collected and eluted peaks were detected at 280 and 420 nm. Fractions isolated were washed from NaCl by ultrafiltration and stored at 4 °C. Protein content was determined by Bradford’s method20 using untreated avidin as standard. Binding Assays. Ligand binding by Ag-Av hybrids was tested by binding of the low-molecular-weight ligand HABA, followed by its displacement by biotin as well as by binding of a highmolecular-weight ligand biotinylated alkaline phosphatase. The binding of small molecules was based on the color change resulting from HABA binding by avidin.17 The protein samples (2.4 μM monomer) in 50 mM HEPES buffer, pH 8 were titrated with increasing HABA (1 mM in DDW) concentrations, followed by titration with 0.5 mM D-biotin (Sigma, cat. no. B4501) dissolved in DDW. The data obtained were subjected to nonlinear curve fitting and analyzed by GraphPad software. Binding of high-molecular-weight ligand was carried out by microplate binding assays as follows: Avidin-PGA and Ag-Av 400 at a concentration of 5 μg mL 1 in phosphate buffer containing 8 g L 1 NaCl, 0.24 g L 1 KH2PO4, 1.44 g L 1 Na2HPO4, and 0.2 g L 1 KCl, equilibrated to pH 7.4 with NaOH, were separately transferred to 96-well flat-bottomed microtiter plate (Costar 3596, Corning) and incubated at 37 °C overnight. The wells were washed three times with 0.05% v/v PBS-Tween blocked with PBS containing 1% w/v bovine serum albumin at 37 °C for 1 h and washed again with PBS-Tween. We added 100 μL of 166 ng mL 1 biotinylated alkaline phosphatase 22696
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Figure 2. Cation exchange chromatography of avidin-silver hybrids. Protein content and silver content were monitored spectrophotometrically at 280 and 420 nm, respectively. (A) Untreated avidin; (B) Ag-Av 200; (C) Ag-Av 300; and (D) Ag-Av 400.
(Sigma cat. no. P-1318) in PBS and incubated the solution at 37 °C for 1 h. The wells were washed with PBS-Tween and 100 μL of a 1 mg mL 1 solution of p-nitrophenyl phosphate (Sigma cat. no. 104-0) dissolved in diethanolamine buffer equilibrated to pH 9.8 with HCl containing 0.5 mM MgCl2 were added. Absorbance at 405 nm was recorded following 10 min of incubation. Physical and Chemical Surface Characterization of Ag-Av Hybrids. Characterization of Ag-Av hybrids was carried out using high-resolution transmission electron microscopy (HRTEM) by a Philips Tecnai F20 field-emission gun electron microscope operating at 200 kV and transmission electron microscopy (TEM) by a JEOL 1200EX electron microscope operating at 80 kV, using SPI carbon-coated 200 MESH copper grids. Thermal Stability and Residual Binding Capability Assays. Aliquots of untreated avidin and Ag-Av 400 were incubated for 20 min at temperatures within the range of 25 100 °C and subjected to SDS-PAGE analysis and biotin-binding assays. For SDS-PAGE analysis, protein samples were preincubated with 1% (v/v) β-mercaptoethnol and subsequently analyzed by 15% SDS-PAGE without boiling. Proteins were visualized by Coomassie brilliant blue staining, and the amounts of oligomeric avidin were quantitated by densitometry. To determine and compare the residual biotin binding capability of Ag-Av 400 following heat treatment to that of untreated avidin, we transferred samples to 96-well microplates, and residual binding capability of biotinylated alkaline phosphatase was determined as described above. Immunolabeling with Ag-Av. EGFR overexpressing A431 human epidermoid carcinoma cells were labeled by a two-step procedure: exposure to biotinylated anti-EGFR antibody (BErbitux), followed by washing to remove nonbound antibody and subsequent specific labeling of the EGFR-biotinylated Erbitux sites by Ag-Av. Synthesis of biotinylated Erbitux was carried out by the addition of 5.2 μL of 10 mM biotin N-hydroxy-succinimide ester (NHS-biotin) dissolved in DMSO to 2 mg mL 1 Erbitux (Cetuximab, Merck) in 200 μL of PBS and incubation at room temperature for 4 h. Nonbound biotin was separated from the antibody by dialysis and stored at 4 °C. The biotin-labeling ratio was determined by the HABA assay as described in ref 21. EGFR immunolabeling was carried out by electron microscopy: 1.5 mL of A431 cells (∼107 cells mL 1) grown in DMEM
Figure 3. Electron micrographs of Ag-Av 400 (obtained without staining): (A) Low magnification TEM micrographs of a field of discrete Ag-Av 400 hybrids (Bar: 100 nm). (B) High magnification HRTEM micrographs of hybrid (Bar: 2 nm). (C) Size distribution of Ag-Av 400 hybrids. (D) EDX-HRTEM analysis of hybrid’s surface composition.
(containing 10% fetal bovine serum, 0.03% L-glutamine, 100 units mL 1 penicillin, and 100 μg mL 1 streptomycin in 5% CO2 at 37 °C) and suspended following 1 min trypsinization was incubated with 3% bovine serum albumin in PBS buffer at pH 7.4 for 0.5 h to block nonspecific antibody binding sites. Erbitux (1 mL, 30 μg mL 1) or biotinylated Erbitux (30 μg mL 1) were subsequently added to the medium and incubated for 2 h at room temperature. Cells were washed three times with PBS, and 0.5 mL of Ag-Av (1 μM) was added for 2 h of incubation at room temperature. The labeled cells were washed three times with PBS-Tween (0.05%) and three times with PBS and fixed with 2.5% glutaraldehyde in PBS overnight at 4 °C, followed by treatment with 1% OsO4 in PBS for 2 h at 4 °C. Dehydration was carried out in increasing ethanol concentrations (25 100%), followed by embedding in glycid ether. Thin sections (80 100 nm) were mounted on 22697
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Figure 4. Retention of low-molecular-weight ligands (HABA and its displacement by biotin) binding by each step of Avidin’s metallization process: Avidin, Avidin-PGA, Avidin-PGA-β alanine, and Ag-Av 400 hybrids. Samples were diluted to 7.2 μM (monomers), and their HABA binding capacity was assayed by monitoring OD500 (A). Subsequently, biotin was added, affecting HABA displacement and its accompanying decrease in OD500 (B).
Figure 5. Time dependence of avidin subunits cross-linking by PGA treatment monitored by SDS-PAGE. (M: molecular mass markers).
Formvar/carbon-coated grids (Ems) and examined by Jeol 1200EX transmission electron microscope. Images were captured using SIS Megaview III and iTEM imaging platform (Olympus).
’ RESULTS AND DISCUSSION Directed Deposition of Silver on the Surface of Single, Soluble Avidin Molecules. Silver deposition directed to the
surface of avidin was readily carried out by controlled conjugation of silver-reducing polymeric chains to avidin surface. Biotin binding sites were preferably protected by prebinding of HABA molecules. Conjugation of PGA onto avidin surface (AvidinPGA) was carried out first using large PGA excess, followed by the removal of nonbound PGA by ultrafiltration. Subsequent Schiff base formation between displayed unreacted aldehyde groups and β-alanine, enabled effective reduction of silver ions into atomic silver nuclei, readily carried out under mild physiological conditions. Enlargement of the reduced silver nuclei thus obtained into partially overlapping silver patches formed on Avidin’s surface was carried out by the addition of soluble PGA-β-alanine conjugate into the silver ions containing solution (Figure 1). The increase in metallic silver content thus obtained on Avidin’s molecules was reflected in the appearance of a new absorption peak at 420 nm (See Figure 1 of Supporting Information). To evaluate the tolerance of the silver-avidin hybrids thus obtained to increasing content of hybrids’ silver without losing their solubility and binding capability, we added different silver ion input concentrations to 12 μM of soluble Avidin-PGA-βalanine. Results obtained indicated that the maximal silver/avidin molar ratio that could be applied without affecting solubility was 600:1. Avidin-silver hybrids were hence prepared at molar ratios of Ag/Av of 200:1, 300:1, and 400:1 (described as Ag-Av 200,
Figure 6. Residual biotinylated alkaline phosphatase binding by untreated avidin and Ag-Av 400 hybrids following exposure for 20 min at elevated temperatures.
Ag-Av 300, and Ag-Av 400, respectively). Hybrid solutions thus obtained were stable in solution for at least 3 weeks at room temperature. Because the deposited metallic layer thus obtained is expected to mask a part of the natural positive surface electrical charge of avidin molecules, gradient ion exchange chromatography was attempted for Ag-Av hybrid purification. Untreated avidin or metallized avidin samples were applied onto SP sepharose cation exchange column, and the eluted fractions were simultaneously detected at 280 and 420 nm, indicating the protein-to-metallicsilver ratio (Figure 2). Protective HABA molecules and the added reducer, PGA-β alanine, were eluted first by the effluent (buffer A, 50 mM HEPES buffer, pH 8), whereas untreated avidin or metallized avidin were retained bound to the column. Metallized avidin was subsequently eluted by 7% content of buffer B (50 mM HEPES buffer containing 1 M NaCl, pH 8), whereas untreated avidin was eluted following the addition of 36% of buffer B. The data of Figure 2 clearly indicate that untreated Avidin was not present in a detectable amount in the solutions resulting from the metallization process. Three Ag-Av hybrids with different protein/silver ratios (Ag-Av 200, Ag-Av 300, and Ag-Av 400) were readily purified, exhibiting OD420/OD280 ratios of 0.1, 0.33, and 1.3, respectively. Ag-Av 400 hybrid fraction, exhibiting the higher silver content within this group, was subjected to further structural and chemical analysis. Structural analysis of the Ag-Av 400 hybrid using electron microscopy (Figure 3) without staining showed single nanoparticles population, 41% of which were within 9 to 10 nm average size and 30% within the 7 to 8 nm size, all exhibiting metal coating (Figure 3 A,B). Because the reported sizes of avidin 22698
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Figure 7. Molecular labeling and imaging of EGFR displaying A431 cells by Ag-Av 400 hybrid following preincubation with untreated Erbitux (A) or biotinylated Erbitux (B,C; Bars: (A,B) 500 nm; (C) 200 nm).
tetramer and monomer crystal structures are 5.4 6.5 and 2.4 3.2 nm, respectively (Protein data bank (PDB) file 1AVD,) it appears that the thickness of the added silver layer was ∼1.5 nm. The main size population of Ag-Av 400 hybrids (Figure 3C) appears to be silver hybrids of avidin tetramers, dimers, and monomers as well as minor population of avidin “bis”’ tetrameric formation. Energy dispersive analysis (EDX) confirmed the presence of metallic silver layer on the surface of these particles (Figure 3D). Effect of Metallization on Low-Molecular-Weight Ligand Binding. Avidin is known to bind biotin in an irreversible manner22 with a binding constant of ca. 1015 M 1, whereas the reversible binding of its ligand analogue 2-(4-hydroxyphenylazo) benzoic acid (HABA) is much weaker (105 M 1). Because HABA binding may be readily spectrophotometrically monitored by measuring OD500, the retention of reversible lowmolecular-weight ligand binding by each step of the metallization procedure was investigated by monitoring first HABA binding by Avidin, Avidin-PGA, Avidin-PGA-β alanine, and Ag-Av 400, followed by bound HABA displacement by biotin binding (Figure 4). Whereas the kinetics of HABA binding was similar for Avidin and its derivatives, the biotin concentration required for full HABA displacement was 6 μM for untreated Avidin, 10 μM for Avidin-PGA/Avidin-PGA-β alanine, and 13 μM for Avidin-silver 400 hybrid, respectively, indicating mild diffusional limitations affected by Avidin’s surface modifications, without blocking ligand access to its binding sites. The data of Figure 4 thus clearly indicate retention of low-molecular-weight ligands specific binding by Avidin metal hybrids obtained by our method. The impact of binding site protection throughout the metallization process by HABA prebinding was investigated by comparing the retained binding capacity of silver-avidin hybrids prepared in the absence of HABA to hybrids prepared following HABA site protection. A 10% higher binding capacity was observed when silver deposition was carried out with siteprotected avidin (data not shown). Avidin’s binding sites were hence protected throughout the silver deposition process in all subsequent experiments by presaturation of the binding sites prior to silver reduction with HABA. This ligand was readily removed from the avidin-silver hybrid throughout the cation exchange chromatography procedure described above. Effect of Metallization on High-Molecular-Weight Ligand Binding. The retention of high-molecular-weight ligand binding by Ag-Av 400 hybrid was tested by binding of biotinylated alkaline phosphatase by Ag-Av 400 hybrid in ELISA assay. Untreated avidin and the interim Avidin-PGA derivative served as controls. The avidin samples were adsorbed on an ELISA plate. Following washing and blocking with PBS-Tween and PBS containing 1% w/v bovine serum albumin, respectively, the adsorbed samples were incubated with biotinylated alkaline phosphatase,
followed by washing and removal of nonbound enzyme. The substrate p-nitrophenyl phosphate was subsequently added to the wells, and the absorbance at 405 nm following 10 min of incubation was measured. Results obtained 0.51 ( 0.1 for untreated Avidin and 1.08 ( 0.05 for Ag-Av 400, clearly indicating that silver-avidin retained its capability to specifically bind a highmolecular-weight biotinylated ligand (biotinylated enzyme). The improved binding capacity of Ag-Av 400 may be partially attributed to the PGA pretreatment of avidin, stabilizing its tetrameric structure by intramolecular subunits cross-linking as previously suggested by Reznik et al.23 To support this assumption, avidin was incubated with PGA and intrasubunit crosslinking monitored by reaction arrest, followed by subjecting the samples to SDS-PAGE. Because avidin monomeric subunit has an apparent molecular mass of 16 kDa, it may be readily distinguished from dimers and tetramers by SDS-PAGE. It appears, however, that most of the cross-linking events involved intracellular cross-linking of monomers to dimers or tetramers (Figure 5). The size distribution of Ag-Av 400 hybrid population recorded by electron microscopy (Figure 3) indicated that 9% of the hybrid particles reflected intermolecular cross-linking into whole avidin dimer (larger aggregates were not recorded; see also figure 2 in Supporting Information). Thermal Stability of Ag-Av Hybrid. The impact of PGA cross-linking of avidin on the hybrid’s thermal stability was subsequently investigated by SDS PAGE analysis and binding assays. Ag-Av 400 hybrid thermal stability was compared with that of untreated avidin: whereas untreated avidin survived incubations up to ∼50 °C, Ag-Av 400 exhibited significantly higher tetramer stability surviving incubations up to ∼90°. Stabilization of the tetrameric form also had an impact on biotinylated alkaline phosphatase-binding capability: whereas untreated avidin incubated at 55 °C for 20 min exhibited residual biotinylated phosphatase binding capacity of 65% of its original biotinylated enzyme binding capability following the same condition (Figure 6). Our results are in agreement with previously reported data on avidin or streptavidin subunit thermostability improvement exhibited by chimeric avidin containing a 21 amino acid segment of another member of the protein family24 or by the introduction of specific covalent bonds between adjacent subunits across the dimer dimer interface of the protein23 or by introducing intermonomeric disulfide bridges between avidin subunits.25 Molecular Labeling and Imaging of EGFR Displaying Cells by Ag-Av Hybrids. The demonstrated capability of Ag-Av hybrids to specifically bind biotinylated proteins paved the way to its application as a marker for electron microscopy: by targeting and binding to cancer cells pretreated with a biotinylated monoclonal antibody, for example, biotinylated-Erbitux prelabeled A431 cancer cell line displaying EGFR receptor. Cells 22699
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The Journal of Physical Chemistry C were treated with bovine serum albumin to block nonspecific sites, followed by incubation with 30 μg mL 1 Erbitux or 30 μg mL 1 biotinylated Erbitux, an anti-EGFR monoclonal antibody. Ag-Av 400 hybrid (1 μM, 0.5 mL) was added to label Erbitux-EGFR displaying sites. Cells were washed thoroughly, and thin sections of cells were prepared for electron microscopy analysis without staining. A clear difference was observed between cells that were prelabeled with the biotinlated Erbitux (Figure 7B,C) and cells pretreated with Erbitux control (Figure 7A). The clear difference observed between cells prelabeled with untreated anti-EGFR antibody and identification of the location of metallic silver labeling of biotinylated anti-EGFR antibodylabeled A431 cells shows that for the Avidin-silver labeling of A431 cells, biotinylated anti-EGFR was specific and predominantly localized where a high level of their transmembrane EGFR expression is usually found.26
’ CONCLUSIONS Directed electroless silver deposition was successfully applied for the preparation of novel binding protein silver hybrids composed of a biologically active binding protein core coated with a thin metallic silver layer. Avidin-silver hybrids thus obtained retained their solubility and specific binding capability of both low-molecular-weight ligands, for example, HABA, and biotin and high-molecular-weight ligands, for example, biotinylated enzyme or antibody. Furthermore, Avidin-silver hybrids exhibited improved thermal stability. The retention of solubility, binding activity, enhanced stability, and compact size of the hybrids obtained paved the way to their application as an effective marker, specifically targeting biotinylated-antibody prelabeling for molecular imaging and targeting applications. These findings pave the way to exploration of the potential inherent in the integration of silver-binding protein hybrids with electrodes and chips for other analytical purposes. ’ ASSOCIATED CONTENT
bS
Supporting Information. UV-visible spectra of Ag-Av 400 hybrids and untreated avidin and dissociation of untreated avidin or avidin-PGA to monomers affected by elevated temperatures. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
ARTICLE
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Corresponding Author
*E-mail:
[email protected]. Tel:+972-3-6409054. Fax:+9723-6409147.
’ ACKNOWLEDGMENT This study was partially supported by the Edouard Seroussi Chair for Protein Nanotechnology. G.M. gratefully acknowledges the support of the ISEF Foundation Ph.D. Program. The skillful help of Dolev Katz in the design of Figure 1 is acknowledged. We would like to thank Dr. Vered Holdengreber for the preparation of cell samples for TEM analysis. ’ REFERENCES (1) Lagziel-Simis, S.; Cohen-Hadar, N.; Moscovich-Dagan, H.; Wine, Y.; Freeman, A. Curr. Opin. Biotechnol. 2006, 17, 569–573. 22700
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