José M. Abad, Marcos Pita, and Víctor M. Fernández
Gold has been a widely used support for protein immobilization in a nonspecific way through electrostatic and hydrophobic interactions. As no tools are available to predict the binding of proteins of biological interest to gold supports—for either nano, micro, or macroscopic sizes—smart, reliable, and reproducible protein immobilization protocols on gold are sought. This chapter will focus on a synthetic strategy which allows for the development of a multiplicity of architectures on gold that may be used for protein immobilization. Because of its simplicity, both from a conceptual and a practical point of view, the strategy demonstrated by this step-by-step synthesis of a functionally self-assembled monolayer (SAM) of thiols on gold is accessible to most laboratories working on enzyme technology, even those with limited organic synthesis facilities.
Key Words: Gold; self-assembled monolayers; SAM; oriented immobilization; nanoparticles.
Gold has been widely used as a support for proteins in different applications. A classic, well-known example is the use of colloidal gold particles as a support for antibodies in immunological studies to detect cell antigens at the optical and electron microscope levels. The most popular variant makes use of gold nanoparticles covered with protein A that specifically interacts with the Fc fragment of immunoglobulin (IgG) molecules; the use of protein A labeled with 125I made this process a rapid quantitative method for estimation of membrane antigens (1). In this arrangement, protein A plays a dual role, as a linker of IgG to gold nanoparticles and as a stabilizer of colloidal gold solutions. The stability of gold nanoparticles in solution is maintained by electrostatic repulsions, and the addition of electrolytes can alter the surface charge of the nanoparticles and produce their flocculation. In addition, positively charged groups of the proteins interact with the negatively charged surface forming noncovalent binding complex and stabilizing the colloid from the effects of electrolytes (2).
From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ
Proteins tend also to adsorb to macroscopic gold surfaces in a nonspecific way by electrostatic and hydrophobic interactions (3). Interestingly, proteins of a certain size that adsorb into a gold material may also bind to a smaller particle of the same material, but less strongly. Moreover, a polypeptide that binds preferentially to a specific crystallographic surface may bind with an altered affinity to another crystallographic surface of the same material (4). Progress made in the understanding of protein adsorption on gold was reported in the recent work by K.S. Shulten and colleagues (5). These authors have simulated the binding of a peptide engineered to match the spacing of the gold atomic lattice. Their calculations indicate that polar side chains of serine and threonine residues can establish contact with gold atoms of Au (111) surfaces, placing a periodic structure of OH- groups on the regular lattice. The same peptides do not bind to Au (112) as tightly, because of the migration of water molecules through this crystallographic surface. However, despite these advances, no tools are available to predict the binding of proteins of biological interest to gold supports. Therefore smart, reliable and reproducible protein immobilization protocols on gold are sought.
Over the past years, there has been a noticeable interest in covering gold surfaces with monolayers of proteins based on the molecular recognition properties of biological systems (6). This strategy for the immobilization of proteins on surfaces while retaining their full activity and stability constitutes a challenge. Most of the common methods are difficult to control and usually yield randomly bound proteins. On the contrary, an ideal immobilization would produce saturation coverage of specifically bound proteins. The formation of protein layers is induced by anchoring the protein molecules to gold surfaces functionalized with active molecules, such as transition metal complexes for the immobilization of proteins with repetitive histidine sequences. A feasible method to uniformly cover gold surfaces consists of the self-assembly of thiols by oxidative-chemisorption over the gold (7). Reversible monolayers of histidine-tagged proteins have been produced using a gold layer covered with a monolayer of a chelator thioalkane (8). Other systems have been described with biotynilated alkanethiols (9) and this biotin-avidin system has been used as a template to direct the binding of monobiotynilated Fab fragments of monoclonal antibodies (10). As an alternative to this methodology, which demands highly complex organic synthetic work, a step-by-step construction of the functional monolayer over a template of thiocarboxylic acid chemi-sorbed onto gold has been worked up (11). In a comparative study of A/-palmitoylcysteamine self-assembled monolayers (SAMs), either from ex situ synthesis of the chain or from in situ acylation of a cysteamine SAM (12), the authors did not find differences in topography and mean roughness between the two monolayers.
These studies have benefited from the development of surface sensitive techniques for the detection of bimolecular interactions between one partner immobilized on a gold surface and the complementary molecule from the solution. The binding effect can be monitored by specific signals associated to the binding event. Representative examples of detection systems on gold-modified surfaces are atomic force microscopy (AFM) (13), quartz crystals microbalance (QCM) (14), and surface plasmon resonance (SPR) (15).
Because of its simplicity, both from a conceptual as well as a practical point of view, the step-by-step synthesis of a functional self-assembled monolayer is accessible to most of the laboratories working on enzyme technology, even those with limited facilities for organic synthesis. This chapter focuses on the synthetic strategy, which by a judicious design of the synthetic route allows the development of a multiplicity of architectures on SAMs (Fig. 1).
We have used this strategy for controlled immobilization of enzymes onto gold surfaces and used them as amperometric electrodes in the characterization of their catalytic performance (14,16,17).
2.1. Preparation of Piranha Solution
1. Sulfuric acid, 95 to 98%, American Chemical Society (ACS)-grade reagent (Aldrich). Caution: Corrosive and toxic.
2. Hydrogen peroxide, 30% solution in water. Caution: Corrosive and toxic.
3. Fume cupboard, disposable gloves, and face protection.
2.2. Gold Support
1. 0.5-mm Gold wire, 99.99% (Goodfellow, Cambridge, UK).
2. Gamma Micropolish Alumina B 0.05 micrometer (Buehler).
2.3. Gold Modification With Thiol Self-Assembled Monolayers Presenting Reactive Ester Intermediates
1. Thioctic acid (6,8-dithiooctanoic acid, TOA) reagent (Sigma).
3. Dimethyl sulfoxide (DMSO; Merck). Caution: Higly flammable; harmful.
4. #-Hydroxysuccinimide (NHS; Sigma).
5. 1-Ethyl-3-[3-(dimethylaminopropyl)] carbodiimide (Sigma).
2.4. Coupling of Spacers
1. 1,8-Diamino-3,6-dioxaoctane (DADOO; Merck).
2. Dimethyl formamide (DMF).
3. Ethylenediamine (Sigma).
4. 1-Chloro-2,3-epoxypropane (epichlorohydrin; Epi) (Fluka). Caution: Toxic by inhalation or contact with the skin; may cause cancer; flammable.
2.5. Coupling of Affinity Ligands
1. Thiocholine (ThC; MP Biochemicals, Irvine, CA).
2. #-(5-Amino-1-carboxypentyl)-iminodiacetic acid (ANTA) (Fluka).
3. 3-Aminophenyl boronic acid (Aldrich).
2.6. Enzymatic Activity
1. (/>-Aminophenyl)-P-D-galactopyranoside (PAPG) (Sigma).
3. P-Aminoethyl ferrocene, synthesized as described by Godillot et al. (18).
4. 4-Aminophenol hydrochloride (Sigma).
5. Thionine (Lauthe Violett, Merck).
1. The fusion protein of P-gal and C-Lyta was produced and purified as reported previously (19).
2. Ferredoxin NADP+ reductase native and mutants purified as described (16).
3. Type 1 horseradish peroxidase (Sigma).
3.1. Preparation of Piranha Solution
1. Using gloves and protective glasses, operate in the fume cupboard.
2. Mix 98% sulfuric acid with 30% hydrogen peroxide solution, in a 3:1 volume ratio. The acid is slowly poured onto the peroxide.
3. This solution is explosive and corrosive. It reacts violently with organic matter, so it should be handled with extreme caution. Be very scrupulous with the safety indications.
4. Keep this solution inside the fume cupboard far from flames.
3.2. Gold Support Preparation
1. Heat a 0.5-mm gold wire directly on a Bunsen burner until the gold reaches light red temperature, but avoid melting.
2. Polish the heated wire with y-alumina suspension of 0.05-pm diameter for about 10 min.
3. Wash the wire with Milli-Q water.
4. Dip the wire in a 2:1 [vol/vol] ethanol-water solution.
5. Sonicate the wire in an ultrasonic bath for 15 min.
3.3. Gold-Surface Modification With Thioctic Acid Monolayers
1. Dip the gold wire in a 1 mM dl-6, 8-TOA solution prepared in ethanol-water solution (2:1 [v/v]) at room temperature for 24 h.
2. The gold electrode, covered with a dithioctic SAM is rinsed in ethanol-water for 5 min and then air-dried.
3.4. Activation of Carboxylic Acid Groups of the Monolayer
1. The TOA-modified gold is immersed for 3 h in a solution of 0.1 M N-hydroxysuccinimide (NHS) in DMSO, containing 0.1 M 1-ethyl-3-[3-(dimethyl-amino)] propyl carbodiimide (EDAC), which catalyzes the esterification reaction.
2. Rinse the gold electrode for 5 min with DMSO while stirring gently with a magnetic stirrer.
Up to this step, the process is general for all the SAMs that will be described further. From now on, the chemical modification is different for each type of SAM that will be synthesized. In the following subheadings the step-by-step synthesis of different monolayers decorated with ligands chosen by their affinity for specific protein motifs is described.
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