Functionality is the most recent advancement in coatings technology that adds significantly to the traditional role of a coatings system. Natural biomaterials, such as proteins and peptides, provide an enormous resource of functional additives that are non-persistent in the environment, non-toxic and renewable. Biomolecules, such as peptides, antibodies and receptors, have provided many distinct non-catalytic activities in natural environments. By focusing on the unique and specific binding properties of these non-catalytic molecules, biobased additives are being created which will provide a new and innovative function to coatings systems: “recharge-ability”. Being able to change or renew a biocide or other functionality, without recoating adds a new dimension to the coated surface. The current state of research in the development of biomaterials for functional coatings, including antifouling and antimicrobial surfaces, with specific examples of recharging will be presented.
Innovation in Materials Science: From Nonselective Absorption & Release to Artificial Receptors
In its most simple embodiment, the concept of a “rechargeable” coating can be a cross-linked polymer or copolymer that would absorb a target molecule, release it under specified conditions and remain receptive to re-absorption of the target . Nature has provided a large number of molecules that already perform highly efficient small molecule recognition and/or catalysis. These molecules, such as enzymes, antibodies and membrane receptors are molecular machines that can contain a combination of small molecule binding, transport, release, and/or catalytic functionalities. Current approaches in material science involve mimicking natural molecules to create artificial receptors and molecularly imprinted polymers that have a release/re-absorption property. Artificial receptors are typically created by “imprinting” binding sites or receptor structures within a polymeric surface, thus creating the ability to selectively and reversibly bind molecules of interest. A target molecule is employed as a template around which interacting and cross-linking monomers are polymerized to form a block that contains a number of cavities each with a template molecule inside (Fig. 1). The resulting binding sites are complementary to Fig 1. A target molecule serves as a template around which a cross-linked polymeric material forms a number of hollows conformed to the target molecule. the target molecule in size and shape . After polymerization, the template is removed and the binding sites that have been imprinted on the polymer are capable of selectively rebinding to the target molecule.
Monomers with vinyl or acrylic groups are most commonly polymerized and cross-linked to form imprinted material or artificial receptors. There are a variety of such monomers which can carry basic (e.g., vinylpyridine) or acidic (e.g., methacrylic acid) functional groups. They can be permanently charged (e.g., 3-acrylamidopropyltrimethyl ammonium chloride) or hydrophobic (e.g., styrene) or be capable of participating in hydrogen bonding (e.g., acrylamide) for participation in charge, hydrophobic, and/or hydrogen bonding interactions in binding sites. Addition of a solvent can be used to induce a porous structure in the polymer to facilitate access of the target molecules to the binding sites. Other materials, such as polyphenols and polyurethanes and sol–gels are also finding use as imprinting matrices. 
Because of the way the binding sites are created, their distribution, accessibility and binding properties are often heterogeneous. This is not always disadvantageous, but it can be difficult for artificial receptors/imprinted polymers to adequately substitute for the flexibility and specificity of natural molecules. In spite of this, imprinted polymers have been used successfully for separations, as catalysts, and as sensing elements [3-4], and as such provide successful examples of one type of re-chargeable polymeric materials.
The next innovation is to design and build re-chargeable coatings that deliver a desired functionality that can be replenished, refreshed or redirected (“re-programmed”) as needed. A novel illustration of this concept is a metal-based antifouling coating that charges and recharges itself using environmental resources. By harnessing the properties of peptides and proteins to selectively bind and release metals, metals that are naturally found in the environment can be utilized as an antifouling biocide.
Metals as Biocides for Antifouling Coatings
Fouling refers to the surface adhesion of molecules or other materials to a surface upon contact with water (e.g., sea water, fresh water). Biofouling is prevalent form of fouling produced by the adhesion of biomolecules (e.g., proteins, glycoproteins) and/or organisms, as opposed to inorganic materials, and can begin within minutes upon contact with water to produce an initial biofilm. Microorganisms such as bacteria, diatoms, algae, marine fungus, protozoan, cyprid and/or rotifer generally incorporate into the fouling biofilm within 24 hrs, though macroorganisms (e.g., barnacles, tunicates, mollusks, bryozoans) may adhere to the surface days or weeks later. Biofouling occurs worldwide in various industries (e.g., offshore oil and gas industries, fishing, power stations, paper and pulp industries) and a variety of locations (e.g., ship hulls, heat exchangers, water-cooling pipes, propellers, ballast water). A fouled surface is typically rougher and/or has a higher frictional resistance property. For example, a fouled ship’s surface may reduce the speed of a vessel in water, reduce a vessel’s maneuverability, increase a vessel’s weight, increase a vessel’s fuel consumption (e.g., up to 40%), increase a vessel’s maintenance time and/or repair cost in dry dock, reduce the use time of a vessel, enhance corrosion, alter a surface’s electrical conductivity, and/or discolor a surface.
One way to prevent biofouling is by appropriate selection of water contacting materials. Both copper metal and copper compounds are used as principal biocides because these substances are able to form Cu2+ ion, which is the biocidal active. Copper-nickel alloys have good biofouling and corrosion resistance, and therefore are often used for surfaces or surface coatings . Copper has been employed as a bactericide, molluscicide, and fungicide and exhibits antifouling activities against organisms such as barnacles, tubeworms and the majority of algal fouling species. The most popular, and successful, of the antifouling coatings, which were banned for all boats by 2007/2008, was tributyl tin (TBT)-coatings. TBT-coatings were effective in reducing/controlling biofouling; however, they are also highly toxic to marine organisms. There is some concern that similar environmental impact will be linked to Cu2+. In the U.K., 21.7% of sampling locations/year exceeded 5 μg/L for copper and in 4.3% the concentrations lay between 10 and 15 μg/L. These data suggest that monitoring of concentrations of copper is important to avoid ecological damage in marinas and mooring areas, where there are intense boating activities and poor tidal exchange. 
Re-chargeable Coatings: A Biobased Approach to Creating Dynamically Functional Coatings
There are many unique mechanisms developed in nature for metal absorption, accumulation or resistance. Currently, about 40% of all known proteins contain a liganded metal, Meq+ , which performs a plethora of essential tasks from protein structure stabilization and enzyme catalysis to photosynthesis and respiration. Metal ions such as Na+, K+, Mg2+, Ca2+, Zn2+, Mn2+, Ni2+, Cu+/2+, Fe2+/3+, and Co2+/3+ are most frequently found to bind to proteins via one or more metal-binding domains under physiological conditions [6,7]. Most metal-binding proteins (“metalloproteins”) bind a given metal with specificity . Using recombinant DNA techniques, it is possible to create protein-based biomaterials with both metal-binding and tunable properties that can be used to selectively “catch-and-release” heavy metals from environmental sources. These biomaterials are environmental friendly, requiring no toxic chemicals for their synthesis, are easily produced in mass quantity. The use of metal-binding domains of proteins has significant advantages over chemical or polymeric chelators, including higher specificity and affinity for selected metals described above. The potential lower limit for successful heavy metal binding from a dilute source of the metal in solution can be as low as approximately 10-10 M, or in the parts per trillion, depending on the specific metal-binding domain employed .
Two common classes of metal binding peptides are metallothioneins and phytochelatins [8-10]. Both of these are cysteine-rich peptides that bind divalent metal ions through sulfhydryl groups (-SH). Metallothioneins are typically formed in mammals, plants and microorganisms in response to the presence of cadmium . Related proteins that are structurally similar can be associated with metal ions like zinc, copper and cadmium . Phytochelatin peptides are produced by enzymatic synthesis in fungi, algae, and some prokaryotic organisms, worms, and plants to bind heavy metals such as Ag+, As3+, Cd2+, Cu+/2+, Hg2+ Ni2+, or Zn2+.
Peptides with unique metal binding properties can either be pulled from nature, designed de novo or selected by screening libraries. His6 is an example of a peptide designed from a template in nature, and which can be used to functionalize metal binding coatings. (Fig 2) Nickel generally provides good binding efficiency to His6, but also tends to bind nonspecifically to endogenous proteins that contain histidine clusters. Cobalt ions, and even more so copper ions, exhibit a more specific interaction with histidine tags, resulting in less nonspecific interaction. The research presented here demonstrates that it is possible to selectively and reversibly bind metals to a coating containing metal binding peptides, thus granting the coating anti-fouling and anti-microbial properties.
Functionalized Coatings with Metal Chelated Protein
It has previously been demonstrated that proteins and short chain peptides can be used to functionalize coatings [14-19]. In those studies, we used bio-based molecules that were themselves inherently antimicrobial, typically impacting the cell wall or membrane of the target microorganisms. The series of studies presented here demonstrate the use of biobased additives which themselves are not inherently antimicrobial to create re-chargeable coatings. The enzyme organophosphorus hydrolase (OPH, E.C. 220.127.116.11) is a metalloenzyme in which four histidine residues coordinate two divalent cations. Although the identity of the divalent metal ions in the active-center influences the activity and stability of the enzyme (Zn2+, Co2+, Cd2+, Ni2+ or Mn2+) all support the catalytic activity of the enzyme. A two Zn2+ center has the greatest effect on stability, while a two Co2+ center increases the activity of the enzyme at the expense of stability. If the enzyme loses either of the liganded metals, the activity is lost. (Fig 3)
The use of OPH (OPDtox, Reactive Surfaces) to functionalize coatings provides a direct measure of metal-chelation by a protein in a coating. To assess whether the enzyme embedded within the film maintained its chelated metal ligands, the prepared films were challenged with the organophosphorus substrate, paraoxon. The OPDtox enhanced coatings catalytic mechanism demonstrated bulk volume dependence (Fig. 4), and clearly demonstrates the ability of metalloenzymes to chelate the required metal ions.
Functionalized Coatings with Metal Chelated Peptide
Nickel, copper and cobalt were selected to evaluate binding to His6 peptide within the vinyl latex coating. Copper was chosen due to the importance of it being used as the main ingredient of anti-fouling coatings. The His6 peptide was blended into the vinyl latex coating and then assessed for impact on the binding properties of the peptide. Binding affinity to the peptide was performed in a solution assay by suspending free films to a metal solution of 200 mM nickel sulfate, 100 mM copper sulfate and 42 mM cobalt chloride hexahydrate in three separate experiments. All of the metal solutions were adjusted to pH 10 to avoid hydrogen ions competing for the binding sites. The exposure time was 24 hours ensure a binding saturation of metals to the peptides in the coating. The liquid in which the free films were suspended was then removed and pH 5 water was added to remove the bound metal from the free films. This elution process occurred for 24 hours to maximize release of the metal ions back into solution, due to the competition of the hydrogen ions with the metals for the binding sites. The wash liquid was then decanted and analyzed for the amount of metals present using a spectrometer. The vinyl latex films containing the metal binding peptide His6 bound more nickel, copper and cobalt than in the control films that did not contain the peptide (Figure 5).
Nickel and copper binding to the films was stronger when compared to cobalt binding which occurred at a much lower concentration. In a polymer system or coating, the peptide is suspended in the polymer matrix and diffusional constraints derived from metal accessibility to the peptides become crucial. One reason for lower cobalt chloride hexahydrate binding to the films may be due to the fact that the cobalt complex is larger than the other metals that were tested which makes it harder to interact with the binding site.
This study demonstrates that it is possible to create re-chargeable coatings for metal ions used as antifouling agents. This platform technology will be used to create anti-microbial coatings that use metals as a deterrent for biological growth. Our experience with other natural peptides that are not toxic to the environment suggests these metal-binding peptides can be used as “drop in” additives for a coating to imbue it with these functionalities. Peptides can be chosen to interact with and bind to specific metals, to change these metals at will, and to use combinations of metals or peptides with increasingly larger binding coefficients, making the system programmable. When a specific metal binding peptide is chosen, an assessment of the polymer system can be performed to optimize the performance of the peptide. Since metals are so abundantly found in seawater, they can be utilized by marine coatings to prevent biological growth from occurring without adding metal ions to the environment (Fig 6). These approaches should reduce the regulatory concerns of using heavy metals as antifouling actives.
This work represents an ongoing effort by our teams (Reactive Surfaces and University of Southern Mississippi) to develop systems using natural additives where enzymes and reactants are dispersed, embedded, and maintained in an active state within a continuous polymer phase, either as solid films or aqueous dispersions. There are many advantages to using this resource to design novel functionalities. Not the least of these advantages is, as demonstrated here, the combination of the use of polymer chemistries with known, easily accessible and renewable functionalities. In the case of recharge-able coatings based upon metal-binding peptides that are extracted from nature, the numbers, types, ease of genetic manipulation, and essential “drop in” characteristics of these additives provides the formulator with palette of functionalizing possibilities. In the near term, our teams will investigate the breadth of suitable resin systems, the range of microbial targets, and the ranges of metal-binding proteins that will create the most efficient recharge-able coatings in the widest variety of applications and commercial products.
Reagents. His6 peptide (21st Century Biochemicals, Product # HIS6-0400) was used. His6 is a peptide composed of six histidine amino acids and was produced chemically by 21st Century Biochemicals. Glidden Vinyl Latex (1424) was used to create the rechargeable coating. Nickel sulfate (Sigma # 656895), copper sulfate (Sigma # 451657) and cobalt chloride hexahydrate (Sigma # 255599) were used for metal assay solutions. The organophosphorus compound paraoxon (Toronto Research Chemicals) was the reactant (substrate) used for OPDtoxTM (Reactive Surfaces Ltd., Texas).
Preparation of Peptide/Enzyme Blended Films and Coatings. To create a coating with 3% dry weight, 93 mg/ml peptide or enzyme was added into water and mixed into Glidden vinyl latex coating at a ratio of 1 part water to 7 parts coating. The coating was mixed until it was evenly dispersed. A draw down bar was used to make films on a polypropylene block at 8 mils thickness (wet). Films were allowed to cure for 48 hrs before use. Control films were made in the same manner without the added peptide or enzyme.
Functional Activity Assessment for Metal Chelating Proteins. Activity was determined using 1-3 cm² free films cut from OPDtox blended coatings that had been applied and dried on a polypropylene sheet. The free films were incubated in the appropriate reaction buffer under agitation and analyzed. The reactions were performed as described for solution phase. Control samples, which included both reactions without films and films without enzyme, were treated identically. To measure the efficiency of the enzymes in solid phase chemistry, the reactive coatings were challenged with substrate under dry conditions using coated aluminum panels. All assays were performed in triplicate.
Functional Activity Assessment For Metal Chelating Peptides. Coupons (3 cm²) were cut from the polypropylene blocks. Individual coupons were placed into labeled microtubes; each test material was tested in triplicate. Nickel sulfate (200 mM, pH 10), 100 mM copper sulfate, or 42 mM cobalt chloride hexahydrate was added into each microtube. Microtubes were placed on a rocker overnight. All of the liquid was removed from the microtubes and distilled water (pH 5) was added into each microtube. Microtubes were again placed on a rocker overnight. All of the liquid was removed (leaving the films behind) from the microtubes and placed into new microtubes. The microtubes with the pH 5 wash were then placed in an oven at 70°C to remove all of the water. Once the microtubes were dry, 200 μl water (pH 10) was added into each tube and transferred into a 96-well plate. The absorbance was read at 340 nm, 450 nm, or 414 nm for the nickel sulfate, copper sulfate and cobalt chloride hexahydrate assay wells, respectively, and metal concentration determined using a standard curve.
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