Article Cite This: J. Am. Chem. Soc. 2019, 141, 1359−1365
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Cooking Chemistry Transforms Proteins into High-Strength Adhesives Jessica K. Romań † and Jonathan J. Wilker*,†,‡ †
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907-2084, United States School of Materials Engineering, Purdue University, Neil Armstrong Hall of Engineering, 701 West Stadium Avenue, West Lafayette, Indiana 47907-2045, United States
‡
J. Am. Chem. Soc. 2019.141:1359-1365. Downloaded from pubs.acs.org by MIDWESTERN UNIV on 01/23/19. For personal use only.
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ABSTRACT: In prior generations, proteins were taken from horses and other animals to make glues. Petroleum-derived polymers including epoxies and cyanoacrylates have since replaced proteins owing to improved performance. These modern materials come at a cost of toxicity as well as being derived from limited resources. Ideally, replacement adhesives will be made from benign, cheap, and renewable feedstocks. Such a transition to biobased materials, however, will not occur until similar or improved performance can be achieved. We have discovered that coupling of proteins and sugars gives rise to strong adhesives. An unexpected connection was made between adhesion and Maillard chemistry, known to be at the heart of cooking foods. Cross-linked proteins bonded metal and wood with high strengths, in some cases showing forces exceeding those withstood by the substrates themselves. Simple cooking chemistry may provide a route to future high-performance materials derived from low-cost, environmentally benign components.
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INTRODUCTION Industrial adhesives are used on massive scales with, for example, 9 billion kilograms produced annually in the United States.1,2 Nearly all of these glues are petroleum-derived and permanent in nature, and almost half off-gas carcinogenic formaldehyde.1,2 Wood composites such as plywood, particle board, medium density fiberboards, and floor laminates give rise to a significant fraction of our daily formaldehyde exposure, be it from houses or furniture.1 Consequently, we have a tremendous demand for new adhesives that are obtained sustainably, not releasing toxins, and of low cost. Proteins might provide one of the best sources of cheap, renewable, and nontoxic organics for adhesives. Early successes with adhesives, centuries ago, did include hide glue comprised of collagen obtained from animal connective tissue.3 Today, horses are marched to the glue factory less often. The twentieth century petrochemical boom has supplanted most use of biobased adhesives in favor of synthetic systems.4 Cyanoacrylates, epoxies, and urethanes often exhibit high structural bond strengths not achievable previously with biomacromolecules, albeit with accompanying toxicity and even some cost concerns. Despite persistent problems, formaldehyde−urea and formaldehyde−phenol glues still dominate within wood products given the low costs. Avoiding the drawbacks of these modern materials will only happen when we can match the performance of petroleum-based materials with cheap and renewable feedstocks such as proteins. © 2018 American Chemical Society
On their own, proteins are not generally too sticky. However, marine biology has developed several clever strategies here. Mussels, barnacles, and oysters bind themselves to rocks quite well using protein-based attachment strategies.5−9 As we learn how marine organisms stick, an emerging theme is the cross-linking of proteins. This chemistry is complex, far from understood well, and somewhat unusual in that cross-linking may be a product of post-translational modifications allowing oxidation reactions to then take place. By analogy to shellfish, cross-linking chemistry will be needed if we wish to make use of such organics available on large scales. Biomimetic design of materials is both exciting and a popular means of gaining access to adhesion. Simple polymers such as polystyrene can be transformed into high strength glues upon incorporation of mussel chemistry in the form of 3,4-dihydroxyphenylalanine (DOPA). 10 Such functional groups have also been appended to, for example, soy protein with excellent adhesion results.11 Yet biomimetics often include synthetic challenges, extensive modifications of proteins,12,13 and increased costs and may only take us so far. We still need affordable and simple chemistry that can be scaled up to millions of tons per year. While exploring other types of cross-linking, we noticed that simple reactions between proteins and ascorbic acid were creating materials with appreciable adhesive properties. We Received: November 12, 2018 Published: December 21, 2018 1359
DOI: 10.1021/jacs.8b12150 J. Am. Chem. Soc. 2019, 141, 1359−1365
Article
Journal of the American Chemical Society
Figure 1. Effects of ascorbic acid concentration (moles of protein to ascorbic acid, A, B) at 25 °C, reaction time (C, D), and protein concentration (E, F) on the lap shear adhesion of bovine serum albumin (A, C, E) and soy protein isolate (B, D, F) on aluminum and wood substrates. The reaction time was tested with the optimal molar ratio of protein to ascorbic acid and concentration tests used the optimal reaction time. The reaction time and concentration experiments used the optimal reaction temperatures of 25 °C (BSA−aluminum, soy−aluminum, and soy−wood) and 37 °C (BSA−wood). Error bars represent 90% confidence intervals, and asterisks identify data sets that were statistically (p ≤ 0.05) different from the control value in each experiment (1:0 protein/ascorbic acid, 0 days, and 0.05 g/mL).
have now applied these reactions to biopolymers that can be sourced on large scales. When conditions were just right, high strength adhesion resulted. In some cases, performance was comparable to the commercial products in most need of replacement yet without requiring the high heat and pressure often needed in composite material manufacturing. Delving into the origin of this adhesion discovered that one of the oldest known means of coupling proteins was at play. Reducing sugars can react with proteins to generate cross-links. We are, perhaps, most familiar with such chemistry when cooking and baking food.14−17 Consider making bread and watching the dough become brown. This Maillard chemistry is a dominant process in all cooking, darkening foods via cross-linking reactions and releasing numerous flavorful compounds. By combining insights from shellfish, biomimetics, and cooking
chemistry, we now have the opportunity to impact how we make the adhesives all around us.
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MATERIALS AND METHODS
Cross-Linking Reactions. All protein + ascorbic acid reactions were completed in 50 mL Falcon tubes with protein dissolved in 3 mL of double-distilled, deionized water. These tubes were then Parafilmed shut and shaken on their side at ∼150 rpm in a New Brunswick Scientific incubator. Bovine serum albumin was purchased from Sigma-Aldrich, and soy protein isolate (92% protein) was received from MP Biomedicals. Protein Characterization. Amino acid analysis was carried out at the Molecular Structure Facility of the University of California, Davis. Hydrolysis was completed at 110 °C over 24 h with the addition of 200 μL, 2 M HCl with 1% phenol. This facility utilizes Hitachi amino acid analyzers, which separate amino acid residues via ion-exchange 1360
DOI: 10.1021/jacs.8b12150 J. Am. Chem. Soc. 2019, 141, 1359−1365
Article
Journal of the American Chemical Society chromatography followed by a ninhydrin reaction and detection system. Viscosity measurements were carried out using Fungilab BS/UTube capillary viscometers (ASTM nos. 4, 5, and 6). Samples for UV−vis and circular dichroism were removed from a reaction vessel at T = 0 with the controls made at the same concentration as their reaction counterpart. UV−vis analysis was carried out on a Varian Cary 100 Bio UV−vis spectrophotometer. Circular dichroism samples were further diluted to protein concentrations of 0.025 mg/mL and analyzed by a JASCO J-1500 spectrophotometer. Samples for FTIR analysis were removed after the optimal reaction times and lyophilized to dryness. These materials were then analyzed by a Thermo Nicolet 6700 FTIR/FT-Raman spectrophotometer. Adhesion Testing. All adhesion testing was carried out using aluminum and pine wood substrates. Sheets of aluminum, 6061-T6 (Farmer’s Copper), were cut into adherends (8.89 cm × 1.27 cm × 0.318 cm) and cleaned following the ASTM D2651-01 standard method. Common pine, purchased from a local hardware store, was cut to 8.89 cm × 1.27 cm × 1.27 cm, holes drilled for testing with rods, and used without any further surface modification. After adhesion testing, the strength was determined by dividing the maximum force at failure (newtons) by the overlap area, determined by a Vernier caliper. Lap shear testing of protein samples on aluminum was done generally as follows. The protein solution (7.5 μL) was spread onto two adherends using a micropipette and overlapped (1.2 × 1.2 cm) in the single lap shear arrangement, and pressure was applied with 55 g weights. Lap shear testing on wood substrates was performed the same way with the exception of the amount of material deposited, 45 μL vs 7.5 μL. In most cases, the adherends were allowed to set for 30 min, after which time the weights were removed and the adherends allowed to cure at 37 °C for 23 h followed by cooling for 30 min. Bulk adhesion was tested in shear using an Instron 5544 Material Testing System with a 2000 N load cell and a crosshead speed of 2 mm min−1. The method used is a modification of the ASTM D1002 standard method. For each study, a data set of at least 10 trials was collected. The average of these data sets and errors at 90% confidence intervals are reported. A statistical difference of p < 0.05 from that of the control in each parameter optimization was considered significant.
ascorbic acid (i.e., vitamin C) was combined with BSA and soy protein at varied molar ratios (protein/ascorbic acid). Parts A and B of Figure 1 show that pronounced influences upon adhesion were found. Bonding near ∼1 MPa was achievable with BSA on aluminum and soy protein on wood. Without the addition of any ascorbic acid, adhesion was in the 0−0.1 MPa range. Ascorbic acid enhanced adhesion, but only up to a point, after which performance decreased. When ascorbic acid was used in excess over protein (i.e., ratios of 1:10, 1:50, 1:100), the solutions became increasingly viscous, possibly due to excessive protein cross-linking. In addition to creating cohesive bonding at the expense of surface adhesive bonding, this viscosity may also have reduced the ability of solutions to penetrate into the wood substrates and create mechanical interlocking. Solution pH also influenced adhesion, although values both higher and lower than pH = 7 decreased performance (Figure S3), possibly due to diminished solubility, with prior reports illustrating decreased soy protein solubility at lower pH.19,20 Reaction times of ascorbic acid plus protein, prior to applying the mixtures between substrates, altered bonding significantly, seen in Figure 1C,D. For BSA, a large jump in wood adhesion came at 14 days. Longer than 2 weeks showed less strong joints, likely a result of over cross-linking. With soy protein, earlier time points up to 3 days had appreciable bonding, but further reaction time decreased solubility as well as adhesion. Achieving a proper viscosity, neither too thin nor too thick, is one of many factors in developing an adhesive, with increased viscosity caused by factors beyond concentration including intramolecular forces from cross-linking. Here, in Figure 1E,F, concentration was varied from a water-like 0.05 g/mL up to a more pastelike 0.2 g/mL for soy protein and 0.6 g/mL for BSA. High bonding at ∼2 MPa was noted for BSA at 0.3 g/mL and wood substrates. Interestingly, when bonding wood, soy protein decreased strengths at higher concentrations, possibly related to increased viscosity and diminished penetration into the surface. On aluminum, by contrast, a middle concentration proved more optimal. Metals ions have been implicated in cross-linking within several contexts including proteins involved in Alzheimer’s disease21 and marine bioadhesives.7 Here, we assessed the effect of added Na+, Cu2+, Fe2+, Zn2+, and Fe3+ salts. Large changes were not observed for most ions (data not shown), although Fe3+ did increase BSA adhesion on wood by ∼0.5 MPa. For soy protein, Fe3+ increased aluminum bonding by ∼0.5 MPa (Figure S4). Harsh oxidants such as dichromate [(Cr2O7)2−] are often used to cross-link proteins when, for example, tanning leather.22 Here, we examined potential reaction with sodium periodate (NaIO4), a strong oxidant without the established carcinogenicity of chromium. Changes in the material failure mode on the aluminum substrates were found with this oxidant. Higher NaIO4 concentrations resulted in more adhesive failure, evidenced by, after the completion of measurements, protein debonded from the substrates. Cohesive failure was exemplified, after pulling apart two substrates, by an even distribution of glue on each substrate and was common in this study up until the point of adding an oxidant. The BSA and periodate bonding increased on wood by ∼1 MPa up to ∼3.8 MPa (Figure S5A). Although periodate decreased soy protein adhesion on wood, there was an increase on aluminum up to ∼0.7 MPa (Figure S5B). Curing of adhesives is often a trade-off between time and elevated temperature. Both loss of solvent and induction of cross-
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RESULTS AND DISCUSSION Two different systems were chosen to explore the transformation of proteins into high-strength adhesives. Bovine serum albumin (BSA) is one of the cheapest proteins that can be purchased on a large scale within a laboratory setting, providing a pure material with which to best understand the chemistry at hand. Soy protein isolate is less pure and not fully soluble but is one of the lowest cost sources of organic biopolymers available today at pennies per kilogram in bulk, in addition to being biodegradable, even following cross-linking.18 Being under a dollar per kilogram may enable soy-based organics to be cost competitive with formaldehyde-based wood adhesives, which comprise nearly half of all industrial glues in use today.1,2 These proteins were subjected to several reaction conditions and adhesive testing in order to explore the possibility of generating high strength, protein-based bonding. Initial screening of conditions found suitable starting points to include protein concentrations of 0.05 g/mL for soy or 0.30 g/mL for BSA, 25 °C, pH = 7, and mixing times of either 1 or 7 days. These formulations were then used for initial bulk adhesive bonding of aluminum and pine (i.e., wood) substrates (Figures S1 and S2). After placement of the protein solutions onto one substrate, a second substrate was added to create a lap shear joint. Typical cure conditions were then 30 min at room temperature followed by 23 h at 37 °C and then 30 min at room temperature. Adhesive bonding was quantified by pulling the substrates apart until failure. The reducing sugar 1361
DOI: 10.1021/jacs.8b12150 J. Am. Chem. Soc. 2019, 141, 1359−1365
Article
Journal of the American Chemical Society
Replacing petroleum-based adhesives with renewable feedstocks is likely to occur only when performance and cost can be comparable. Methods for generating adhesives from proteins are being discovered constantly. One visible example is the incorporation, via a multistep synthesis, of marine mussel adhesive chemistry into soy protein.11 Animal-derived hide glues, casein adhesives from milk, and the biomedical BioGlue all have proteins for the main components, although performance of these systems tends to not be in league with more modern petroleum-derived systems. How did the performance of these new materials compare to analogous commercially available products? Conditions vary from study to study, thereby making correlations between literature reports not terribly insightful. Thus, side-by-side testing under identical conditions was carried out here. Several protein-based commercial glues were examined (LD Davis Industries NW139C, Superset, and CM261C as well as Cargill Prolia). Further comparisons were made to starch-based glues (Grain Processing Corp. Sealmaster P30L and LD Davis Industries AP240) as well as a modern, petroleum-derived polyurethane (Gorilla Glue). Table 2 shows how the cross-linked proteins performed relative to these products. For aluminum bonding, the highest measured strength was for BSA at ∼2.8 MPa, with performance similar to Gorilla Glue. The highest lap shear bond strengths measured were when joining wood substrates. One commercial product (Sealmaster) and the new BSA protein system both yielded strengths of ∼4 MPa. Figure 2 shows that the joint created between wood pieces could be stronger than the wood itself. When trying to pull apart bonded substrates, the wood failed while the adhesive joint remained intact (Figures 2B,C). Likewise, adhesion was strong enough with plywood that substrate failure was seen during experiments (Figure 2D). Initial testing of the optimized soy and BSA protein adhesives with polyvinyl chloride (PVC) and polytetrafluoroethylene (Teflon) substrates resulted in weak bonding (
