Creating Anti-icing Surfaces via the Direct Immobilization of Antifreeze Proteins on Aluminum

Cryoprotectants such as antifreeze proteins (AFPs) and sugar molecules may provide a solution for icing problems. These anti-icing substances protect cells and tissues from freezing by inhibiting ice formation. In this study, we developed a method for coating an industrial metal material (aluminum, Al) with AFP from the Antarctic marine diatom, Chaetoceros neogracile (Cn-AFP), to prevent or delay ice formation. To coat Al with Cn-AFP, we used an Al-binding peptide (ABP) as a conjugator and fused it with Cn-AFP. The ABP bound well to the Al and did not considerably change the functional properties of AFP. Cn-AFP-coated Al (Cn-AFP-Al) showed a sufficiently low supercooling point. Additional trehalose coating of Cn-AFP-Al considerably delayed AFP denaturation on the Al without affecting its antifreeze activity. This metal surface–coating method using trehalose-fortified AFP can be applied to other metals important in the aircraft and cold storage fields where anti-icing materials are critical.

Ice formation is a major problem in industries and applications such as air conditioners, transportation (including aircraft), and power generation [1][2][3][4] , because it reduces the cold resistance of equipment, leading to high energy loss 4 . To resolve this problem, surface-coating techniques based on thermal, chemical, and mechanical methods have been implemented to attain anti-icing properties; however, most of these rely on complicated processes that require expensive equipment and labor-intensive procedures 4 . To this end, new cryoprotectants that can be used as effective anti-icing materials have drawn the attention of the food, plant, military, and electronics industries. Whereas several cryoprotectants, such as glycols, polyols including glycerol, and sugars have been used, antifreeze proteins (AFPs) produced by psychrophilic organisms under freezing conditions 5 have been recognized as one of the most exciting materials for use in various industrial and biological fields including food, medical, and cryopreservation 6-10 , due to their distinct ability to inhibit ice or frost formation in a concentration-independent manner 11,12 . It is important to note that AFPs have been used to coat glass surfaces via polymer-linked conjugation to prevent ice formation 13 . Although the polymer-coating method can be used to improve the stability of AFPs, this strategy is generally limited to surface-bound proteins, because chemical modification of the surfaces is labor-intensive.
Aluminum (Al) is the third most abundant element in earth's crust and has many properties that make it useful in a wide range of industrial fields including the transportation, construction, electronics, freezer and cryostat industries 14 . Al surfaces with anti-icing properties are of particular interest, because the frost that forms on Al-coated compressors in the outdoor units of air conditioners at cool temperatures causes critical damage to air circulation, which is inevitably accompanied by high energy Results AFP as a novel anti-icing material. To produce aluminum-binding Cn-AFP, we fused its N-terminus with an ABP sequence (VPSSGPQDTRTT), which was previously identified via the phage display method 22 . Since Cn-AFP allows the diatom to survive under freezing conditions by lowering the freezing point via ice binding 12,15 , we used wild-type Cn-AFP (Cn-AFP WT ) and its mutant Cn-AFP G124Y , and their ABP-fused forms (ABP-Cn-AFP WT and ABP-Cn-AFP G124Y ). Cn-AFP G124Y has been reported to exhibit a thermal hysteresis (TH) value 1.5 times higher than that of Cn-AFP WT 16 . This means a wider temperature gap between the melting and freezing points in the presence of Cn-AFP G124Y than in the presence of Cn-AFP WT , due to the enhanced ability of Cn-AFP G124Y to lower the freezing point of a liquid in a non-colligative way. To check whether ABP-fused Cn-AFPs retain their antifreeze activities, we assessed their effects on TH and ice crystal morphology (Fig. 1). We observed a slight reduction in the TH values of ABP-Cn-AFP WT (0.9 °C) and ABP-Cn-AFP G124Y (1.2 °C) in comparison with the reported values (1.2 °C and 2.0 °C, respectively) 16 . The ice crystals exhibit burst patterns in the presence of both proteins, appearing when a hyperantifreeze protein is present in the solution 23 . To further verify the antifreeze activity of ABP-Cn-AFP, the supercooling point of ABP-Cn-AFP G124Y was measured via differential scanning calorimetry (Fig. 2). The supercooling point of ABP-Cn-AFP G124Y was −20.5 °C, whereas that of water (negative control) was −5.79 °C. Taking into consideration that the supercooling point of AFP from the beetle Dendroides canadensis ranges from −16 to −26 °C 24 , this result indicates that ABP-Cn-AFP G124Y , despite a reduction in its TH value, has antifreeze activity.

Construction and characterization of surface-immobilized AFPs on Al.
To determine whether ABP-Cn-AFP can be effectively immobilized on the Al surface, a simple coating method was developed based on dipping an Al plate into a reaction solution containing Cn-AFP WT or ABP-Cn-AFP WT ( Fig. 3A). The immobilization of AFP on Al was monitored using a colorimetric assay, where Cn-AFP WT or ABP-Cn-AFP WT expressed with His 6 -tag at its N-terminus was detected by Ni (II)/horseradish peroxidase (HRP), which produced a strong blue coloration because of HRP-catalyzed oxidation of its  In the presence of HRP, TMB and peroxide present in the substrate solution react to produce a blue product. The color intensity is proportional to the HRP activity. (C) Confirmation of ABP-Cn-AFP binding to an Al substrate using the TMB assay. Al alone or after "coating" with Cn-AFP (without ABP) did not result in blue coloration. In the presence of ABP-Cn-AFP-coated Al, the solution color changed to blue, which indicated ABP-Cn-AFP binding to the Al. substrate (tetramethylbenzidine, TMB) (Fig. 3B). As shown in Fig. 3C, a strong blue color was significantly observed on the Al surface coated with ABP-Cn-AFP WT , while bare Al and ABP-free Cn-AFP WT produced no coloration. To further verify the ABP-mediated binding of Cn-AFP to Al, three surfaces (bare Al, Cn-AFP WT immobilized on Al, and ABP-Cn-AFP WT immobilized on Al) were characterized via Fourier transform-infrared (FT-IR) spectroscopy (Fig. 4). While the Al− OH regions were commonly observed for all three tested surfaces, as indicated by three bands in the FT-IR spectra at ~955, ~1033, and 3535-3580 cm −1 , the intensities of protein-specific peaks corresponding to N-H stretching (3700-3500 cm −1 ), amide C = O stretching (1690-1630 cm −1 ), and amide C-N stretching (1000-1250 cm −1 ) were higher for ABP-Cn-AFP WT immobilized on the Al surface (blue line in Fig. 3) than those for the other two surfaces (black and red lines in Fig. 4). However, the red line (AFP without ABP tag) also showed an amide bond peak, which is presumed to be denatured after surface adsorption, taking consideration into Fig. 3. Taken together, these results indicate that ABP-fused Cn-AFP effectively binds to the Al surface via ABP without denaturation.

Anti-icing surfaces via trehalose-coated AFPs on Al. Despite successful immobilization, denatur-
ing of the AFP on the Al surface may reduce its antifreeze ability. To gain insight into AFP denaturation, we examined the stability of surface-immobilized Cn-AFP over time using the HRP/TMB colorimetric assay. We employed the trehalose-coating method to prevent protein denaturation, because trehalose is reported to make proteins resistant to dehydration when dried. Protein conformation can be preserved during drying through the hydrogen-bonded interaction of trehalose and water molecules, which efficiently competes with the interaction between protein and water molecules inside a trehalose-entrapped network 25 . As depicted in Fig. 5A, trehalose treatment on AFP-immobilized-on-Al surfaces was performed by incubating the surfaces in a trehalose-loaded solution for 1 h. When the ABP-Cn-AFP G124Y was incubated at room temperature for up to 12 days after immobilization on Al, its denaturation was monitored via colorimetric analysis; the surface not treated with trehalose showed a rapid reduction in color (left panel in Fig. 5B and black bar in Fig. 5C). The colorimetric signal gradually decreased and the coloration declined to 34% at day 12, compared to the initial signal intensity at day 0 (Fig. 5B). It is likely that the His 6 -tagged regions of ABP-Cn-AFPs are susceptible to a rapid conformational change upon binding to Al, probably due to protein denaturation on the Al surface. Mass spectrometric surface analysis of amino acids revealed that histidine-rich regions of both ABP-Cn-AFPs disappeared after 30 days on the Al surface (data not shown). In contrast, color remained stable in the presence of trehalose over the tested time period (right panel in Fig. 5B and white bar in Fig. 5C). This result suggests that trehalose coating effectively inhibits AFP denaturation on the Al surface, and this method may be suitable for preserving the activity of surface-immobilized AFPs.
To investigate the effect of trehalose on the anti-icing activity of ABP-Cn-AFP, the supercooling point of ABP-Cn-AFP G124Y was measured via differential scanning calorimetry both with and without trehalose treatment of the Al surface. Figure 6 shows a representative thermogram of the ABP-Cn-AFP G124Y with and without trehalose on Al cooled at 0.1 °C min −1 from 0 °C to −25 °C. The supercooling point of ABP-Cn-AFP G124Y in the presence of trehalose (−7.92 °C, Fig. 6A) is similar to that in the absence of trehalose (−8.43 °C, Fig. 6B). This result indicates that trehalose treatment did not affect the anti-icing activity of ABP-Cn-AFP G124Y . To further explore the possibility that directly immobilized AFP on Al prevents ice or frost formation, we examined time-dependent ice formation in a cold chamber for 3 h. Notably, Cn-AFP-immobilized on the Al surface (ABP-Cn-AFP G124Y with trehalose) prevented ice formation, whereas ice formed on bare Al and hydrophilic Al surfaces coated with a thin ZrO 2 film, which is a universal surface-coating method for Al [26][27][28][29][30] (Fig. 7). This result demonstrates that direct immobilization of ABP-CnAFP G124Y followed by trehalose treatment inhibits ice formation on an Al surface.

Discussion
The present study proposes an environmentally-friendly approach to anti-icing or anti-frosting through the coating of metals with AFP, which is further fortified by trehalose. Ice or frost formation causes serious economic and safety issues in various applications 31 leading to burst power lines, shortened lifespans in aircraft wings, and the prevention of air circulation in refrigerators and freezers 32 . Ice problems can be solved via traditional hydrophilic polymer coatings incorporated with BaO 2 or ZrO 2 , or via other cryoprotectant coatings such as AFPs, sugars, and polyols. AFPs have been used in cryosurgery, the storage and fermentation of foods, prevention of cellular or tissue damage via freeze/thaw cycles, and to increase the storage time of red blood cells and oocytes 6,10,[33][34][35][36] . Despite the study of potential applications for AFPs, a practical one-step method to coat metal surfaces with AFPs for industrial applications has not been available to date, and is reported in this study for the first time.
A method of coating glass surfaces with AFPs using a chemical polymer as a protein conjugator was previously reported 19 ; however, this method requires a series of complicated reaction steps and the stability of the resulting protein coating has not been verified. Therefore, we developed a one-step The TMB assay, six days after coating, the proteins were denatured in the absence of trehalose coating. Secondary trehalose coating preserved the protein structure. (C) Spectrophotometry of the TMB solutions. Without trehalose coating, proteins on the Al plate were denatured after 6 days. In the presence of trehalose coating, the protein was still not denatured twelve days after coating. Asterisks (* and **) denote statistical significance of the differences in colorimetric intensity of surface-immobilized ABP-Cn-AFP G124Y with and without trehalose over the time course (p < 0.01, paired ttest, n = 4). Error bars indicate standard deviation. method to coat aluminum (which is universally used in industrial fields) with AFPs. We used mutant AFP (Cn-AFP G124Y ), because it is 1.5 times more efficient in lowering freezing point 16 than the wild-type protein, Cn-AFP WT . To immobilize the AFP on Al, we used an Al-binding peptide (ABP). The ABP did not considerably decrease Cn-AFP activities (Figs 1 and 2) and allowed Cn-AFP binding to Al (Fig. 3). Other metal-binding peptides such as those binding to silver, gold, gallium arsenide, and mild steel 22,37-39  can also be used for the protein immobilization of respective metal surfaces. Development of fusion proteins consisting of AFPs and other metal-binding peptides could enable numerous industrial applications of AFPs.
Proteins can be easily denatured via temperature 40 . Indeed, the Cn-AFPs immobilized on Al were partially denatured 6 days after coating (Fig. 5B). When used in industrial applications, the denaturation of AFPs must be prevented and their stability maintained. Since trehalose is known to prevent protein denaturation and to stabilize protein structures under freezing conditions via the formation of multiple hydrogen bonds between the hydroxyl groups of trehalose and polar residues in proteins 20,41 , we performed trehalose coating of Al coated with Cn-AFP. In addition to measuring the TH activity, we measured the supercooling points of AFP proteins to further analyze their anti-icing function (Fig. 2). Mutant AFP considerably lowered the supercooling point of solution in comparison with water, clearly confirming the previously-reported TH value of Cn-AFP G124Y in comparison with the wild-type protein 16 . Additional trehalose coating dramatically increased the stability of the immobilized protein (Fig. 5B,C). The supercooling point of ABP-Cn-AFP G124Y -Al was not considerably affected by trehalose coating (Fig. 6). Thus, trehalose coating clearly improves protein durability on metal surfaces. Frost and ice were not formed on the surface of a Tre-ABP-Cn-AFP G124Y -Al, even though large amounts of ice accumulated on the surface of a traditional hydrophilic-coated Al (Fig. 7).
In summary, our newly-developed AFP coating on Al fortified with trehalose offers remarkable benefits and advantages for the industrial application of AFPs. First, recombinant ABP-Cn-AFP proteins are viable for production on an industrial scale. This is an environmentally-friendly system because the production of recombinant proteins is bio-based. Secondly, Al surface coating with AFPs is accomplished rapidly via a one step-dipping method without complicated surface modification. ABP-fused AFPs are capable of maintaining the appropriate orientation of the AFP on a surface, which allows for application of their full anti-icing properties. Most importantly, AFPs clearly impeded ice formation on the Al surface when compared to bare Al and traditional hydrophilic Al coatings. This effect was due to the TH activity of Cn-AFP and its ability to lower the freezing point. The results of this study will provide numerous opportunities for applications in the cryostat, refrigerator, and freezer industries to protect against frost and ice formation.

Construction and purification of Cn-AFP and Al-binding peptide (ABP)-fused Cn-AFP.
To prepare an expression construct to produce the ABP-Cn-AFP fusion protein, the 5′ -forward primer encoding the aluminum-binding peptide (VPSSGPQDTRTT; shown in bold in Table 1) was used. The 5′ -and 3′ -primers included the XhoI and SalI restriction sites, respectively (underlined in Table 1). The open reading frame (ORF) encoding the active form of Cn-AFP (without a signal peptide) was amplified by PCR using the genomic DNA of C. neogracile, and was used to produce Cn-AFP G124Y via site-directed mutagenesis, as described in our previous report 16 . Cn-AFP genes were amplified by PCR, digested with XhoI and SalI, and ligated into the pColdI expression vector (Takara, Japan). The expression vectors were transformed into E. coli BL21 (DE3), and the pColdI expression method (Takara, Japan) was used to induce protein production. Cells were disrupted by sonication and soluble recombinant proteins were purified by His-tag affinity chromatography (Qiagen, U.S.A) 16 . Purified proteins were concentrated by Centricon microconcentrators (Millipore, U.S.A.) and the protein concentration was measured using the Bradford reagent (BioRad, U.S.A.).

AFP immobilization on
Al. An Al substrate 1 mm in thickness was cut into 10 mm × 10 mm sections and cleaned via subsequent immersion in the following solutions: i) 10% Na 2 CO 3 (pH 11) for 1-2 min at 40-45 °C, ii) deionized water (washing three times), iii) 5-10% NaOH (pH 11) for 1-2 min at RT, and iv) deionized water (washing three times). The substrates were then dried in air prior to use. For the formation of an AFP-coated surface, Al substrates were immersed in a 10 μ M solution containing either Cn-AFP G124Y or ABP-Cn-AFP G124Y fusion proteins (dissolved in 10 mM phosphate-buffered saline (PBS), pH 7.4) for 2 h at RT Al surface, followed by thorough rinsing with distilled water (three times). To minimize protein denaturation, each AFP-coated Al substrate was immersed in 0.1% (w/v) trehalose solution in 10 mM PBS (pH 7.4) for 1 h at RT in a conventional 12-well plate. Trehalose-treated substrates were then air-dried prior to use.
Characterization of surface-immobilized AFPs on an Al surface. For direct detection of the His 6 -tag at its N-terminal region of the AFP or ABP-AFP fusion proteins on Al, protein-coated Al surfaces were immersed in a nickel-activated HisProbe-HRP solution (at a final concentration of 1 μ g mL −1 dissolved in 100 mM PBS containing 0.01% Tween-20, pH 7.2) for 2 h at RT, followed by thorough rinsing with a washing buffer (100 mM PBS containing 0.01% Tween-20, pH 7.2) (three times), according to the manufacturer's instructions. The surfaces were then immersed in a 1 mL stock solution of Scientific RepoRts | 5:12019 | DOi: 10.1038/srep12019 1-Step Turbo TMB (Thermo, U.S.A.) as a soluble colorimetric substrate for HRP. After 30-60 min of incubation at RT, an aliquot (100 μ L) from the reactant blue solution was transferred to a new 96-well plate. The absorbance spectrum of the solution was measured using a micro-plate reader equipped with a UV-spectrophotometer (Varioskan Flash Spectral Scanning Multimode Reader, Thermo, U.S.A.). The absorption maxima were observed at 370 nm, as defined elsewhere 42 .
To further verify the AFP immobilization on Al, surface infrared (IR) analysis was performed in attenuated total reflectance mode using a Fourier transform (FT)-IR spectrophotometer (IFS66V/S & Hyperion 3000, Bruker Optiks, Germany) equipped with the Smart Apertured Grazing Angle (SAGA) accessory. A total of 64 scans on average were performed to yield the IR spectrum at a resolution of 4 cm −1 . All spectra of ABP-Cn-AFP WT -Al and control surfaces (bare Al and Cn-AFP WT -Al) were displayed in the absorption mode, and ranged from 4,000 to 400 cm −1 .
Characterization of the anti-icing activity of AFPs immobilized on Al. The supercooling points of an Al substrate coated with ABP-Cn-AFP G124Y and an Al substrate coated with Tre-ABP-Cn-AFP G124Y were measured using a differential scanning calorimeter (DSC Q100; TA instruments, U.S.A.). The temperature of the sample stage was lowered by 0.1 °C min −1 from 0 °C to −25 °C. The antifreeze activities of the fusion proteins (5 mg/mL) were assayed on the basis of thermal hysteresis and ice crystal morphology using a nanoliter osmometer (Otago Osmometer, New Zealand). Briefly, the recombinant proteins were placed at the sample stage, and the temperature of the sample stage was lowered to a rate of 0.01 °C min −1 . During cooling, the TH values were measured and the ice crystals were observed under a light microscope equipped with a CCD camera (BX53, Olympus, Japan). The anti-icing activity of Cn-AFP-Al was observed in an isothermal-isohumidity chamber. The temperature and relative humidity of the chamber were maintained at −3.5 °C and 84%, respectively, while air (flow velocity, 0.5 m s −1 ) and a refrigerant (brine; flow velocity 2 L min −1 ; −12 °C) flowed for 3 h. Tre-ABP-Cn-AFP G124Y -Al, bare Al, and Al with hydrophilic ZrO 2 coating were used in these experiments.