Bioinspired Anti-Icing Hydrogel Enabled by Ice-Nucleating Protein

Abstract

Open AccessCCS ChemistryCOMMUNICATION1 Jan 2022Bioinspired Anti-Icing Hydrogel Enabled by Ice-Nucleating Protein Zhanhui Wang†, Baixue Lin†, Siyu Sheng, Sicong Tan, Pengchao Wang, Yong Tao, Zhang Liu, Zhiyuan He and Jianjun Wang Zhanhui Wang† Key Laboratory of Green Printing, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 , Baixue Lin† CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 , Siyu Sheng Key Laboratory of Green Printing, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 , Sicong Tan Heat and Mass Transfer Center, Institute of Engineering Thermophysics, Chinese Academy of Sciences, Beijing 100190 , Pengchao Wang CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 , Yong Tao CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 , Zhang Liu Key Laboratory of Green Printing, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 , Zhiyuan He *Corresponding author: E-mail Address: [email protected] Key Laboratory of Green Printing, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 and Jianjun Wang Key Laboratory of Green Printing, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 School of Future Technology, University of Chinese Academy of Sciences, Beijing 100049 https://doi.org/10.31635/ccschem.021.202000648 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Ice-nucleating proteins (INPs) are the most effective ice-nucleating agents that play a significant role in preventing freeze injuries in freeze-tolerant organisms. INPs promote ice nucleation in the extracellular space, harvesting water from cells due to the low vapor pressure of ice compared with water, thereby protecting freeze-tolerant organisms from intracellular freezing. The antifreeze mechanism of INPs offers a unique opportunity to inhibit large-scale freezing by localized control of ice formation, with valuable enlightenment in anti-icing material sciences. By learning from nature, we transferred the excellent ice nucleation-facilitating capability of INPs along with an antifreeze concept of spatially controlled ice nucleation to anti-icing material design, fabricating icephobic coatings that consisted of patterned hydrogel-encapsulated INP (PHINP). The ice patterns were templated by patterned PHINPs via the tuning of ice nucleation so that the ice coverage fraction could be controlled by <30% on almost all PHINP-coated surfaces. Combining PHINP with solar-thermal conversion surfaces endowed the composite coatings with high anti-icing performances at any time of the day. Download figure Download PowerPoint Introduction Undesired ice formation is ubiquitous and poses many serious problems to a broad range of applications, including vehicle transportation, power transmission systems, shipping, aviation, and outdoor facilities.1–6 Currently, various anti-icing methods have been developed to inhibit or delay ice nucleation since the process is the controlling step of water to ice transition.7–11 Despite intensive studies of fabricating ice nucleation-inhibiting surface intrinsically, in real-live environments, ice nucleation has so far been inevitable due to unavoidable surface defects or contamination.1,12,13 However, some plants and animals, which live in cold regions on earth, rely entirely on different yet very effective ways to enable antifreezing. This is not done by inhibiting but instead promoting ice nucleation.14–16 Ice-nucleating proteins (INPs) are found in many freeze-tolerant species, including winter rye,17 citrus fruit,18 and insects.15 INPs can trigger an ice formation at high subzero temperatures, which help freeze-tolerant organisms to coexist with ice at low supercool temperatures while avoiding freeze injury.16 For instance, INPs are produced in the extracellular fluid of freeze-tolerant insects, promoting ice nucleation in the extracellular spaces predominantly (Figure 1a). Then the ice crystals pull water from the intracellular space into the extracellular space because the vapor pressure of ice is lower than that of liquid water.19 Subsequently, the cytoplasmic (fluid within the cell, between the cell membrane and membrane of the nucleus) solute concentration increases, thereby diminishing supercooling points. As a result, the insects are protected from intracellular freezing.20,21 Figure 1 | (a) Schematic illustration showing the antifreeze mechanism of freeze-tolerant insects via promoting ice nucleation in the extracellular spaces with the help of INPs. (b) Fabrication of the insect-shaped PHINP coatings using a visible light projection printing method. (c) Optical images of water condensation before freezing at −6 °C (left row) and formation of the dry area after condensation freezing at −8 °C (right row) on insect-shaped PHINP coating with ice supersaturation of 109%. Scale bar = 1 mm. Download figure Download PowerPoint Recent studies have indicated that integrating these active INPs into icephobic materials provided promising anti-icing strategies for scientific research and enable widespread anti-icing applications under different conditions.22,23 Herein, we report an anti-icing coating of patterned hydrogel-encapsulated INP (PHINP) based on a simple projection-printing method.24 The INPs triggered freezing of the freezable interfacial water inside the hydrogel preferentially at high subzero temperatures, generating patterned frozen PHINP coatings exclusively on substrates. The patterned frozen PHINP coatings could efficiently harvest surrounding water droplets, leading to drying of the uncoated surface areas. The INPs promote ice nucleation more efficiently than any other material known, ensuring the priority of ice formation of patterned frozen gel coatings on almost all material surfaces. The availability of coating PHINP on a wide range of materials would strengthen the general applications of this distinct anti-icing strategy. Experimental Methods Experimental methods are available in the Supporting Information. Results and Discussion The Pantoea ananatis INP (PA-INP) was expressed in Escherichia coli. The gene encoding PA-INP was amplified by polymerase chain reaction (PCR) using P. ananatis genomic DNA as the template (for details, see the Experimental Section of the Supporting Information). The proposed projection-printing method to encapsulate INPs in hydrogel matrices is schematically illustrated in Figure 1b. To maintain the activity of INPs during the formation of the patterned hydrogel, we developed visible light-reactive precursor-containing monomers of acrylamide and 2-hydroxyethyl methacrylate, a cross-linking agent of N,N-methylenebis(acrylamide), PA-INPs, and visible light initiator of TPO-Li on a water basis. The covalently cross-linked network of the poly(acrylamide-co-2-hydroxyethyl methacrylate) poly(AAm-co-HEMA) hydrogel offered a scaffold to encapsulate INPs and water ( Supporting Information Figures S1 and S2). The swelling ratio and INP content in the hydrogel could be tuned simply by changing the initial concentrations of cross-linker of N,N-methylenebis(acrylamide), and INPs in the precursor. Any arbitrary patterns of PHINP with a resolution as high as ∼20 μm could be printed on various vinyl-functionalized substrates, including glass, metal, and plastic surfaces ( Supporting Information Figures S3–S9). The film thickness of PHINP could be tuned from a few microns to several hundred microns via regulating the movable stage. As shown in Figure 1c, an insect-shaped PHINP coating was printed on a silicon substrate. The water condensation occurred at −6 °C before freezing (images in the left row in Figure 1c) as the temperature was lowered from room temperature at a cooling rate of 0.1 °C/min. The ice formation preferentially occurred on the insect-shaped PHINP coating at −8 °C (images in the right row in Figure 1c) due to the excellent ice nucleation-facilitating capability of INPs. The ice grew exclusively via desublimation on the insect-shaped PHINP pattern. Additionally, the surrounding condensed water droplets were absorbed by patterned ice because the saturated vapor pressure of water is higher than ice. Accordingly, the condensation freezing surrounding the insect-shaped PHINP coating was inhibited, leading to a dry area formation of the uncoated surface. Unlike the freezing properties of pure aqueous INP solution, ice formation inside hydrogel not only depends on the INP content but also on the interaction between water molecules and highly hydratable polymer networks.7,25 Water inside the hydrogel can be subdivided roughly into "free water" and "bound water," respectively.26 Polymer chains can capture nearby water molecules through hydrogen bonding to form bound water (Figure 2a, pink area). The water molecules that have almost no interaction with polymer chains are termed free water, exhibiting similar properties to those of bulk water (Figure 2a, blue area). It is proposed that the freezing behaviors of PHINP film are strongly determined by the states of free water and bound water, as well as INP contents in the PHINP.7 As shown in Figure 2b, we first measured the water contents in the fully swollen samples of PHINP with different cross-linking densities using thermogravimetric analysis (TGA). The water content (weight fraction) ranged from 30% to 83% as the cross-linking density decreased from 0.6% to 0.01% mol/cm3, respectively. To gain a molecular level understanding of the water molecules in the PHINP, the nuclear magnetic resonance (NMR) proton spin–spin relaxation time (T2) of the water molecules was studied, as shown in Figure 2c. There were two types of water molecules with different mobilities inside the PHINP film, that is, free water with high mobility having a long T2,free water (∼1800–2500 ms), and bound water having a short T2,bound water (∼30–700 ms), respectively. Notably, even the longest T2,free water is smaller than T2 of pure water (∼2850 ms). Both the T2,free water and T2,bound water were plotted against the cross-linking densities of PHINP, as shown in Supporting Information Figure S10. It was apparent that the values of T2, free water, and T2, bound water decrease with increasing cross-linking densities, showing that the mobilities of both free water and bound water decreased as the cross-linking densities increased. This observation was ascribed to the effects of confinement of the PHINP network.26 Figure 2 | (a) Schematic illustration showing that the PHINP contains two types of water, that is, "free water" and "bound water." (b) The TGA results show the equilibrium water weight ratio of PHINP with different cross-linking densities ranging from 0.01% to 0.6% mol/cm3. (c) T2 inversion spectra for water protons of free water and bound water inside PHINP films with various cross-linking densities. DSC investigation of freezable water molecules inside PHINP films with various (d) cross-linking densities, and (e) WINP, respectively. (f) TIN of water inside PHINP films with different WINP was measured from the onset points of the DSC peaks. Download figure Download PowerPoint TGA could detect all water molecules (i.e., "freezable water" and "nonfreezable water") within the PHINP films, while differential scanning calorimetry (DSC) detects only the freezable water, including "freezable free water" and "freezable bound water." Thus, the ice-nucleating effects of INPs inside the PHINP with various cross-linking densities were studied by DSC measurements, as shown in Figure 2d. We found that the ice-nucleating temperature (TIN) did not change with water content or the initial cross-linking densities of PHINP. However, as the cross-linking densities decrease from 0.6% to 0.01% mol/cm3, the relative fraction of freezable water inside PHINP increased from 50% to 72% (for details, see Supporting Information Figure S11). Figures 2e and 2f show that TIN values are strongly dependent on INP content. As the INP content (WINP, the weight fraction of INP) increased from 1 × 10−5 to 15 × 10−5, TIN increased from −16.1 to −8.3 °C. The TIN values of pure water and INP aqueous solution (0.2 mg/mL) were −19.0 and −6.6 °C, respectively. The freezing of pure water droplets atop of the hydrogel film could be triggered by the ice formed inside the hydrogel because the interfacial water was interconnected from atop and inside the hydrogel ( Supporting Information Figure S12).7 The heterogeneous ice-nucleation temperature (TH) of water droplets atop PHINP-coated surface was studied using an optical microscope coupled with a high-speed camera. The formation of ice was signified by the sudden change of water droplet opacity, as shown in Figure 3a. When the temperature was lowered from room temperature at a rate of 2.0 °C/min, the water droplet atop-uncoated silicon surface did not freeze until the temperature reached −23.3 °C (left column images of Figure 3a). In strong contrast, the water droplet atop the PHINP-coated surface froze at −8.6 °C (right column images of Figure 3a). By comparing the TH of 200 independent IN events on PHINP-coated and -uncoated silicon surfaces, as shown in Figure 3b, it was evident that TH of water droplets was raised significantly by the PHINP coating. The INP content has a profound effect on TH. The effect of cooling rates on TH was also investigated, as shown in Supporting Information Figure S13. We found that TH decreased gradually from −8.6 ± 0.4 °C to −23.3 ± 1.7 °C as WINP decreased, exhibiting a temperature window as large as 14.7 °C (Figure 3c). TH on PHINP surfaces is independent of the film thickness (from 2 to 350 μm), as shown in Supporting Information Figure S14. Figure 3 | (a) In situ-polarized optical microscopic images of a water droplet (1.0 μL) freezing on PHINP-coated and -uncoated silicon surfaces at a cooling rate of 2.0 °C/min. The cross-linking densities of PHINP are 0.02% mol/cm3, and WINP is 15 × 10−5, respectively. Scale bar = 100 μm. (b) TH of 200 individual freezing events on PHINP-coated and -uncoated silicon surfaces. (c) TH of water droplets on PHINP-coated surfaces with different WINP. Each value of TH represents the mean of 200 freezing events. Time-resolved optical images of condensation freezing on (d) PHINP-coated and (e) -uncoated silicon surfaces at −20 °C with ice supersaturation of 118%. "W" and "I" are denoted as "water" and "ice," respectively. Scale bar = 1 mm. Download figure Download PowerPoint The stripe-patterned PHINP coating on a silicon substrate was printed through the projection lithography system. The condensation and freezing behaviors at −20 °C on a patterned PHINP with cross-linking densities of 0.02% mol/cm3 and WINP of 15 × 10−5 are shown in Figure 3d. The ice nucleation preferentially occurred on stripe-patterned PHINP coating due to its high ice nucleation-promoting efficiency, forming ice stripes within 1 min. The ice stripes began to attract nearby water vapor due to the low saturation vapor pressure of ice, compared with that of water.27 As the ice stripe grew exclusively, a dry area surrounding the stripe was observed. The ice coverage on the patterned PHINP surface was still below 12%, even though the condensation freezing lasted more than 160 min. The inhibition of condensation freezing on PHINP surfaces with various patterns was also studied, as shown in Supporting Information Figures S15 and S16. In strong contrast, the uncoated silicon surface was wholly covered with condensed water for 70 min (the freezing delay time is ∼ 70 min), as shown in Figure 3e. After that, the freezing occurred, and the entire-uncoated surface was covered by thick ice/frost. Both condensation freezing and ice coverage can be spatially controlled on various PHINP-coated substrates by local tuning the ice nucleation efficiency of PHINP ( Supporting Information Figures S17 and S18). However, the PHINP-coated surface would still be covered wholly by ice when the freezing time is long enough (>24 h). Hence, developing a multifunctional anti-icing surface, which can meet the all-time anti-icing demands, is highly desirable.13,28,29 Compared with the previously reported patterned polyelectrolyte brush surface based on complex surface-initiated atom transfer radical polymerization methods,30 the possibility of coating PHINP on various icephobic surfaces can provide us with more opportunities to build multifunctional anti-icing materials based on this simple and large-scale producing projection-printing method. Moreover, the highest ice nucleation-promoting efficiency of INP (TH ∼ 8 °C) ensures the effective anti-icing performance of PHINP coating on all material classes. Combining the PHINP coatings with solar-thermal material of polypyrrole (PPy) endowed the composite surface of PHINP-PPy with high anti-icing performances at any time of the day, as shown in Figure 4a. The dry area formed on the PHINP-PPy surface (at night) can adsorb and utilize solar energy for heating material surfaces in the daytime, thus, melting the covered ice. The reflection spectra measurements (295–2500 nm) showed that the PPy surface exhibits good antireflection performance, such as average reflectivity of 7.2% in UV–vis region and 9.0% in the NIR region ( Supporting Information Figure S19). As the solar illumination intensities were raised from 20 to 70 mW/cm2, the average surface temperature of PPy increased from 34 to 58 °C, as shown in Figure 4b and Supporting Information Figures S20 and S21. A simulated heat transfer model was applied to predict the equilibrium temperatures on the PHINP-PPy surface with various ice coverages, as shown in Figure 4c. At an ambient temperature of −20 °C, the simulated equilibrium temperature on the PHINP-PPy surface exceeded 0 °C under one sun illumination even if the ice coverage reached 80%. This result was confirmed through quantitative simulated analysis on various substrates with different ice coverage ( Supporting Information Figures S22 and S23). Figure 4 | (a) Schematic illustration of spatial inhibition of freezing on ICCAS-patterned PHINP-PPy surface at night. The ice on PHINP can be melted, thanks to the solar-to-thermal effect during the day. (b) The temperature changing plots of PHINP-PPy and silicon surface under solar illumination of qi = 22, 33, 45, 60, and 67 mW/cm2 on and off. Inset is the infrared (IR) image of ICCAS-patterned PHINP-PPy surface under solar illumination of qi = 10 mW/cm2 for 300 s. (c) Numerical-simulated temperature changing curve of PHINP-PPy surface with an ice coverage of 80% under one sun illumination. The ambient temperature is at −20 °C. The inset of (c) is a side view of the temperature distribution of PHINP-PPy from simulations. (d) Time-resolved images of frost formation on PHINP-PPy and PPy surfaces at −15 °C with ice supersaturation of 142% without solar light (left row of images) and under solar illumination of qi = 70 mW/cm2. (e) Ice coverage on PHINP-PPy and PPy surfaces at −15 °C with ice supersaturation of 158% obtained by switching the light on and off many times. (f) Solar anti-icing performance of PHINP-PPy surface for an extended period. The testing temperature and solar intensity are set at –17.3 °C and 61 mW/cm2, respectively, based on Changchun's realistic environment (for details, see the Supporting Information). The inset images show the ice coverage change for days 1 and 100. Download figure Download PowerPoint Figure 4d and Supporting Information Figure S24 show the condensation-freezing behaviors on PPy and Institute of Chemistry, Chinese Academy of Sciences (ICCAS)-patterned PHINP-PPy surface under different light conditions, without light (images in left rows), and under solar illumination of 70 mW/cm2 (images in right rows). The uncoated PPy surface was wholly covered with frost/ice at −15 °C with ice supersaturation of 158% within 30 min. The covered frost/ice could not be melted and continued to grow even under solar illumination because most of the light was reflected or adsorbed by frost/ice. In strong contrast, ice formation preferentially occurred on ICCAS-patterned PHINP coating without solar illumination, forming a dry area within 30 min, which was more than 70% the amount of the entire surface area. The covered ice was melted within 5 min under solar illumination of 70 mW/cm2. Furthermore, water was evaporated under solar illumination in an hour, leaving a dry surface without ice or water. By switching the light on and off several times, the ice coverage on the PHINP coating could be controlled to <30% at all times (Figure 4e). In nature, air temperature, humidity, and sunlight intensity change frequently, which created increased challenges for the anti- and de-icing applications during different seasons and times of the day.31–33 We chose Changchun in northeast China as the research city; the average solar intensities, humidity, and temperatures of Changchun were collected in the winter seasons from 1985 to 2005.33 The ice coverage on PHINP-PPy surface in the daytime and at night was studied in laboratories by simulating the real-life conditions of Changchun. The ice coverage on the PHINP-PPy surface is below 60% at night, and the whole ice cover could be melted and removed in the daytime, as shown in Figure 4f. PHINP-PPy surface exhibited good anti- and de-icing performances after 100 repeated diurnal cycle tests over a long period of 100 days. These results confirmed the long-term anti- and de-icing capability and good durability of PHINP-PPy surfaces. Conclusions Being bioinspired by the antifreeze strategy of INPs in the freeze-tolerant insects, we fabricated PHINP coatings based on a simple projection-printing method. Thanks to the high ice nucleation-facilitating capability of INPs, global freezing on different PHINP-coated surfaces could be successfully controlled via localized tuning of the ice nucleation. Despite the high anti-icing efficiency, PHINP-coated surfaces could still be covered completely by ice if the freezing times were long enough. Establishing an effective anti-icing surface with multiple functions that could meet all-time anti-icing requirements would be highly desirable. However, little attention has been paid to the design of ice-phobic materials with high anti-icing properties during both night and day. Herein, we developed a PHINP coating on a solar-thermal conversion material PPy that endowed composite coatings with high anti-icing properties for any time of the day. At night, the freezing-driven formation of the dry area was observed on the PHINP-PPy surface, which benefited from the high activity of ice nucleation-facilitating PHINP coatings. During the day, the dry area adsorbed sunlight and converted solar energy to heat, thus melting the covered ice. This cyclic process ensured low ice coverage and high anti- and de-icing efficiency on the PHINP-PPy surface at all times. The broad applicability of PHINP coatings on different functional anti-icing surfaces provides many new possibilities to design multifunctional ice-phobic materials. Compared with the highest ice nucleation-promoting efficiency of INP, the long-term durability of PHINP might be less prominent, especially in outdoor environments due to the possible degradation of INPs. The introduction of more durable ice-nucleating agents, including feldspar, soot, and graphene oxide, is still highly needed. Supporting Information Supporting Information is available and includes experimental section and supporting figures. Conflict of Interest The authors declare no competing financial interest. Acknowledgments The authors gratefully acknowledge the financial support from The National Key Research and Development Program of China (grant no. 2020YFE0100300), Chinese National Nature Science Foundation (grant and National Key Program of China (grant no. Key Research Program of Sciences, CAS (grant no. and the of the Chinese Academy of Sciences (grant no. of Anti-Icing or for He Liu Wang for Ice and of We to Ice and He Wang Research on Anti-Icing He Wang Wang Wang Ice with Zhang Liu of Ice and Wang Ice of with P. of Ice by He Liu Wang at the for Ice China He Wang of Ice by He Wang He Anti-Icing and in of and Future for Ice in and and of Ice in and of Ice with of and P. of of by 22, of Ice-Nucleating on to of and Ice the of Ice He with Liu Wang the for Ice with T2 in as a of Water Ice of and Anti-Icing the of Tao Fabrication of and for Anti-Icing Water and Ice He Wang on Solar for Ice Solar for Tan Wang He Wang Solar via a Wang and Ice by The of Chemistry, Zhang Zhang and of the of Hydrogel Chemistry, , Information Chinese authors gratefully acknowledge the financial support from The National Key Research and Development Program of China (grant no. 2020YFE0100300), Chinese National Nature Science Foundation (grant and National Key Program of China (grant no. Key Research Program of Sciences, CAS (grant no. and the of the Chinese Academy of Sciences (grant no. times