Water deep supercooling via surface sealing

When water molecules aggregate on the water surface (water/air interface) to form an ice nucleus, they need to overcome an energy barrier γia − γwa (γ: interfacial tension, symbols i, w, and a refer to ice, water, oil, and air, respectively) per unit area as the ice/air interface replaces original water/air interface. In comparison, the energy barrier for homogeneous ice nucleation within bulk water is proportional to the water/ice interfacial tension, γwi. This interfacial tension can be expressed via the Young’s equation as γwi = γia − γwa cosθ iwa ≥ γia − γwa (θ iwa : water contact angle on ice/water/air interface, Supplementary Note 1 and Supplementary Figure 1a). This inequality indicates that heterogeneous ice nucleation on the surface is thermodynamically more favorable than homogeneous nucleation in bulk as complete wetting (θ iwa = 0°) is not generally observed27, and a receding contact angle of 12° has been reported31. Therefore, if the water surface is sealed by an oil phase, the energy barrier of ice nucleation at the water-oil interface would be γio − γwo (symbol o refers to oil phase). Similarly, the homogenous nucleation energy barrier can be now expressed in terms of another triple interface, namely the oil/water/ice as γwi = γio − γwo cos θ iwo , where θ iwo , for many oils can be nearly 0° as they are very repellent to ice (Supplementary Note 1 and Supplementary Figure 1b)28,32. In the case of θ iwo ≅ 0, the energy barrier approaches the limiting case γio − γwo ≅ γwi. This analysis indicates that the energy barrier of heterogeneous crystallization at the surface is elevated almost to the level of homogeneous one when the water/air interface is replaced by an oil/water interface. Accordingly, we hypothesized that surface sealing of water with an appropriate oil phase could suppress primary heterogeneous ice nucleation at the surface and enable extended storage of deeply supercooled water.

Water deep supercooling via surface sealing with oils

First, we cooled a large ensemble of polystyrene tubes containing 1 ml of ultra-pure water to −13 °C (Fig. 1a, b). This resulted in >90% of samples to be frozen after 24 h and nearly all samples to be frozen after 5 days. In contrast, the ultra-pure water samples could be kept in the liquid phase for a week, at the same temperature, if their surfaces were sealed by various types of immiscible oils, such as light mineral oil (MO), olive oil (OO), heavy paraffin oil (PO), and nutmeg oil (NO). Interestingly, the curdling of OO during DSC does not trigger water freezing, though the cumulative freezing frequency (f f , f f = 1 − f s ) increases significantly compared to water sealed by other oils (Fig. 1a). In supplementary experiments, we observed that the water degassed by vacuuming for 24 h, has similar f f as normal water, with or without oil sealing (Supplementary Figure 2). These experiments indicate that air dissolved in the water does not play a major role in ice nucleation in our experiments. Given this result and the consistent efficacy of surface sealing by different oils on freezing reduction, we infer that the air-water interface is the primary nucleation site.

Fig. 1 Deep supercooling of pure water enabled by surface sealing with oils. a Cumulative freezing frequency (f f ) for 1 ml water at −13 °C over 7-day deep supercooling (DSC), without sealing (W/O seal), with surface sealing by light mineral oil (MO), olive oil (OO), heavy paraffin oil (PO), and nutmeg oil (NO). Number of independent experiments n = 6, number of total tested samples for each case N = 56. NS: p > 0 .05; *0.005 < p < 0.05; **1.0 × 10−6 < p < 0.005, ***p < 1.0 × 10−6. b Corresponding samples of (a) post 1-day storage. c f f of DSC water of various volumes post 1-day storage at −13 and −16 °C. n = 7, N = 272, 145, 336, 123, and 125 for 3, 30, 200, 1000, and 10000 μl water, respectively. d f f of 100,000 μl water with different sealing oils and temperatures post 1-day storage, n = 7, N = 35. Error bars represent standard deviations Full size image

We also examined the influence of water volume on the efficacy of oil sealing for freezing inhibition. We studied the two most promising oils, MO and PO, at −13 and −16 °C for ultra-pure water ranging from 100–105 µl (Fig. 1c, d, Supplementary Figure 3). We found that MO sealing can effectively suppress water freezing for water volumes up to 104 µl at −13 and −16 °C. PO sealing was even more effective with a low f f throughout the entire volume range at −13 °C, and only 45.8% of samples frozen at −16 °C for the 105 µl samples. In addition, 8 out of 35 (22.8%) samples of 105 µl water were kept in the supercooled state at −16 °C for 100 days without any freezing event after Day-3 (Supplementary Figure 3b). While further investigations might be necessary, these observations are incompatible with conventional stochastic freezing processes (Supplementary Note 1), which implies exponential decrease of f s with time21,22. Alternatively, the freezing of DSC water sealed by oil could be depicted as “case-specific” that some of sealed water samples are more susceptible to crystallization than others. A reconciliation of these two cases might lie in the fact that those samples that do not freeze within our observation period have much fewer impurities and thus a much smaller exponential for the decay of f s than those that freeze within 3 days.

In order to further support our hypothesis that the water/air interface plays a dominant role in ice nucleation and subsequent freezing, we measured water freezing frequencies under differential degrees of surface sealing by MO, ranging from (I) unsealed (0 oil), (II) ring sealed along the contact line between water and tube wall (0.01 ml), (III) partially sealed with partial exposure to air (0.1 ml), (IV) critically sealed with water surface just completely covered (0.15 ml), (V) normally sealed (0.5 ml), and (VI) over sealed with excessive oil mounted on water surface (3.5 ml) (Fig. 2a, b and Supplementary Figure 4). The results indicate that the capacity of freezing inhibition increases with the degree of sealing, with a statistically maximum plateau achieved by critical sealing (Fig. 2a). Ring sealing (0.01 ml) that nullifies the triple solid/water/air contact line has a mild effect on freezing inhibition at high temperatures (−10 °C) but is not effective below −13 °C. Taken together with partial sealing results (0.1 ml), this result implies that the contact nucleation at the air/water/solid triple interface is not as dominant as that at water/air interface especially at low temperatures. Considering the crystallization efficiency depends on the integration of nucleation probability J and nucleation length (or area), the triple contact line of short length would provide smaller crystallization efficiency than the air/water interface even though it has higher J33,34. Overall, we confirmed that the water/air interface is the primary ice nucleation site for DSC water, and surface oil sealing that removes the water/air interface can effectively inhibit ice nucleation and water freezing.

Fig. 2 Dependence of water freezing efficiency on the volume and viscosity of sealing agents. a Effect of sealing oil (MO) volume on f f post 1-day DSC at different temperatures. n = 6, N = 70. b Side view of corresponding samples of a. MO includes Oil Red O for staining and imaging. 0 oil, 0.01 ml, 0.1 ml, 0.15 ml, 0.5 ml, and 3.5 ml indicate no seal, ring seal, partial seal, critical seal (just complete surface seal), standard seal, and over seal by MO, respectively. c Effect of viscosity of sealing agents on f f post 1-day DSC at −16 °C. The sealing agents are hydroxy (OH) terminated polydimethylsiloxane (PDMS) of different chain lengths and viscosities. n = 5, N = 56. Error bars represent standard deviations Full size image

We also observed that oil addition beyond the critical sealing has a statistically negligible effect on freezing suppression. This indicates that additional pressure and dampening effects, associated with a long-column of viscous oil phase, have a negligible effect on freezing inhibition. In order to further test this, we examined the effects of viscosity of the sealing agents where we used hydroxy (-OH) terminated polydimethylsiloxane (PDMS) of different chain lengths (Fig. 2c). In a similar fashion, we did not observe statistically significant differences in the capacity of freezing inhibition of PDMS with a viscosity range of 1–5 × 105 cP, with the exception of 3500 cP PDMS that has almost no freezing suppression effect. We hypothesize that this odd behavior is likely due to the formation of an ordered structure between water and this particular PDMS on the interface through hydrogen bonding, which closely matches the lattice of hexagonal ice35.

Water deep supercooling via surface sealing with alkanes and alcohols

Most oils are complex mixtures of alkanes, saturated cyclic alkanes, alkylated aromatic groups, and fatty acids among other hydrocarbon compounds. In an effort to more systematically study the observed freezing inhibition effect of supercooled water sealed with an immiscible hydrocarbon phase, we studied two prototypical families of hydrocarbons: linear alkanes and their corresponding primary alcohols of different lengths (Fig. 3). Specifically, we have studied alkanes (C m H 2m+2 , denoted C m , m = 5 – 11) and primary alcohols (C m H 2m+1 OH, denoted C m OH, m = 4 – 8) as the sealing agents for DSC water at −20 °C. Since linear alkanes have very low polarity, they have weak interaction with polar water molecules. On the other hand, the primary alcohols, which are amphipathic, can form strong hydrogen bonds with water through their hydroxyl group (hydrophilic end) and even stable ordered interfacial structures. However, a binary combination of an alkane and an alcohol should not be utilized as a sealing agent as it forms cooperative hydrogen bonding, heterogenous microdomains, and interfaces36,37, pumping water into the mixture to produce milky emulsions on top of water (Supplementary Figure 5).

Fig. 3 Deep-supercooled water sealed with linear alkanes and primary alcohols. a f f of 1 ml deep-supercooled (DSC) water at −20 °C. n = 7, N = 87. Error bars represent standard deviations. When m > 11 for linear alkanes and m > 8 for primary alcohols, the sealing agents are frozen at −20 °C and cause DSC water frozen. When m < 5, the linear alkanes are gaseous under atmospheric condition and not suitable for sealing. When m < 4, the primary alcohols are miscible with water and not suitable for sealing either. b, c Schematic configurations of alkane/water (b) and alcohol/water interface (c), respectively. The alkane and alcohol molecules are displayed without aliphatic hydrogen atoms and colored in light green. The O and H atoms in hydroxyl group of alcohol and water are shown in red and white dots, respectively Full size image

We found that f f of DSC water, at −20 °C, sealed with alkanes decreases monotonically with increasing carbon number m and chain length l (Fig. 3a). The capacity of alkanes in freezing inhibition matches that of MO (Fig. 2a at −20 °C) at m > 9. This coincides with the observation that mineral oils tend to have hydrocarbons with alkane chain lengths above 10. While oils comprise of many different hydrocarbons, alkanes make up a major fraction of their composition. Accordingly, the trend of higher freezing inhibition with longer alkane chain lengths, might also partially explain the differences in f f between PO and MO (Fig. 1d) among other effects from other hydrocarbon groups that we have not yet studied. PO likely consists of longer carbon chain alkanes than MO based on their densities (PO ~ 0.855 – 0.88 vs MO ~ 0.838 g ml^−1) and dynamic viscosities (PO ~34 vs MO ~23 cP38). On a molecular level, the mechanism for this trend might lie in the structure of the alkane/water interface. It has been observed that an interfacial electron depletion layer with a thickness δ exists between water and hydrophobic alkane chains by both X-ray reflectivity (XR) measurements39,40,41 and atomistic molecular dynamics (MD) simulations42,43. The few water molecules in the depletion layer (electron density < 40% that of bulk water44) can buckle in the intermolecular space near the ends of alkane molecules (Fig. 3b), and create a template for the formation of an ice nucleus29. The alkane chains adjacent to the water molecules preferentially have their longest axis parallel to the water interface with a tilt angle β39. This tilt angle increases with m and l, resulting in a more parallel orientation for longer alkanes39. Accordingly, longer alkane chains are expected to reduce the corrugation and roughness of the interface on the side of alkanes. This, consequently, would decrease the number of buckled water molecules and nucleus templates, and thus lower the probability of heterogeneous ice nucleation on that layer29. These expectations are in line with our observations of decreasing freezing frequencies for longer alkane chain lengths. From the perspective of thermodynamics, longer and flatter-oriented alkanes results in fewer and sparser buckled water molecules in the interface serving as nucleation template, which implies smaller contact region between ice embryo and sealing alkanes, smaller θ iwo (even though they are already much smaller than θ iwa ), and higher energy barrier for heterogeneous ice nucleation (Supplementary Figure 1b).

On the other hand, f f of DSC water, at −20 °C, sealed with alcohols increases with m and l. For example, f f equals 4.4% and 21.4% after 1-day DSC for C 4 OH and C 8 OH, respectively. C 4 OH has a higher freezing inhibition capacity compared to C 5 OH (f f = 4.4% for C 4 OH, f f =12.8% for C 5 OH sealing). Nevertheless, C 4 OH has a small but relatively higher solubility in water than C 5 OH, and accordingly C 5 OH might be the optimal choice for sealing. The different behavior of alcohols with respect to chain length might be due to the different structures of the alcohol/water interface compared to that of alkane/water interface (Fig. 3c). Unlike the alkanes which prefer a parallel orientation, the primary alcohols orient perpendicularly to the interface with a small β (usually less than 30°)45,46. The primary alcohols align their hydroxyl (-OH) heads toward the interface to form hydrogen bonds with water molecules. Accordingly, no depletion layer of interfacial water exists as in the alkane/water interface. The 2D layer of interfacial water molecules are strongly hydrogen-bonded to the hydroxyl groups, with their H atoms pointing toward alcohol as revealed by heterodyne-detected vibrational sum frequency spectroscopy (SFG)47. Therefore, structures and dimensions of the contacting layer of amphilic alcohols essentially determine the distribution and arrangement of interfacial water molecules, and the formation of heterogeneous ice nucleus35,48.

Experimental measurements via grazing incidence X-ray diffraction (GIXD) and MD simulation of ice nucleation in droplets under monolayers of long primary alcohol chains with 16 ≤ m ≤ 31, revealed a very low tilt angle β (~7.5 – 12°) and a very good lattice match between hexagonal ice and the alcohol structure for 29 ≤ m ≤ 3145,46,49, resulting T f as high as −1 °C for these longest chains. As m and l decrease, β increases up to ~19° for m = 1646. In conjunction, a greater lattice mismatch between hexagonal ice lattice and ordered alcohol layer at the interface along with a lower ice nucleation efficiency and T f were observed35,45,46,49. For shorter alcohols (4 ≤ m ≤ 8) in this study, larger tilt angles would ensue as evidenced by β = 28° for m = 6 and β = 30° for m = 546, causing greater lattice mismatches between hexagonal ice and ordered alcohol structure given the general structural similarity of primary alcohols. Compared to longer alcohol chains, the interfacial -OH groups anchored to smaller alcohols have stronger in- and out-of-plane fluctuations at the same temperature. We, therefore, expect that lattice mismatch and the –OH group fluctuations can destabilize any ordered domaine of crystalline water and impede the formation of ice nucleus of critical size49. Given that both effects are larger with smaller chain lengths, we expect that higher nucleation inhibition can be achieved by smaller primary alcohols, in line with our experimental observations. From the perspective of thermodynamics, greater lattice mismatch and interface fluctuation associated with shorter alcohol molecules directly reduce the probability of the formation of icing template of critical size for successful nucleation, which indicates smaller stable contact area between ice nucleus and sealing alcohols and thus, higher free energy barrier for heterogeneous ice nucleation at the interface. Once again, we observed that there is no significant difference of f f between 1-day and 7-day storage when sealed by either alkanes or alcohols. This further suggests the case-specific, rather than stochastic, nature of water freezing with oil sealing that we have previously discussed.

Stability tests for deep-supercooled water

Having established the efficacy of the DSC approach using either oils or pure alkane and alcohol phases, we then studied its stability under vibrational, thermal, and ultrasonic disturbances. Vibrational disturbances were introduced by placing DSC water onto a shaking plate with various shaking speeds and frequencies. When the DSC water (−20°) is sealed by MO, its f f is 0% and 5.6%, respectively, under 0.84 g and 2.1 g centrifugal acceleration (Fig. 4a), which are much higher than ac/deceleration forces of a commercial airliner (0.2 – 0.4 g) during potential transporation. Thermal disturbances were induced by putting the DSC samples into 37 °C incubator or plunging them into 37 °C water bath with warming rate of 100 C min-1 (heated by natural convection in air) or 102 C min−1 (heated by forced convection in water), respectively. Very few (0% for gas warming, 2.5% for water warming) of the samples freeze under these thermal fluctuations. In contrast, these samples cannot endure ultrasonication in 40 kHz ultrasonic water bath (Fig. 4a and Supplementary Movie 1), with f f of ~ 84%. This is probably due to the vigorous collapse of cavitation bubbles in water during ultrasonication18, which would cause ultrahigh local pressure (>1 GPa)50, and therefore, significantly increase equilibrium temperature T e and the degree of supercooling ΔT.

Fig. 4 Stability tests for 1 ml deep-supercooled water at −20 °C. a f f of deep-supercooled (DSC) water sealed by MO under various disturbances. Vibrational disturbance was imposed by shaking plate with different shaking frequencies and centrifugal forces (i.e., 0.84 g or 2.1 g). Thermal disturbance was imposed by placing or plunging the DSC tubes into 37 °C incubator (37 °C gas) or water bath (37 °C water). Ultrasonic disturbance was introduced by putting the DSC tubes into 40 kHz ultrasonic water bath. n = 6, N = 48. Error bars represent standard deviations. b f f of DSC water sealed by linear alkanes and primary alcohols under 40 kHz ultrasonic disturbance. n = 3, N = 24 (except for C 5 , N = 8). c Representative image sequences of ultrasonication tests for DSC water sealed by linear alkanes or primary alcohols Full size image

Upon the instability of DSC water sealed by MO under ultrasonication, we further tested its stability sealed by pure alkanes and primary alcohols. DSC water sealed by alkanes freeze immediately upon being ultrasonicated (Fig. 4b–c and Supplementary Movie 2), which is consistent with previous observation of MO sealed water since MO has a high content of various alkanes. On the contrary, none of the samples freeze upon ultrasonification if they were sealed by any of the primary alcohols (Fig. 4b). Instead, the sealing alcohols would be emulsified with supercooled water, starting from the interface and then evolving toward supercooled water (Fig. 4c and Supplementary Movie 3). The exact mechanism of the freezing resistance of DSC water sealed by alcohols to ultrasonic disturbance is still unknown, and one hypothesis would be that ultrasound preferentially transduces its energy into joint molecular motion at interface due to the hydrogen bonding between water and amphilic alcohols to form nanoemulsion51, rather than cavitation bubbles for ice nucleation in DSC water.

Deep supercooling of hRBCs for extended preservation

In addition to DSC of pure water, we have utilized the surface sealing method to achieve DSC for aqueous solutions and cell suspensions to demonstrate extended supercooling preservation of biological specimens (Fig. 5). The current clinical standard for preservation of hRBCs is via conventional cold storage at 4 °C with the CP2D + AS-3 (anticoagulant citrate phosphate double dextrose supplemented with additive solution 3) solution52. This standard approach for hRBC preservation can provide storage for a maximum of 42 days53, beyond which the cells experience irreversible storage lesions including hemolysis as shown in Fig. 5a. According to the Arrhenius relationship, preservation at deep subzero temperatures would slow down cellular metabolism and decay rates, extending this biopreservation period. Accordingly, we have preserved hRBCs at as low as −16 °C for an extended period of 100 days. Specifically, using surface sealing by PO, we successfully supercooled 1 ml suspensions of hRBCs in either conventional cold storage solution, CP2D + AS−352, or University of Wisconsin solution supplemented with 5% (w/v) trehalose (UW + Tre), for as long as 100 days at −7, −10, −13, and even −16 °C (see Supplementary Figure 6 for f f ). The suspension volume could be extended to the magnitudes of 101 even 102 ml as 5 out of 6 vials of 30 ml hRBC suspension (500 million cells) kept unfrozen over 365-day supercooling at −13 °C in our pilot experiments. In CP2D + AS-3 solution, hRBCs experience remarkable hemolysis as shown by the presence of dark spots (RBC debris without holding hemoglobin) in the micrographs of Fig. 5a and reduced recovery rates of hemoglobin in Fig. 5b, c, especially at deep subzero temperatures. Surviving hRBCs become spherically shaped, losing the distinctive biconcave disc form of fresh hRBCs (Supplementary Figure 7). However, in UW + Tre solution, hRBCs do not undergo noticeable hemolysis, and the recovery rates of hemoglobin at −16 °C (94%) is higher than that of conventional storage at 4 °C in CP2D + AS-3 solution post 100-day storage (76%, Fig. 5c). In addition, they can also maintain their intact, shiny, and discoid-shaped phenotype at DSC temperatures (the second and fourth rows of Fig. 5a), resulting in higher percentages of normal morphology (>88%, exclusion of serrated, spherical, swollen, or shrunk cells) than cold storage at 4 °C (49%, Fig. 5d). These results demonstrate that DSC via surface sealing can effectively prolong the storage time of hRBCs to 100 days combined with the optimization of preservation solutions.