The inhibition of ice growth has been the focus of significant recent research. This inhibition is important for many fields, including (a) cryotherapy [1
], (b) the use of ice slurries (the mixtures of ice and water) to cool the brain during cardiac arrest, (c) the preservation of organs for transplantation in hospitals [2
], (d) the improvement of storage and transportation of fresh foods by using ice slurries, and (e) the development of distributed air-conditioning systems by using ice slurries. In these cases, there exists the need to control ice growth, at least in the cases of blood plasma and ice slurries.
One of the most promising methods for controlling the growth of ice is to use a substance which has the specific purpose of controlling ice growth. Antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) are appropriate substances for controlling ice growth, because they lower the temperature at which a seed crystal grows but do not alter the temperature at which the crystal is stable during melting at very low cooling rates. Furthermore, an appropriate concentration of AF(G)Ps is much lower than that of other solutes [3
]. This minimizes the effect of AF(G)Ps on osmotic pressure.
Among various AF(G)Ps discovered to date, HPLC6, the major fraction of winter flounder AFP, has been widely investigated. Measurements of ice crystal growth and ice crystal morphology in solutions of this protein are classified into the following two types according to the cooling rates: (i) at a very low cooling rate (≤−1 °C/min) and (ii) at a low cooling rate (>−1.5 °C/min). In type (i) measurements, an AFP solution of 0.01 mm3
in oil or liquid paraffin in osmometers are typically frozen at −40 °C, and gradually heated until only a single crystal of approximately 7 μm in diameter is obtained [3
]. The solution with the tiny crystal is maintained at this temperature for 1 min to allow the crystal to stabilize, and this temperature is defined as the ‘melting point’ (mp), although in some cases the mp is taken to be the temperature at which the crystal finally disappears. The solution with the crystal is then gradually cooled at −1.0 °C/min. Chao et al. [5
] considered the ‘freezing point’ to be the temperature at which the ice growth velocity exceeded 0.2 μm/s. It can be considered from this low ice-growth velocity that thermodynamically quasi-equilibrium conditions are held. In the case of type (ii) measurements, there are several other experimental methods that can help to elucidate the activities of AF(G)P: Wilson et al. [6
] developed an automated lag-time apparatus where a 200 mm3
sample solution of winter flounder AFP was held in a glass tube, and they measured the nucleation rate in the tube. Coger et al. [7
], Furukawa et al. [8
], Butler [9
] Hagiwara and Yamamoto [10
], Hagiwara and Aomatsu [11
] and Miyamoto et al. [12
] carried out experiments on the unidirectional freezing of AF(G)P solutions in narrow spaces between two glass plates. They measured the velocities and morphologies of the ice/solution interfaces, the concentration of solutes, temperature distribution and the dimension of AFP aggregates. The ice growth velocity was in the range of 0–89 μm/s. Serrated interfaces were produced by not only the adsorption of AF(G)P but also the approach of AFP aggregates or high concentration region of solute to the interfaces. The concentration distributions of AFP and ions in the mixed solution were changed by the other solute, which is a possible reason for the synergistic effect of solutes.
Although these findings are valid, it has not yet been confirmed whether they hold in the situation where the AFP solution flows along with ice crystals. Furthermore, experimental results for such flows are limited. Grandum et al. [13
] showed that the pressure drop for the ice slurry flow was higher than that for water flow in a pipe of 6 mm in inner diameter. They also observed the growth of seed crystals in the direction of c
-axis in the quiescent solution and the crystals being transported in the core region of flow. Onishi et al. [14
] found that HPLC6 inhibited the aggregation of ice particles in ice slurry flow in a mini-channel.
With this in mind, in the present study, we measure the growth of ice particles in the flow of HPLC6 solution in mini-channels. We discuss the effects of preheating or ultrafiltration of the solution on the ice-particle growth.
2. Materials and Methods
The sequence of amino-acid residues for HPLC6 is as follows: DTASDAAAAAALTAANAKAAAELTAANAAAAAAATAR (A: Alanine, D: Aspartic acid, E: Glutamic acid, K: Lysine, L: Leucine, N: Asparagine, R: Arginine, S: Serine, T: Threonine). It consists of 37 amino acid residues and has a molecular weight of 3242 Da. The secondary structure of HPLC6 is an α-helix. Four threonine residues are positioned at nearly identical distances on one line parallel to the helical axis. The distance between the oxygen atoms on the pyramidal faces of ice crystals, which were observed in the supercooled solution of HPLC6, is nearly identical to the distance between the threonine residues. It had thus been hypothesized that the hydrogen atoms of the threonine residues were bonded permanently to the oxygen atoms on the pyramidal faces in the ice crystal and that the water molecules were prevented from bonding to the ice surface by the Kelvin (or Gibbs–Thomson) effect [5
]. On the other hand, discussion was conducted on the alanine-rich surfaces of HPLC6 in the ice-growth inhibition mechanism [15
We purchased the synthetic polypeptide from GenScript Inc. (Taito, Tokyo, Japan).
The HPLC6 solution was preheated at a constant temperature for a predetermined time and cooled in the temperature-controlled room before the measurements were carried out. A polypropylene bottle containing the sample liquid was installed in a drying chamber (As One Co., Ltd., Osaka, Japan, ETTAS ONW-450S). The temperature was set to 80 °C and the duration was one hour. The HPLC6 concentration was 0.5 mg/mL. The preheating condition was determined based on our previous study on the inhibition of unidirectional freezing of HPLC6 solution [12
The preheated HPLC6 solution was filtered to increase the solution concentration. We used a centrifuge (Koki Holdings Co., Ltd., Tokyo, Japan, Himac CF15D2) and ultrafiltration membranes. The molecular weight cut-offs of membranes were 104 and 105. The centrifugal force was 14,000× g.
2.4. Production of Ice Slurry
shows the apparatus for producing ice slurry. Saline solution of 100 mL was contained in a beaker made of polymethylpentene. The beaker was installed in the constant-temperature liquid bath (Yamato Scientific Co., Ltd., Tokyo, Japan, BB301). The bath was filled with ethylene glycol as a coolant. The free surface of the coolant was set higher than that of the saline solution so that the solution was cooled from surroundings.
First, the temperature of the solution was maintained at −3 °C. Secondly, seed crystals of ice were added to the supercooled solution. The solution with ice particles was stirred with an agitator (As One Co., Ltd., Osaka, Japan, K-2RFN) for one hour. The temperature of the mixture was maintained at −1.2 °C.
The apparatus consisted of an inverted biological microscope (Nikon Instech Co., Ltd., Tokyo, JapanNikon, C2+), a monochrome charge-coupled device (CCD) video camera (Hamamatsu Photonics K. K., Hamamatsu, Japan, ORCA-ER), a bench-top cooling section and a syringe pump (Harvard Apparatus, Holliston, Massachusetts, USA, Model 11 Elite). The apparatus was placed in a temperature-controlled room maintained at 2 °C.
shows the mini-channel. The channel was made of a grooved polydimethylsiloxane (PDMS) plate of thickness 1 mm, width 10 mm and length 70 mm, and a flat glass plate of thickness 1 mm, width 52 mm and length 76 mm. These plates were glued to each other. The following two mini-channels were used: tThe dimensions of the mini-channel 1 were depth 1.0 mm, width 2.0 mm and length 50 mm, whereas the dimensions of the mini-channel 2 were depth 0.70 mm, width 1.0 mm and length 50 mm. These channels are models for blood vessels in the applications of (a)–(d) mentioned in the introduction. We arranged the coordinates as follows: The X
-axis was in the slurry-flow direction, the Y
-axis was in the transverse direction, and the Z
-axis was in the vertical (viewing) direction. The origin of the coordinates was located at the corner of the channel bottom surface.
A silicone tube of inner diameter 2 mm and outer diameter 3 mm was attached to the channel exit. The tube was connected to the syringe pump to draw ice slurry from the channel. A syringe, which consisted of a polypropylene cylinder and a polyethylene piston, was used. The volume of the syringe was 1 mL. The flow rate of slurry was set at 0, 20 and 40 μL/min.
The ice slurry with or without HPLC6 was introduced into the mini-channels from the cone-shaped entrance. The ice slurry was mixed manually with a small stirrer at the entrance.
2.7. Cooling of Channels
We adopted the following two types of channel cooling: The ambient air temperature was 0.5 °C lowered to subzero degrees (type A), and copper plates surrounding the channel were cooled by a thermoelectric cooler (i.e., Peltier cooler) (type B). In the case of type B cooling, the channel axis was in parallel with the axis of conductive heat transfer of the copper plates. The flow direction was opposite to the conduction. The copper plates and the thermoelectric cooler were adhered with heat conduction grease. The copper plates had slits of width 2 mm to illuminate and observe the channel. The temperature inside the cooler was set at −2.0 °C.
2.8. Image-Capturing Condition
The image-capturing condition is shown in Table 1
. In the case of quiescent ice slurry containing HPLC6, we used the mini-channel 1 under the type A cooling condition. The concentration of HPLC6 was varied as 0, 0.125, 0.25 and 0.50 mg/mL. The measured temperature around the observation area was −1.2 °C.
In the case of ice slurry flow containing HPLC6, we adopted the identical mini channel, the cooling condition and the image-capturing condition. The concentration of HPLC6 was 0.25 mg/mL. The flow rate was 40 μL/min. The observation area was moved downstream so that the images of identical ice particles could be captured. The measured temperature around the observation area was −1.4 °C.
In the case of flow of the mixture of ice slurry with the preheated solution of HPLC6, we used the mini-channel 2 under the type B cooling condition. We adopted the identical image-capturing condition. The concentration of HPLC6 was 0.25 or 0.50 mg/mL. The concentration of sodium chloride was 0.45 wt%. The measured temperature around the observation area was 1.0 °C.
In the case of flow of the mixture of ice slurry with the ultra-filtrated solution of preheated HPLC6, the mini-channel and the cooling condition were identical with those used in the case of flow of the mixture of ice slurry with the preheated solution of HPLC6. We changed the volume of HPLC6 solution for its mixing with the ice slurry and the molecular-weight limit of ultrafiltration.