冷冻保护剂浓度和降温速率对水溶液玻璃化冷冻的影响
Effects of cryoprotectant concentration and cooling rate on vitrification of aqueous solutions
摘要：
含水冷冻保护剂混合物的玻璃化在蛋白质和其他生物样品的冷冻保存中是必不可少的。我们报告的临界低温保护剂（CPA）的玻璃化所需的浓度在插入冷却从T=295 K到T=77 K在液氮中的系统测量。包括醇类在内的14种常见CPA的测定（甘油、甲醇、异丙醇）、糖（蔗糖、木糖醇、右旋糖、海藻糖）、PEG（乙二醇，PEG 200，PEG 2 000，PEG 20 000），二醇对从约nL至20 mkL的四个数量级的体积范围内进行DMSO、MPD和盐（NaCl）的分离，并且覆盖蛋白质晶体学中感兴趣的范围。X-射线衍射测量水甘油混合物证实，多晶到玻璃体的转变发生在CPA浓度小于2% w/v的跨度内，并且多晶冰的形式（六边形或立方体）取决于CPA浓度和冷却速率。对于大多数研究的冷冻保护剂，临界浓度随体积在约5 mkL至约0.1 mkL范围内强烈降低，通常为2倍。通过结合测量的临界浓度与体积与冷却时间与体积，我们得到的功能最大的内在物理利益：在闪蒸冷却过程中的临界CPA浓度与冷却速率。这些结果提供了一个基础，更合理的设计冷冻保护协议，并应产生洞察物理玻璃形成的含水混合物。
英文摘要：
Vitrification of aqueous cryoprotectant mixtures is essential in cryopreservation of proteins and other biological samples. We report systematic measurements of critical cryoprotective agent (CPA) concentrations required for vitrification during plunge cooling from T=295 K to T=77 K in liquid nitrogen. Measurements on fourteen common CPAs including alcohols (glycerol, methanol, isopropanol), sugars (sucrose, xylitol, dextrose, trehalose), PEGs (ethylene glycol, PEG 200, PEG 2 000, PEG 20 000), glycols (DMSO, MPD), and salt (NaCl) were performed for volumes ranging over four orders of magnitude from ~nL to 20 mkL, and covering the range of interest in protein crystallography. X-ray diffraction measurements on aqueous glycerol mixtures confirm that the polycrystalline-to-vitreous transition occurs within a span of less than 2% w/v in CPA concentration, and that the form of polycrystalline ice (hexagonal or cubic) depends on CPA concentration and cooling rate. For most of the studied cryoprotectants, the critical concentration decreases strongly with volume in the range from ~5 mkL to ~0.1 mkL, typically by a factor of two. By combining measurements of the critical concentration versus volume with cooling time versus volume, we obtain the function of greatest intrinsic physical interest: the critical CPA concentration versus cooling rate during flash cooling. These results provide a basis for more rational design of cryoprotective protocols, and should yield insight into the physics of glass formation in aqueous mixtures.
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