Abstract：
BACKGROUND: Psychrophiles can survive under cryogenic conditions because of various biomolecules. These molecules interact with cells, ice crystals, and lipid bilayers to enhance their functionality. Previous studies typically measured these interactions by thawing frozen samples and conducting biological assays at room temperature; however, studying these interactions under cryogenic conditions is crucial. This is because these biomolecules can function at lower temperatures. Therefore, a platform for measuring chemical interactions under sub-zero temperature conditions must be established.
RESULTS: The chemical interactions between biomolecules under sub-zero temperature conditions were evaluated within ice grain boundaries with a channel-like structure, which circumvents the need for thawing. An aqueous solution of sucrose was frozen within a microfluidic channel, facilitating the formation of freeze-concentrated solutions (FCSs) that functioned as size-tunable electrophoretic fields. Avidin proteins or single-stranded DNA (ssDNA) were introduced into the FCS in advance. Probe micro/nanospheres whose surfaces were modified with molecules complementary to the target analytes were introduced into the FCS. If the targets have functionalities under sub-zero temperature conditions, they interact with complementary molecules. The chemical interactions between the target molecules and nanospheres led to the aggregation of the particles. The size tunability of the diameter of the FCS channels enabled the recognition of aggregation levels, which is indicative of interaction reactivity. The avidin-biotin interaction and ssDNA hybridization served as models for chemical interactions, demonstrating interactivity under sub-zero temperature conditions. The results presented herein suggest the potential for in situ measurement of biochemical assays in the frozen state, elucidating the functionality of bio-related macromolecules at or slightly below 0 °C.
CONCLUSIONS: This is the first methodology to evaluate chemical interactions under sub-zero temperature conditions without employing the freeze-and-thaw process. This method has the advantage of revealing the chemical interactions only at low temperatures. Therefore, it can be used to screen and evaluate the functionality of cryo-related biomolecules, including cold-shock and antifreeze proteins.
RESULTS: The chemical interactions between biomolecules under sub-zero temperature conditions were evaluated within ice grain boundaries with a channel-like structure, which circumvents the need for thawing. An aqueous solution of sucrose was frozen within a microfluidic channel, facilitating the formation of freeze-concentrated solutions (FCSs) that functioned as size-tunable electrophoretic fields. Avidin proteins or single-stranded DNA (ssDNA) were introduced into the FCS in advance. Probe micro/nanospheres whose surfaces were modified with molecules complementary to the target analytes were introduced into the FCS. If the targets have functionalities under sub-zero temperature conditions, they interact with complementary molecules. The chemical interactions between the target molecules and nanospheres led to the aggregation of the particles. The size tunability of the diameter of the FCS channels enabled the recognition of aggregation levels, which is indicative of interaction reactivity. The avidin-biotin interaction and ssDNA hybridization served as models for chemical interactions, demonstrating interactivity under sub-zero temperature conditions. The results presented herein suggest the potential for in situ measurement of biochemical assays in the frozen state, elucidating the functionality of bio-related macromolecules at or slightly below 0 °C.
CONCLUSIONS: This is the first methodology to evaluate chemical interactions under sub-zero temperature conditions without employing the freeze-and-thaw process. This method has the advantage of revealing the chemical interactions only at low temperatures. Therefore, it can be used to screen and evaluate the functionality of cryo-related biomolecules, including cold-shock and antifreeze proteins.
摘要：
背景：由于各种生物分子，嗜冷物可以在低温条件下存活。这些分子与细胞相互作用，冰晶,和脂质双层来增强它们的功能。以前的研究通常通过解冻冷冻样品并在室温下进行生物测定来测量这些相互作用;然而，在低温条件下研究这些相互作用是至关重要的。这是因为这些生物分子可以在较低的温度下发挥作用。因此,必须建立一个在零度以下温度条件下测量化学相互作用的平台。
结果：在零度以下温度条件下，在具有通道状结构的冰粒边界内评估了生物分子之间的化学相互作用，这避免了解冻的需要。蔗糖水溶液在微流体通道内冷冻，促进冷冻浓缩溶液（FCS）的形成，该溶液充当大小可调的电泳场。将抗生物素蛋白或单链DNA(ssDNA)预先引入FCS中。将其表面用与靶分析物互补的分子修饰的探针微/纳米球引入FCS中。如果目标在零下温度条件下具有功能，它们与互补分子相互作用。靶分子和纳米球之间的化学相互作用导致颗粒的聚集。FCS通道直径的大小可调性使得能够识别聚合级别，这表明相互作用反应性。抗生物素蛋白-生物素相互作用和ssDNA杂交作为化学相互作用的模型，证明在零度以下温度条件下的交互性。本文提供的结果表明，在冷冻状态下原位测量生化测定的潜力，阐明生物相关大分子在0℃或略低于0℃时的功能。
结论：这是在零度以下温度条件下评估化学相互作用而不采用冻融过程的第一种方法。该方法具有仅在低温下显示化学相互作用的优点。因此,它可用于筛选和评估冷冻相关生物分子的功能，包括冷休克和防冻蛋白。
结果：在零度以下温度条件下，在具有通道状结构的冰粒边界内评估了生物分子之间的化学相互作用，这避免了解冻的需要。蔗糖水溶液在微流体通道内冷冻，促进冷冻浓缩溶液（FCS）的形成，该溶液充当大小可调的电泳场。将抗生物素蛋白或单链DNA(ssDNA)预先引入FCS中。将其表面用与靶分析物互补的分子修饰的探针微/纳米球引入FCS中。如果目标在零下温度条件下具有功能，它们与互补分子相互作用。靶分子和纳米球之间的化学相互作用导致颗粒的聚集。FCS通道直径的大小可调性使得能够识别聚合级别，这表明相互作用反应性。抗生物素蛋白-生物素相互作用和ssDNA杂交作为化学相互作用的模型，证明在零度以下温度条件下的交互性。本文提供的结果表明，在冷冻状态下原位测量生化测定的潜力，阐明生物相关大分子在0℃或略低于0℃时的功能。
结论：这是在零度以下温度条件下评估化学相互作用而不采用冻融过程的第一种方法。该方法具有仅在低温下显示化学相互作用的优点。因此,它可用于筛选和评估冷冻相关生物分子的功能，包括冷休克和防冻蛋白。
