Kimijima Junya, Inagawa Arinori, Miyagawa Akihisa, Nasuno Eri, Uehara Nobuo
School of Engineering, Utsunomiya University, 7-1-2, Yoto, Utsunomiya, Tochigi, 321-8585, Japan.
School of Engineering, Utsunomiya University, 7-1-2, Yoto, Utsunomiya, Tochigi, 321-8585, Japan.
Anal Chim Acta. 2024 Jul 4;1311:342713. doi: 10.1016/j.aca.2024.342713. Epub 2024 May 9.
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.
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.
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.
嗜冷菌能够在低温条件下存活，这得益于多种生物分子。这些分子与细胞、冰晶和脂质双层相互作用，以增强其功能。以往的研究通常通过解冻冷冻样本并在室温下进行生物学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至关重要。这是因为这些生物分子能在较低温度下发挥作用。因此，必须建立一个用于测量零下温度条件下化学相互作用的平台。
在具有通道状结构的冰粒边界内评估了零下温度条件下生物分子之间的化学相互作用，这避免了解冻的需要。蔗糖水溶液在微流控通道内冷冻，促进了作为尺寸可调电泳场的冷冻浓缩溶液（FCS）的形成。预先将抗生物素蛋白或单链DNA（ssDNA）引入FCS中。将表面用与目标分析物互补的分子修饰的探针微球或纳米球引入FCS中。如果目标在零下温度条件下具有功能，它们会与互补分子相互作用。目标分子与纳米球之间的化学相互作用导致颗粒聚集。FCS通道直径的尺寸可调性使得能够识别聚集水平，这表明了相互作用反应性。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，证明了在零下温度条件下的相互作用性。本文给出的结果表明了在冷冻状态下原位测量生化分析的潜力，阐明了在0℃或略低于0℃时生物相关大分子的功能。
这是第一种在不采用冻融过程的情况下评估零下温度条件下化学相互作用的方法。该方法的优点是仅在低温下揭示化学相互作用。因此，它可用于筛选和评估与低温相关的生物分子的功能，包括冷休克蛋白和抗冻蛋白。
