Probing the interaction between biomolecules under sub-zero temperature conditions by electrophoresis in ice grain boundaries.

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. SIGNIFICANCE: 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.

Incorporation of a Morphologically Controlled Ice Grain Boundary into a Microfluidic Device for Size-Selective Separation of Micro/Nanospheres.

A frozen aqueous solution was integrated into a microfluidic device as a size-tunable separation field for the size-selective separation of micro/nanospheres. The width of the ice grain boundaries formed in frozen aqueous solutions could be altered by controlling the operating temperature. A freezing chamber was placed adjacent to the microfluidic channel. A sample-dispersing aqueous sucrose solution was injected into the chamber and frozen, allowing the freeze-concentrated solution (FCS) to run vertically to the microfluidic channel, where the eluting solution flows. The operating temperature can be used to control the physical interaction between the ice wall and micro/nanospheres, enabling size-selective migration. The eluted micro/nanospheres in the microchannel were passed through the eluting solution collected from the outlet. We achieved size-selective separation and collection of microspheres and nanospheres. We separated the exosomes and yeast cells to demonstrate their applicability in bioseparation. The present method is suitable not only for size-selective separation but also for evaluating the biological expression of extracellular vesicles under cryogenic conditions.