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In this paper, using hawthorn leaves as raw materials, the effect of extracting total flavonoids from hawthorn leaves by aqueous two-phase system (ATPS) combined with ultrasonic assisted was studied. Through single factor test and response surface method Box-Behnken design (response surface method, BBD), according to the regression equation, the optimal process conditions were obtained as follows: the ratio of solid to liquid 1:37, ultrasonic time 40 min, ultrasonic power 360 w, ultrasonic temperature 65°C, and the yield of flavonoids was 2.869±0.0004%. This study shows that ultrasound combined with aqueous two-phase system (ATPS) is an effective method to extract total flavonoids from hawthorn leaves.

The efficient and stable capture of cells within microfluidic platforms is essential for cellular biology analyses, offering insights into the heterogeneity of cell properties and cellular processes, for example, among cancer cells. However, conventional microfluidic cell confinement modalities, such as water‐in‐oil emulsions and microstructure trapping, face inherent limitations in biological applicability and precise control. Here an approach is introduced to confine cells in dead‐end microstructures leveraging a dextran concentration gradient. This method allows for the fine‐tuned capture of cells, reaching the precision of single‐cell culture, as demonstrated for yeast and leukemia cells. By incorporating polyethylene glycol (PEG) solutions, phase separation is induced within the microfluidic environment, encapsulating single cells within dextran droplets. The technique is distinguished by its stability, control, and adaptability, paving a new way for innovations not only in cellular biology, but broadly in chemical and biological applications, including the synthesis of bio‐oriented particles, microcarrier production, and advancements in tissue engineering. This work introduces a method to trap cells in dead‐end microstructures using dextran diffusiophoresis. Captured cells can be further encapsulated in dextran droplets via polyethylene glycol (PEG)‐dextran aqueous two‐phase separation. The stable, controllable technique offers new opportunities in cellular biology and bio‐oriented applications.

Membraneless organelles (MLOs) are fundamental to cellular organization, enabling biochemical processes by concentrating biomolecules and regulating reactions within confined environments. While micrometer‐scale synthetic droplets are extensively studied as models of MLOs, submicron droplets remain largely unexplored despite their potential to uniquely regulate biomolecular processes. Here, submicron droplets are generated by a polyethylene glycol (PEG)/dextran aqueous two‐phase system (ATPS) as a model to investigate their effect on DNA assembly in crowded environments. The findings reveal that submicron droplets exhibit distinct advantages over microdroplets by acting as submicron catalytic centers that concentrate DNA and accelerate assembly kinetics. This enhancement is driven by a cooperative mechanism wherein global crowding from PEG induces an excluded volume effect, while local crowding from dextran provides weak but nonspecific interactions with DNA. By exploiting both the confinement and phase properties of submicron droplets, a rapid and sensitive assay is developed for miRNA detection using confined fluorescent readouts. These findings highlight the unique ability of submicron droplets to amplify biomolecular assembly processes, provide new insights into the interplay between global and local crowding effects in cellular‐like environments, and present a platform for biomarker detection and visualization. Submicron droplets, formed by a polyethyleneglycol (PEG)/dextran aqueous two‐phase system·(ATPS), can act as catalytic centers that concentrate DNA and accelerate assembly kinetic in crowded environments. It highlights a cooperative mechanism wherein global crowding from PEG induces an excluded volume effect, while local crowding from dextran provides weak but nonspecific interactions with DNA. Additionally, the study develops a rapid and sensitive assay for miRNA detection using confined fluorescent readouts.

A carbohydrate‐based aqueous two‐phase system (ATPS) containing sorbitol/fructose and acetonitrile was utilized to separate constituent amino acids of glatiramer acetate (GA), including alanine, glutamic acid, lysine, and tyrosine. Purification of GA from its amino acid contaminants can be seen as a pretreatment step prior to chromatography‐based techniques. The study evaluated how the type and concentration of carbohydrates affect the partitioning of amino acids and glatiramer acetate. In all cases, these compounds were found to partition to the carbohydrate‐rich phase. The influence of pH on partitioning was examined, showing that a sorbitol‐based system at pH 6 resulted in better GA partitioning among other pHs and fructose‐based systems. Additionally, quantitative structure–activity relationship analysis was used to predict the partitioning behavior of components in ATPSs, with results indicating that components in sorbitol‐based systems can be accurately predicted. For each ATPS, the optimum feed composition, based on selectivity, was found to be (fructose 15 wt% + acetonitrile 40 wt%) and (sorbitol 17 wt% + acetonitrile 35 wt%), respectively. The optimal partition coefficients for GA were determined as 0.213 for the system of (fructose 15 wt% + acetonitrile 40 wt%) and 0.119 for the mixture of (sorbitol 19 wt% + acetonitrile 35 wt%). The partitioning behavior of GA demonstrated distinct characteristics compared to some of its constituent amino acids, indicating promising potential for the application of ATPS in separation processes. The partitioning coefficient and recovery of glatiramer acetate and its constituent amino acids were evaluated using aqueous two‐phase systems based on sorbitol and fructose. Additionally, the selectivity of glatiramer acetate partitioning relative to each amino acid was examined. The influence of the amino acids' physicochemical and structural properties on their partitioning behavior was analyzed, revealing consistent partitioning of amino acids into the carbohydrate‐rich phase. The proposed system is effective for pre‐treatment or downstream processing applications.

The interaction of perfluorinated molecules, also known as “forever chemicals” due to their pervasiveness, with their environment remains an important yet poorly understood topic. In this work, the self‐assembly of perfluorinated molecules with multivalent hosts, pillar‐[5]‐arenes, is investigated. It is found that perfluoroalkyl diacids and pillar‐[5]‐arenes rapidly and strongly complex with each other at aqueous interfaces, forming solid interfacially templated films. Their complexation is shown to be driven primarily by fluorophilic aggregation and assisted by electrostatic interactions, as supported by the crystal structure of the complexes, and leads to the formation of quasi‐2D phase‐separated films. This self‐assembly process can be further manipulated using aqueous two‐phase system microdroplets, enabling the controlled formation of 3D micro‐scaffolds. A class of cyclic compounds, pillar‐[5]‐arenes, can rapidly form unusual complexes with perfluorinated molecules, and this formation is facilitated by size matching between the two. Through microfluidics and aqueous two‐phase systems, these can be shaped into shape‐retaining vesicles at water–water interfaces.

Design of reversible organelle‐like microcompartments formed by liquid–liquid phase separation in cell‐mimicking entities has significantly advanced the bottom‐up construction of artificial eukaryotic cells. However, organizing the formation of artificial organelle architectures in a spatiotemporal manner within complex primitive compartments remains scarcely explored. In this work, thermoresponsive hybrid polypeptide‐polymer conjugates are rationally engineered and synthesized, resulting from the conjugation of an intrinsically disordered synthetic protein (IDP), namely elastin‐like polypeptide, and synthetic polymers (poly(ethylene glycol) and dextran) that are widely used as macromolecular crowding agents. Cell‐like constructs are built using droplet‐based microfluidics that are filled with such bioconjugates and an artificial cytoplasm system that is composed of specific polymers conjugated to the IDP. The distinct spatial organizations of two polypeptide‐polymer conjugates and the dynamic assembly and disassembly of polypeptide‐polymer coacervate droplets in response to temperature are studied in the cytomimetic protocells. Furthermore, a monoblock IDP with longer length is concurrently included with bioconjugates individually inside cytomimetic compartments. Both bioconjugates exhibit an identical surfactant‐like property, compartmentalizing the monoblock IDP coacervates via temperature control. These findings lay the foundation for developing hierarchically structured synthetic cells with interior organelle‐like structures which could be designed to localize in desired phase‐separated subcompartments. Two elastin‐like polypeptide‐block‐polymer conjugates as building blocks for artificial organelles are individually encapsulated inside cytomimetic cell‐like microdroplets. Regulation of temperature enables dynamic assembly/disassembly and programmable position of organelle‐mimics in crowded medium, as well as rapid compartmentalization of hydrophobic coacervates from a monoblock ELP.

The isolation of extracellular vesicles (EVs) using currently available methods frequently compromises purity and yield to prioritize speed. Here, we present a next‐generation aqueous two‐phase system (next‐gen ATPS) for the isolation of EVs regardless of scale and volume that is superior to conventional methods such as ultracentrifugation (UC) and commercial kits. This is made possible by the two aqueous phases, one rich in polyethylene glycol (PEG) and the other rich in dextran (DEX), whereby fully encapsulated lipid vesicles preferentially migrate to the DEX‐rich phase to achieve a local energy minimum for the EVs. Isolated EVs as found in the DEX‐rich phase are more amenable to biomarker analysis such as nanoscale flow cytometry (nFC) when using various pre‐conjugated antibodies specific for CD9, CD63 and CD81. TRIzol RNA isolation is further enabled by the addition of dextranase, a critical component of this next‐gen ATPS method. RNA yield of next‐gen ATPS‐isolated EVs is superior to UC and other commercial kits. This negates the use of specialized EV RNA extraction kits. The use of dextranase also enables more accurate immunoreactivity of pre‐conjugated antibodies for the detection of EVs by nFC. Transcriptomic analysis of EVs isolated using the next‐gen ATPS revealed a strong overlap in microRNA (miRNA), circular RNA (circRNA) and small nucleolar RNA (snoRNA) profiles with EV donor cells, as well as EVs isolated by UC and the exoRNeasy kit, while detecting a superior number of circRNAs compared to the kit in human samples. Overall, this next‐gen ATPS method stands out as a rapid and highly effective approach to isolate high‐quality EVs in high yield, ensuring optimal extraction and analysis of EV‐encapsulated nucleic acids.

It remains a challenge to produce soft robots that can mimic the responsive adaptability of living organisms. Rather than fabricating soft robots from bulk hydrogels,hydrogels are integrated into the interfacial assembly of aqueous two‐phase systems to generate ultra‐soft and elastic all‐aqueous aquabots that exhibit responsive adaptability, that can shrink on demand and have electrically conductive functions. The adaptive functions of the aquabots provide a new platform to develop minimally invasive surgical devices, targeted drug delivery systems, and flexible electronic sensors and actuators. All‐water‐based aquabots functionalized with responsive hydrogel membranes are ultrasoft and elastic. They can adaptively shrink their size to navigate through much narrower spaces. Also, their electrically conductive membranes enable the fabrication of flexible electronic sensors and actuators.

Enriching trace biomarkers (e.g., proteins, nucleic acids) is critical for biomedical applications; yet conventional methods often lack versatility, limiting their effectiveness to specific biomarker types. To address this, the phase separation‐assisted pre‐enrichment (PSAP) technology is presented that exploits differential polymer‐polymer partitioning to achieve 47‐fold antigen and 44‐fold RNA enrichment simultaneously. Through systematic optimization of interfacial chemistry, including pH modulation, polymer hydrophilicity, mass fraction, and molecular weights, the protocol is refined to enable direct integration with commercial diagnostics. PSAP‐boosted rapid antigen ests (RATs) detected SARS‐CoV‐2 and Influenza viruses at tenfold and fivefold lower limits, respectively. In clinical validation, 53 clinical specimens (containing PCR undetectable samples as controls) are analyzed. The PSAP method significantly enhanced detection accuracy for both viral antigens and RNA, particularly improving positivity rates in low viral load specimens (27 < Ct < 31) compared to conventional approaches while maintaining specificity in high‐Ct and negative controls. With its universality and tunability, PSAP demonstrates universal applicability across respiratory pathogens and lays the foundation for next‐generation point‐of‐care diagnostics. Conventional biomarker enrichment lacks versatility and specificity. Phase separation‐assisted pre‐enrichment (PSAP) is introduced for concurrent antigen (47×) and RNA (44×) enrichment. Tenfold and fivefold lower detection limits for SARS‐CoV‐2 and Influenza, respectively, were achieved. Enhanced sensitivity (Ct: 27–31) without cross‐reactivity was demonstrated in 53 clinical specimens. PSAP's universality positions it as transformative for point‐of‐care diagnostics.

Ionic liquid‐based ultrasonic‐assisted extraction (IL‐UAE) was developed to extract and separate the isochlorogenic acid C (ICGA) from a cultivar of Chrysanthemum morifolium (Chrysanthemummorifolium Ra Tnat.). The influencing parameters, including IL concentration, liquid‐to‐solid ratio, and ultrasonic time, were optimized using response surface methodology. Of the ILs studied, 1‐butyl‐3‐methylimidazolium bromide [(Bmim)Br] exhibited the best extraction ability. The optimized conditions included liquid‐to‐solid ratio of 23.44:1, ultrasonic time of 48.99 min, and IL concentration of 0.65 mol/L. Under the optimal conditions, the extraction yield of ICGA could reach to 4.20 mg/g. An aqueous two‐phase system was applied for purification and separation of ICGA. The maximum extraction efficiency of 98.18% was obtained under the conditions of (NH4)2SO4 of 4.5 g, pH of 3.0, and a temperature of 20°C at aqueous solution. Furthermore, the thermodynamic parameters showed that the purification of ICGA from salt‐rich phase to IL‐rich phase was a spontaneous and exothermic process. The results indicated that the proposed system is simple, rapid, and effective to serve as a viable and sustainable platform for the extraction and purification of ICGA from Chrysanthemum morifolium flowers. Chemical structures of (Bmim)Br 1‐Butyl‐3‐methylimidazolium bromide (A) and IGCA (B).