Zath, G. K. ; Sperling, R. A. ; Hoffman, C. W. ; Bikos, D. A. ; Abbasi, R. ; Abate, A. R. ; Weitz, D. A. ; Chang, C. B. Rapid parallel generation of a fluorescently barcoded drop library from a microtiter plate using the plate-interfacing parallel encapsulation (PIPE) chip. Lab on a Chip 2022, 22, 4735-4745. Publisher's VersionAbstract

In drop-based microfluidics, an aqueous sample is partitioned into drops using individual pump sources that drive water and oil into a drop-making device. Parallelization of drop-making devices is necessary to achieve high-throughput screening of multiple experimental conditions, especially in time-sensitive studies. Here, we present the plate-interfacing parallel encapsulation (PIPE) chip, a microfluidic chip designed to generate 50 to 90 μm diameter drops of up to 96 different conditions in parallel by interfacing individual drop makers with a standard 384-well microtiter plate. The PIPE chip is used to generate two types of optically barcoded drop libraries consisting of two-color fluorescent particle combinations: a library of 24 microbead barcodes and a library of 192 quantum dot barcodes. Barcoded combinations in the drop libraries are rapidly measured within a microfluidic device using fluorescence detection and distinct barcoded populations in the fluorescence drop data are identified using DBSCAN data clustering. Signal analysis reveals that particle size defines the source of dominant noise present in the fluorescence intensity distributions of the barcoded drop populations, arising from Poisson loading for microbeads and shot noise for quantum dots. A barcoded population from a drop library is isolated using fluorescence-activated drop sorting, enabling downstream analysis of drop contents. The PIPE chip can improve multiplexed high-throughput assays by enabling simultaneous encapsulation of barcoded samples stored in a microtiter plate and reducing sample preparation time.

Shang, L. ; Cao, Y. ; Weitz, D. A. Multidisciplinary endeavors make future medicine smart. Smart Medicine 2022, 1 e20220031. Publisher's VersionAbstract

Humans suffer from thousands of diseases, and many of them lack effective methods for diagnosis and treatment. To address these unmet medical needs, basic research in multiple areas can provide important clinical contributions. The breathtaking discoveries in fundamental life sciences not only enhance our understanding of the human body but also lead to the creation of new diagnostics and pharmaceuticals. Innovations in medically relevant technologies, drawing on a wide range of disciplines, bring hope to previously intractable illnesses. All these lead to the dramatic transformation of medicine toward “Smart Medicine.” We foresee the future of medicine to be nourished with cutting-edge ideas from many different fields. We also anticipate specially designed methods that help translate these ideas and make them fit for specific healthcare purposes.

Xiao, Y. ; Yang, C. ; Zhai, X. ; Zhao, L. ; Zhao, P. ; Ruan, J. ; Chen, D. ; Weitz, D. A. ; Liu, K. Bioinspired Tough and Strong Fibers with Hierarchical Core-Shell Structure. Advanced Materials Interfaces 2022, 2201962. Publisher's VersionAbstract

Strong and tough bio-based fibers are attractive for both fundamental research and practical applications. In this work, strong and tough hierarchical core–shell fibers with cellulose nanofibrils (CNFs) in the core and regenerated silk fibroins (RSFs) in the shell are designed and prepared, mimicking natural spider silks. CNF/RSF core–shell fibers with precisely controlled morphology are continuously wet-spun using a co-axial microfluidic device. Highly-dense non-covalent interactions are introduced between negatively-charged CNFs in the core and positively-charged RSFs in the shell, diminishing the core/shell interface and forming an integral hierarchical fiber. Meanwhile, shearing by microfluidic channels and post-stretching induce a better ordering of CNFs in the core and RSFs in the shell, while ordered CNFs and RSFs are more densely packed, thus facilitating the formation of non-covalent interactions within the fiber matrix. Therefore, CNF/RSF core–shell fibers demonstrate excellent mechanical performances; especially after post-stretching, their tensile strength, tensile strain, Young's modulus, and toughness are up to 635 MPa, 22.4%, 24.0 GPa, and 110 MJ m−3, respectively. In addition, their mechanical properties are barely compromised even at −40 and 60 °C. Static load and dynamic impact tests suggest that CNF/RSF core–shell fibers are strong and tough, making them suitable for advanced structural materials.

Shiba, K. ; Zhuang, C. ; Minami, K. ; Imamura, G. ; Tamura, R. ; Samitsu, S. ; Idei, T. ; Yoshikawa, G. ; Sun, L. ; Weitz, D. A. Visualization of Flow-Induced Strain Using Structural Color in Channel-Free Polydimethylsiloxane Devices. Advanced Science 2022, 10, 2204310. Publisher's VersionAbstract

Measuring flow of gases is of fundamental importance yet is typically done with complex equipment. There is, therefore, a longstanding need for a simple and inexpensive means of flow measurement. Here, gas flow is measured using an extremely simple device that consists of an Ar plasma-treated polydimethylsiloxane (PDMS) slab adhered on a glass substrate with a tight seal. This device does not even have a channel, instead, gas can flow between the PDMS and the glass by deforming the PDMS wall, in other words, by making an interstice as a temporary path for the flow. The formation of the temporary path results in a compressive bending stress at the inner wall of the path, which leads to the formation of well-ordered wrinkles, and hence, the emergence of structural color that changes the optical transmittance of the device. Although it is very simple, this setup works sufficiently well to measure arbitrary gases and analyzes their flow rates, densities, and viscosities based on the change in color. It is also demonstrated that this technique is applicable to the flow-induced display of a pattern such as a logo for advanced applications.

Zhang, Y. ; Zhao, X. - Z. ; Han, P. - H. ; Zhang, L. - Y. ; Weitz, D. A. ; Feng, Y. - J. F. Visualization of adaptive polymer flow and displacement in medium-permeable 3D core-on-a-chip. Petroleum Science 2022. Publisher's VersionAbstract

Polymer flooding has been witnessed an effective technology for enhancing oil recovery from medium-to low-permeability reservoirs; however, direct visualization of polymer solution flow in such reservoir condition is still lacking. In this work, a three-dimensional (3D) core-on-a-chip device with a permeability of around 200 mD was prepared and employed to visualize the pore-scale flow and displacement of a self-adaptive polymer (SAP, 8.7 × 106 g∙mol−1)−whose microscopic association structure and macroscopic viscosity can reversibly change in response to shear action−versus partially hydrolyzed polyacrylamide (HPAM), by recording their flow curves, monitoring dynamic transportation process via particle imaging velocimetry, and building 3D structure of remaining oil. The results show that, in single-phase flow, all polymer solutions exhibit flow thinning and then thickening regions as flow rate increases, but the transition between two regimes occurs at a small Weissenberg number (10−3−10−1) in this medium-permeable condition. In contrast to HPAM-1 with close weight-average molecular weight (Mw), the adaptive character not only extends SAP's shear-govern region, allowing SAP to propagate piece by piece and achieve higher accessible pore volume, but it also enhances the elastic resistibility of polymer in the extension-dominated regime, increasing the microscopic displacement efficiency. These two effects result in 1.5–3 times more oil recovery factor for SAP than for HPAM-1. Regarding ultra-high-Mw HPAM-2 (25 × 106 g∙mol−1), plugging and chain degradation do occur, thus producing lower oil recovery than SAP. This work provides a direct approach for in-situassessment of polymer-based displacing system under a more authentic condition of practical reservoirs.

Wang, X. ; Blumenfeld, R. ; Feng, X. - Q. ; Weitz, D. A. ‘Phase transitions’ in bacteria – From structural transitions in free living bacteria to phenotypic transitions in bacteria within biofilms. Physics of Life Reviews 2022, 43, 98-138. Publisher's VersionAbstract

Phase transitions are common in inanimate systems and have been studied extensively in natural sciences. Less explored are the rich transitions that take place at the micro- and nano-scales in biological systems. In conventional phase transitions, large-scale properties of the media change discontinuously in response to continuous changes in external conditions. Such changes play a significant role in the dynamic behaviours of organisms. In this review, we focus on some transitions in both free-living and biofilms of bacteria. Particular attention is paid to the transitions in the flagellar motors and filaments of free-living bacteria, in cellular gene expression during the biofilm growth, in the biofilm morphology transitions during biofilm expansion, and in the cell motion pattern transitions during the biofilm formation. We analyse the dynamic characteristics and biophysical mechanisms of these phase transition phenomena and point out the parallels between these transitions and conventional phase transitions. We also discuss the applications of some theoretical and numerical methods, established for conventional phase transitions in inanimate systems, in bacterial biofilms.

Wu, Q. ; Pan, M. ; Zhang, S. ; Sun, D. ; Yang, Y. ; Chen, D. ; Weitz, D. A. ; Gao, X. Research Progress in High-Throughput Screening of CO2 Reduction Catalysts. Energies 2022, 15, 6666. Publisher's VersionAbstract

The conversion and utilization of carbon dioxide (CO2) have dual significance for reducing carbon emissions and solving energy demand. Catalytic reduction of CO2 is a promising way to convert and utilize CO2. However, high-performance catalysts with excellent catalytic activity, selectivity and stability are currently lacking. High-throughput methods offer an effective way to screen high-performance CO2 reduction catalysts. Here, recent advances in high-throughput screening of electrocatalysts for CO2reduction are reviewed. First, the mechanism of CO2 reduction reaction by electrocatalysis and potential catalyst candidates are introduced. Second, high-throughput computational methods developed to accelerate catalyst screening are presented, such as density functional theory and machine learning. Then, high-throughput experimental methods are outlined, including experimental design, high-throughput synthesis, in situ characterization and high-throughput testing. Finally, future directions of high-throughput screening of CO2 reduction electrocatalysts are outlooked. This review will be a valuable reference for future research on high-throughput screening of CO2 electrocatalysts.

Weitz, D. A. Soft materials evolution and revolution. Nature Materials 2022, 21, 986-988. Publisher's VersionAbstract

Soft matter has evolved considerably since it became recognized as a unified field. This has been driven by new experimental, numerical and theoretical methods to probe soft matter, and by new ways of formulating soft materials. These advances have driven a revolution in knowledge and expansion into biological and active matter.

Shen, Y. ; Weitz, D. A. ; Forde, N. R. ; Shayegan, M. Line optical tweezers as controllable micromachines: techniques and emerging trends. Soft Matter 2022, 18, 5359-5365. Publisher's VersionAbstract

In the past three decades, the technology of optical tweezers has made significant contributions in various scientific areas, including optics, photonics, and nanosciences. Breakthroughs include manipulating particles in both static and dynamic ways, particle sorting, and constructing controllable micromachines. Advances in shaping and controlling the laser beam profile enable control over the position and location of the trap, which has many possible applications. A line optical tweezer (LOT) can be created by rapidly moving a spot optical tweezer using a tool such as a galvanometer mirror or an acousto-optic modulator. By manipulating the intensity profile along the beam line to be asymmetric or non-uniform, the technique can be adapted to various specific applications. Among the many exciting applications of line optical tweezers, in this work, we discuss in detail applications of LOT, including probing colloidal interactions, transporting and sorting of colloidal microspheres, self-propelled motions, trapping anisotropic particles, exploring colloidal interactions at fluid-fluid interfaces, and building optical thermal ratchets. We further discuss prospective applications in each of these areas of soft matter, including polymeric and biological soft materials.

Zheng, W. ; Zhao, S. ; Yin, Y. ; Zhang, H. ; Needham, D. M. ; Evans, E. D. ; Dai, C. L. ; Lu, P. J. ; Alm, E. J. ; Weitz, D. A. High-throughput, single-microbe genomics with strain resolution, applied to a human gut microbiome. Science 2022, 376, eabm1483. Publisher's VersionAbstract

Single-cell methods are the state of the art in biological research. Zheng et al. developed a high-throughput technique called Microbe-seq designed to analyze single bacterial cells from a microbiota. Microbe-seq uses microfluidics to separate individual bacterial cells within droplets and then extract, amplify, and barcode their DNA, which is then subject to pooled Illumina sequencing. The technique was tested by sequencing multiple human fecal samples to generate barcoded reads for thousands of single amplified genomes (SAGs) per sample. Pooling the SAGs corresponding to the same bacterial species allowed consensus assemblies of these genomes to provide insights into strain-level diversity and revealed a phage association and the limits on horizontal gene-transfer events between strains.

Koveal, D. ; Rosen, P. C. ; Meyer, D. J. ; Diaz-Garcia, C. M. ; Wang, Y. ; Cai, L. - H. ; Chou, P. J. ; Weitz, D. A. ; Yellen, G. A high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors. Nature Communications 2022, 13, 2919. Publisher's VersionAbstract

Genetically encoded fluorescent biosensors are powerful tools used to track chemical processes in intact biological systems. However, the development and optimization of biosensors remains a challenging and labor-intensive process, primarily due to technical limitations of methods for screening candidate biosensors. Here we describe a screening modality that combines droplet microfluidics and automated fluorescence imaging to provide an order of magnitude increase in screening throughput. Moreover, unlike current techniques that are limited to screening for a single biosensor feature at a time (e.g. brightness), our method enables evaluation of multiple features (e.g. contrast, affinity, specificity) in parallel. Because biosensor features can covary, this capability is essential for rapid optimization. We use this system to generate a high-performance biosensor for lactate that can be used to quantify intracellular lactate concentrations. This biosensor, named LiLac, constitutes a significant advance in metabolite sensing and demonstrates the power of our screening approach.

Zhang, H. ; Zhang, L. ; An, C. ; Zhang, Y. ; Zhao, F. ; Gao, Y. ; Zhang, Y. ; Li, H. ; Zhang, Y. ; Ren, C. ; et al. Large-scale single-cell encapsulation in microgels through metastable droplet-templating combined with microfluidic-integration. Biofabrication 2022, 14, 035015. Publisher's VersionAbstract

Current techniques for the generation of cell-laden microgels are limited by numerous challenges, including poorly uncontrolled batch-to-batch variations, processes that are both labor- and time-consuming, the high expense of devices and reagents, and low production rates; this hampers the translation of laboratory findings to clinical applications. To address these challenges, we develop a droplet-based microfluidic strategy based on metastable droplet-templating and microchannel integration for the substantial large-scale production of single cell-laden alginate microgels. Specifically, we present a continuous processing method for microgel generation by introducing amphiphilic perfluoronated alcohols to obtain metastable emulsion droplets as sacrificial templates. In addition, to adapt to the metastable emulsion system, integrated microfluidic chips containing 80 drop-maker units are designed and optimized based on the computational fluid dynamics simulation. This strategy allows single cell encapsulation in microgels at a maximum production rate of 10 ml h−1 of cell suspension while retaining cell viability and functionality. These results represent a significant advance toward using cell-laden microgels for clinical-relevant applications, including cell therapy, tissue regeneration and 3D bioprinting.

Saad, A. M. ; Aime, S. ; Mahavadi, S. C. ; Song, Y. - Q. ; Yutkin, M. P. ; Weitz, D. A. ; Patzek, T. W. Adsorption of Polar Species at Crude Oil-Water Interfaces: the Chemoelastic Behavior. Langmuir 2022, 38, 6523–6530. Publisher's VersionAbstract

We investigate the formation and properties of crude oil/water interfacial films. The time evolution of interfacial tension suggests the presence of short and long timescale processes reflecting the competition between different populations of surface-active molecules. We measure both the time-dependent shear and extensional interfacial rheology moduli. Late-time interface rheology is dominated by elasticity, which results in visible wrinkles on the crude oil drop surface upon interface disturbance. We also find that the chemical composition of the interfacial films is affected by the composition of the aqueous phase that it has contacted. For example, sulfate ions promote films enriched with carboxylic groups and condensed aromatics. Finally, we perform solution exchange experiments and monitor the late-time film composition upon the exchange. We detect the film composition change upon replacing chloride solutions with sulfate-enriched ones. To the best of our knowledge, we are the first to report the composition alteration of aged crude oil films. This finding might foreshadow an essential crude oil recovery mechanism.

King, E. M. ; Wang, Z. ; Weitz, D. A. ; Spaepen, F. ; Brenner, M. P. Correlation Tracking: Using simulations to interpolate highly correlated particle tracks. Physical Review E 2022, 105, 044608. Publisher's VersionAbstract

Despite significant advances in particle imaging technologies over the past two decades, few advances have been made in particle tracking, i.e., linking individual particle positions across time series data. The state-of-the-art tracking algorithm is highly effective for systems in which the particles behave mostly independently. However, these algorithms become inaccurate when particle motion is highly correlated, such as in dense or strongly interacting systems. Accurate particle tracking is essential in the study of the physics of dense colloids, such as the study of dislocation formation, nucleation, and shear transformations. Here, we present a method for particle tracking that incorporates information about the correlated motion of the particles. We demonstrate significant improvement over the state-of-the-art tracking algorithm in simulated data on highly correlated systems.

Elkeles, T. ; Park, S. ; Werner, J. G. ; Weitz, D. A. ; Yossifon, G. Dielectrophoretic Characterization of Dynamic Microcapsules and Their Magnetophoretic Manipulation. ACS Appl. Mater. Interfaces 2022, 14, 15765–15773. Publisher's VersionAbstract

In this work, we present dielectrophoresis (DEP) and in situ electrorotation (ROT) characterization of reversibly stimuli-responsive “dynamic” microcapsules that change the physicochemical properties of their shells under varying pH conditions and can encapsulate and release (macro)molecular cargo on demand. Specifically, these capsules are engineered to open (close) their shell under high (low) pH conditions and thus to release (retain) their encapsulated load or to capture and trap (macro)molecular samples from their environment. We show that the steady-state DEP and ROT spectra of these capsules can be modeled using a single-shell model and that the conductivity of their shells is influenced most by the pH. Furthermore, we measured the transient response of the angular velocity of the capsules under rotating electric field conditions, which allows us to directly determine the characteristic time scales of the underlying physical processes. In addition, we demonstrate the magnetic manipulation of microcapsules with embedded magnetic nanoparticles for lab-on-chip tasks such as encapsulation and release at designated locations and the in situ determination of their physicochemical state using on-chip ROT. The insight gained will enable the advanced design and operation of these dynamic drug delivery and smart lab-on-chip transport systems.

Xia, J. ; Liu, Z. - Y. ; Han, Z. - Y. ; Yuan, Y. ; Shao, Y. ; Feng, X. - Q. ; Weitz, D. A. Regulation of cell attachment, spreading, and migration by hydrogel substrates with independently tunable mesh size. Acta Biomaterialia 2022, 141, 178-189. Publisher's VersionAbstract

Hydrogels are widely used as substrates to investigate interactions between cells and their microenvironment as they mimic many attributes of the extracellular matrix. The stiffness of hydrogels is an important property that is known to regulate cell behavior. Beside stiffness, cells also respond to structural cues such as mesh size. However, since the mesh size of hydrogel is intrinsically coupled to its stiffness, its role in regulating cell behavior has never been independently investigated. Here, we report a hydrogel system whose mesh size and stiffness can be independently controlled. Cell behavior, including spreading, migration, and formation of focal adhesions is significantly altered on hydrogels with different mesh sizes but with the same stiffness. At the transcriptional level, hydrogel mesh size affects cellular mechanotransduction by regulating nuclear translocation of yes-associated protein. These findings demonstrate that the mesh size of a hydrogel plays an important role in cell-substrate interactions.

Wu, H. ; Shen, Y. ; Sivagurunathan, S. ; Weber, M. S. ; Adam, S. A. ; Shin, J. H. ; Fredberg, J. J. ; Medalia, O. ; Goldman, R. ; Weitz, D. A. Vimentin intermediate filaments and filamentous actin form unexpected interpenetrating networks that redefine the cell cortex. PNAS 2022, 119, e2115217119. Publisher's VersionAbstract
The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryo-electron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin.
Battat, S. ; Weitz, D. A. ; Whitesides, G. Nonlinear Phenomena in Microfluidics. Chemical Reviews 2022, 122, 6921-6937. Publisher's VersionAbstract

This review focuses on experimental work on nonlinear phenomena in microfluidics, which for the most part are phenomena for which the velocity of a fluid flowing through a microfluidic channel does not scale proportionately with the pressure drop. Examples include oscillations, flow-switching behaviors, and bifurcations. These phenomena are qualitatively distinct from laminar, diffusion-limited flows that are often associated with microfluidics. We explore the nonlinear behaviors of bubbles or droplets when they travel alone or in trains through a microfluidic network or when they assemble into either one- or two-dimensional crystals. We consider the nonlinearities that can be induced by the geometry of channels, such as their curvature or the bas-relief patterning of their base. By casting posts, barriers, or membranes─situated inside channels─from stimuli-responsive or flexible materials, the shape, size, or configuration of these elements can be altered by flowing fluids, which may enable autonomous flow control. We also highlight some of the nonlinearities that arise from operating devices at intermediate Reynolds numbers or from using non-Newtonian fluids or liquid metals. We include a brief discussion of relevant practical applications, including flow gating, mixing, and particle separations.

Wu, Y. ; Pegoraro, A. F. ; Weitz, D. A. ; Janmey, P. ; Sun, S. X. The correlation between cell and nucleus size is explained by an eukaryotic cell growth model. PLOS Computational Biology 2022, 18, e1009400. Publisher's VersionAbstract

In eukaryotes, the cell volume is observed to be strongly correlated with the nuclear volume. The slope of this correlation depends on the cell type, growth condition, and the physical environment of the cell. We develop a computational model of cell growth and proteome increase, incorporating the kinetics of amino acid import, protein/ribosome synthesis and degradation, and active transport of proteins between the cytoplasm and the nucleoplasm. We also include a simple model of ribosome biogenesis and assembly. Results show that the cell volume is tightly correlated with the nuclear volume, and the cytoplasm-nucleoplasm transport rates strongly influence the cell growth rate as well as the cell/nucleus volume ratio (C/N ratio). Ribosome assembly and the ratio of ribosomal proteins to mature ribosomes also influence the cell volume and the cell growth rate. We find that in order to regulate the cell growth rate and the cell/nucleus volume ratio, the cell must optimally control groups of kinetic and transport parameters together, which could explain the quantitative roles of canonical growth pathways. Finally, although not explicitly demonstrated in this work, we point out that it is possible to construct a detailed proteome distribution using our model and RNAseq data, provided that a quantitative cell division mechanism is known.

Chu, J. - O. ; Choi, Y. ; Kim, D. - W. ; Jeong, H. - S. ; Park, J. P. ; Weitz, D. A. ; Kee, S. - J. ; Lee, H. ; Choi, C. - H. Cell-Inspired Hydrogel Microcapsules with a Thin Oil Layer of Enhanced Retention of Highly Reactive Antioxidants. ACS Appl. Mater. Interfaces 2022, 14, 2597–2604. Publisher's VersionAbstract

In nature, individual cells are compartmentalized by a membrane that protects the cellular elements from the surrounding environment while simultaneously equipped with an antioxidant defense system to alleviate the oxidative stress resulting from light, oxygen, moisture, and temperature. However, this mechanism has not been realized in cellular mimics to effectively encapsulate and retain highly reactive antioxidants. Here, we report cell-inspired hydrogel microcapsules with an interstitial oil layer prepared by utilizing triple emulsion drops as templates to achieve enhanced retention of antioxidants. We employ ionic gelation for the hydrogel shell to prevent exposure of the encapsulated antioxidants to free radicals typically generated during photopolymerization. The interstitial oil layer in the microcapsule serves as an stimulus-responsive diffusion barrier, enabling efficient encapsulation and retention of antioxidants by providing an adequate pH microenvironment until osmotic pressure is applied to release the cargo on-demand. Moreover, addition of a lipophilic reducing agent in the oil layer induces a complementary reaction with the antioxidant, similar to the nonenzymatic antioxidant defense system in cells, leading to enhanced retention of the antioxidant activity. Furthermore, we show the complete recovery and even further enhancement in antioxidant activity by lowering the storage temperature, which decreases the oxidation rate while retaining the complementary reaction with the lipophilic reducing agent.