One-incubation one-hour multiplex ELISA enabled by aqueous two-phase systems.

This work describes a convenient one-hour enzyme-linked immunosorbent assay (ELISA) formulated with conventional antibodies and horseradish peroxidase (HRP) reagents. The method utilizes aqueous two-phase system (ATPS) droplet formation based on poly(ethylene glycol) (PEG)-containing sample solution-triggered rehydration of dehydrated dextran (DEX) spots that contain all antibody reagents. Key advances in this paper include development of a formulation that allows a quick 1-hour overall incubation time and a procedure where inclusion of the HRP reagent in the PEG solution reduces the number of washing and incubation steps required to perform this assay. As an assay application, a 5-plex cytokine test compares cytokine secretion of differentially-treated human ThP-1 macrophages. Given the use of only readily available reagents and a common Western blot imaging system for the readout, this method is envisioned to be broadly applicable to a variety of multiplex immunoassays. To facilitate broader use, companion image processing software as an ImageJ plugin is also described and provided.

Determination of Aqueous Two-Phase System Binodals and Tie-Lines by Electrowetting-on-Dielectric Droplet Manipulation.

Handling the aqueous two-phase systems (ATPSs) formed by liquid-liquid phase separation (LLPS) relies on the accurate construction of binodal curves and tie-lines, which delineate the polymer concentrations required for phase separation and depict the properties of the resulting phases, respectively. Various techniques to determine the binodal curves and tie-lines of ATPSs exist, but most rely on manually pipetting relatively large volumes of fluids in a slow and tedious manner. We describe a method to determine ATPS binodals and tie-lines that overcomes these disadvantages: microscale droplet manipulation by electrowetting-on-dielectric (EWOD). EWOD enables automated handling of droplets in an optically transparent platform that allows for in situ droplet observation. Separated phases are clearly visible, and the volumes of each phase are readily determined. Additionally, in considering the molecular crowding present in living cells, this work examines the role of a macromolecule in prompting LLPS. These results show that EWOD-driven droplet manipulation effectively interrogates the phase dynamics of ATPSs and macromolecular crowding in LLPS.

Combined Reflectometric Interference Spectroscopy and Quartz Crystal Microbalance Detect Differential Adsorption of Lipid Vesicles with Different Phase Transition Temperatures on SiO2, TiO2, and Au Surfaces.

Quantitative analysis of biomolecular adsorption on a substrate is crucial for understanding biomolecular interactions. A quartz crystal microbalance (QCM) is a highly sensitive device to detect such interactions based on mass. However, the physicochemical analysis by the QCM alone often leads to overestimation of the actual adsorbed mass. Here, a combined reflectometric interference spectroscopy (RIfS) and QCM is developed to simultaneously analyze adsorption of biomolecules. RIfS detects the adsorbed mass based on the reflectance and predicts the adsorbed condition by modeling the reflection spectra using the transfer matrix method. In contrast, the QCM detects physicochemical characteristics of the adsorbed molecules along with the adsorbed mass. The combined RIfS-QCM successfully detected the adsorption of proteins with different surface properties and lipid vesicles with different phase transition temperatures. The initial stage of adsorption revealed distinct individual properties of the adsorbates. Moreover, the RIfS-QCM revealed differential adsorption of the vesicles on silicon dioxide, titania, and gold surfaces, and the differences in adsorption were further interrogated by atomic force microscopy. The results demonstrate that the RIfS-QCM serves as a useful tool to quantitatively analyze molecular adsorption on various surfaces.

Nanoparticle Assemblies into Luminescent Dendrites in Shrinking Microdroplets.

The self-assembly of nanoparticles (NPs) is essential for emerging dispersion-based energy-conscious technologies. Of particular interest are micro- and macro-scale self-organizing superstructures that can bridge 2D/3D processing scales. Here we report the spontaneous assembly of CdTe NPs within an aqueous microdroplet suspended in soybean oil. The gradual diffusion of the water into the surrounding medium results in shrinking of the microdroplet, and a concomitant formation of branched assemblies from CdTe NPs that evolve in size from ∼50 µm to ∼1000 µm. The fractal dimension of NP assemblies increases from ∼1.7 to ∼1.9 during the assembly process. We found that constituents of the soybean oil enter the aqueous solution across the microdroplet interface and affect NP assembly. The obtained NP dendrites can be further altered morphologically by illumination with light that results in the disassembly of the NP dendrites. The use of this microheterogeneous dispersion platform with partially soluble hydrophilic and hydrophobic solvents highlights the sensitivity of the NP assembly process to environment and presents an opportunity to explore droplet-confined NP assembly.

Microscale determination of aqueous two phase system binodals by droplet dehydration in oil.

This paper analyzes the use of a dehydrating oil system to determine binodal curves of an aqueous two phase system (ATPS). Aqueous droplets containing phase-forming polymers are dehydrated at the interface between two immiscible oils. The droplets shrink due to diffusion of water into the oil phase while constantly maintaining a spherical shape. Upon sufficient dehydration, dilute one-phase solutions of phase-forming polymers separate into two phases. Comparison of the droplet diameter at this phase separation point and at the beginning allows facile calculation of the concentration of polymers that determine the binodal curve. The miniaturized droplet dehydration-based binodals obtained in this manner matched the binodals determined by the conventional diluting method but using several orders of magnitude less sample volume (150 nL droplets versus 10 mL vials).

Patchy surfaces stabilize dextran-polyethylene glycol aqueous two-phase system liquid patterns.

This paper analyzes surface chemistry effects to stably pattern aqueous two-phase system (ATPS) droplets on chemically modified poly(dimethylsiloxane) (PDMS). Polyethylene glycol (PEG) and dextran (DEX) are used as phase-forming polymers for the ATPS. PDMS surface modifications studied include primary amine groups, carboxylic acid groups, and neutral polymer surfaces. The PDMS surfaces were characterized by fluorescent measurement, water and DEX contact angle measurements, and X-ray photoelectron spectroscopy (XPS) analysis to confirm surface properties. While homogeneous surfaces of different functional groups affect DEX droplet pinning somewhat, the most stable patterns were realized using surfaces with chemical heterogeneity. Arbitrary DEX solution patterning was achieved on a chemically patchy surface.

Small mass-change detectable quartz crystal microbalance and its application to enzymatic one-base elongation on DNA.

A highly sensitive 27 MHz quartz crystal microbalance instrument with an automatic flow injection system was developed to obtain realistic minimal frequency noise (±0.05 Hz) and to obtain a stable signal baseline (±1 Hz/h) by controlling the temperature of each part in the quartz crystal microbalance (QCM) system using three Peltier devices with a resolution of ±0.001 °C and by optimizing the flow system to prevent fluctuation of the internal pressure of the QCM. The improved QCM with an automatic flow injection system enabled detection of small mass changes such as binding of biotin to a streptavidin-immobilized QCM with a high signal-to-noise ratio. We also applied this device to enzyme reactions of one-base elongation by DNA polymerase (Klenow fragment, KF). We immobilized dsDNAs including the protruding end of dA, dG, dT, or dC on the QCM electrode and ran complementary dNTP monomers with KF into the QCM flow cell. We could directly detect the enzymatic one-base elongation of DNA as a small mass increase, and we found the difference in the reaction rate for each monomer.

Preparation of catalytically active, covalent α-polylysine-enzyme conjugates via UV/vis-quantifiable bis-aryl hydrazone bond formation.

Covalent UV/vis-quantifiable bis-aryl hydrazone bond formation was investigated for the preparation of conjugates between α-poly-d-lysine (PDL) and either α-chymotrypsin (α-CT) or horseradish peroxidase (HRP). PDL and the enzymes were first modified via free amino groups with the linking reagents succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic, at pH 7.6) and succinimidyl 4-formylbenzoate (S-4FB, at pH 7.2), respectively. The modified PDL and enzymes were then conjugated at pH 4.7, whereby polymer chains carrying several enzymes were obtained. Kinetics of the bis-aryl hydrazone bond formation was investigated spectrophotometrically at 354 nm. Retention of the enzymatic activity after conjugate formation was confirmed by using the substrates N-succinimidyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide (for α-CT) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, for HRP). Thus, not only a mild and efficient preparation and convenient quantification of a conjugate between the polycationic α-polylysine and enzymes could be shown, but also the complete preservation of the enzymatic activity.

Aqueous Two-Phase Systems and Microfluidics for Microscale Assays and Analytical Measurements.

Phase separation is a common occurrence in nature. Synthetic and natural polymers, salts, ionic liquids, surfactants, and biomacromolecules phase separate in water, resulting in an aqueous two-phase system (ATPS). This review discusses the properties, handling, and uses of ATPSs. These systems have been used for protein, nucleic acid, virus, and cell purification and have in recent years found new uses for small organics, polysaccharides, extracellular vesicles, and biopharmaceuticals. Analytical biochemistry applications such as quantifying protein-protein binding, probing for conformational changes, or monitoring enzyme activity have been performed with ATPSs. Not only are ATPSs biocompatible, they also retain their properties at the microscale, enabling miniaturization experiments such as droplet microfluidics, bacterial quorum sensing, multiplexed and point-of-care immunoassays, and cell patterning. ATPSs include coacervates and may find wider interest in the context of intracellular phase separation and origin of life. Recent advances in fundamental understanding and in commercial application are also considered.

Protocell arrays for simultaneous detection of diverse analytes.

Simultaneous detection of multiple analytes from a single sample (multiplexing), particularly when done at the point of need, can guide complex decision-making without increasing the required sample volume or cost per test. Despite recent advances, multiplexed analyte sensing still typically faces the critical limitation of measuring only one type of molecule (e.g., small molecules or nucleic acids) per assay platform. Here, we address this bottleneck with a customizable platform that integrates cell-free expression (CFE) with a polymer-based aqueous two-phase system (ATPS), producing membrane-less protocells containing transcription and translation machinery used for detection. We show that multiple protocells, each performing a distinct sensing reaction, can be arrayed in the same microwell to detect chemically diverse targets from the same sample. Furthermore, these protocell arrays are compatible with human biofluids, maintain function after lyophilization and rehydration, and can produce visually interpretable readouts, illustrating this platform's potential as a minimal-equipment, field-deployable, multi-analyte detection tool.

DNA-Based Biomaterials for Immunoengineering.

Man-made DNA materials hold the potential to modulate specific immune pathways toward immunoactivating or immunosuppressive cascades. DNA-based biomaterials introduce DNA into the extracellular environment during implantation or delivery, and subsequently intracellularly upon phagocytosis or degradation of the material. Therefore, the immunogenic functionality of biological and synthetic extracellular DNA should be considered to achieve desired immune responses. In vivo, extracellular DNA from both endogenous and exogenous sources holds immunoactivating functions which can be traced back to the molecular features of DNA, such as sequence and length. Extracellular DNA is recognized as damage-associated molecular patterns (DAMPs), or pathogen-associated molecular patterns (PAMPs), by immune cell receptors, activating either proinflammatory signaling pathways or immunosuppressive cell functions. Although extracellular DNA promotes protective immune responses during early inflammation such as bacterial killing, recent advances demonstrate that unresolved and elevated DNA concentrations may contribute to the pathogenesis of autoimmune diseases, cancer, and fibrosis. Therefore, addressing the immunogenicity of DNA enables immune responses to be engineered by optimizing their activating and suppressive performance per application. To this end, emerging biology relevant to the generation of extracellular DNA, DNA sensors, and its role concerning existing and future synthetic DNA biomaterials are reviewed.

Stigmatic Microscopy Enables Low-Cost, 3D, Microscale Particle Imaging Velocimetry in Rehydrating Aqueous Two-Phase Systems.

This paper describes the construction of a novel stigmatic microscope and image analysis algorithm to simultaneously analyze convective mixing both inside and outside of rehydrating µL-scale aqueous two-phase system (ATPS) droplets. Stigmatic microscopy is inexpensive and advantageous because it modifies the point-spread function of fluorescent particles to enable measurement of their 3D positions from single 2D images, without needing to take slices. In one application of the technique, the convection patterns captured clarify how different ATPS formulations succeed or fail to exclude cells for patterning. Particle flow traces reveal speed and directionality of circulation, indicating temporary eddies at the outer edge of the rehydrating droplet. In another application, the speed of circulation during rehydration was analyzed for different ATPS formulations and the results used to develop a new fast ELISA procedure. While this paper focuses on ATPS rehydration, the microscope and algorithm should be applicable to a broad range of microfluidic flows where microscale 3D convection is important.

Extracellular Trap-Mimicking DNA-Histone Mesostructures Synergistically Activate Dendritic Cells.

Extracellular traps (ETs), such as neutrophil extracellular traps, are a physical mesh deployed by immune cells to entrap and constrain pathogens. ETs are immunogenic structures composed of DNA, histones, and an array of variable protein and peptide components. While much attention has been paid to the multifaceted function of these structures, mechanistic studies of ETs remain challenging due to their heterogeneity and complexity. Here, a novel DNA-histone mesostructure (DHM) formed by complexation of DNA and histones into a fibrous mesh is reported. DHMs mirror the DNA-histone structural frame of ETs and offer a facile platform for cell culture studies. It is shown that DHMs are potent activators of dendritic cells and identify both the methylation state of DHMs and physical interaction between dendritic cells and DHMs as key tuning switches for immune stimulation. Overall, the DHM platform provides a new opportunity to study the role of ETs in immune activation and pathophysiology.

Surface Modification Enhanced Reflection Intensity of Quartz Crystal Microbalance Sensors upon Molecular Adsorption.

Molecular adsorption on a sensing surface involves molecule-substrate and molecule-molecule interactions. Combining optical systems and a quartz crystal microbalance (QCM) on the same sensing surface allows the quantification of such interactions and reveals the physicochemical properties of the adsorbed molecules. However, low sensitivity of the current reflection-based techniques compared to the QCM technique hinders the quantitative analysis of the adsorption events. Here, a layer-by-layer surface modification of a QCM sensor is studied to increase the optical sensitivity. The intermediate layers of organic-inorganic molecules and metal-metal oxide were explored on a gold (Au) surface of a QCM sensor. First, polyhedral oligomeric silsesquioxane-derivatives that served as the organic-inorganic intermediate layer were synthesized and modified on the Au-QCM surface. Meanwhile, titanium oxide, fabricated by anodic oxidation of titanium, was used as a metal-metal oxide intermediate layer on a titanium-coated QCM surface. The developed technique enabled interrogation of the molecular adsorption owing to the enhanced optical sensitivity.

Membraneless Compartmentalization Facilitates Enzymatic Cascade Reactions and Reduces Substrate Inhibition.

Living cells possess membraneless organelles formed by liquid-liquid phase separation. With the aim of better understanding the general functions of membraneless microcompartments, this paper constructs acellular multicompartment reaction systems using an aqueous multiphase system. Membraneless coacervate droplets are placed within a molecularly crowded environment, where a larger dextran (DEX) droplet is submerged in a polyethylene glycol (PEG) solution. The coacervate droplets are capable of sequestering reagents and enzymes with a long retention time, and demonstrate multistep cascading reactions through the liquid-liquid interfaces. The ability to change phase dynamics is also demonstrated through salt-mediated dissolution of coacervate droplets, which leads to the release and mixing of separately sequestered reagents and enzymes. Finally, as phase-separated materials in membraneless organelles are often substrates and substrate analogues for the enzymes sequestered or excluded in the organelles, this paper explores the interaction between DEX and dextranase, an enzyme that hydrolyzes DEX. The results reveal that dextranase suffers from substrate inhibition when partitioned directly in a DEX phase but that this inhibition can be mitigated and reactions greatly accelerated by compartmentalization of dextranase inside a coacervate droplet that is adjacent to, but phase-separated from, the DEX phase. The insight that compartmentalization of enzymes can accelerate reactions by mitigating substrate inhibition is particularly novel and is an example where artificial membraneless organelle-like systems may provide new insights into physiological cell functions.

Dispersible oxygen microsensors map oxygen gradients in three-dimensional cell cultures.

Phase fluorimetry, unlike the more commonly used intensity-based measurement, is not affected by differences in light paths from culture vessels or by optical attenuation through dense 3D cell cultures and hydrogels thereby minimizing dependence on signal intensity for accurate measurements. This work describes the use of phase fluorimetry on oxygen-sensor microbeads to perform oxygen measurements in different microtissue culture environments. In one example, cell spheroids were observed to deplete oxygen from the cell-culture medium filling the bottom of conventional microwells within minutes, whereas oxygen concentrations remained close to ambient levels for several days in hanging-drop cultures. By dispersing multiple oxygen microsensors in cell-laden hydrogels, we also mapped cell-generated oxygen gradients. The spatial oxygen mapping was sufficiently precise to enable the use of computational models of oxygen diffusion and uptake to give estimates of the cellular oxygen uptake rate and the half-saturation constant. The results show the importance of integrated design and analysis of 3D cell cultures from both biomaterial and oxygen supply aspects. While this paper specifically tests spheroids and cell-laden gel cultures, the described methods should be useful for measuring pericellular oxygen concentrations in a variety of biomaterials and culture formats.

Aqueous Two Phase System Assisted Self-Assembled PLGA Microparticles.

Here, we produce poly(lactide-co-glycolide) (PLGA) based microparticles with varying morphologies, and temperature responsive properties utilizing a Pluronic F127/dextran aqueous two-phase system (ATPS) assisted self-assembly. The PLGA polymer, when emulsified in Pluronic F127/dextran ATPS, forms unique microparticle structures due to ATPS guided-self assembly. Depending on the PLGA concentration, the particles either formed a core-shell or a composite microparticle structure. The microparticles facilitate the simultaneous incorporation of both hydrophobic and hydrophilic molecules, due to their amphiphilic macromolecule composition. Further, due to the lower critical solution temperature (LCST) properties of Pluronic F127, the particles exhibit temperature responsiveness. The ATPS based microparticle formation demonstrated in this study, serves as a novel platform for PLGA/polymer based tunable micro/nano particle and polymersome development. The unique properties may be useful in applications such as theranostics, synthesis of complex structure particles, bioreaction/mineralization at the two-phase interface, and bioseparations.

Surface-templated hydrogel patterns prompt matrix-dependent migration of breast cancer cells towards chemokine-secreting cells.

This paper describes a novel technique for fabricating spatially defined cell-laden collagen hydrogels, using patterned, non-adhesive polyacrylamide-coated polydimethylsiloxane (PDMS) surfaces as a template. Precisely patterned embedded co-cultures of breast cancer cells and chemokine-producing cells generated with this technique revealed matrix-dependent and chemokine isoform-dependent migration of cancer cells. CXCL12 chemokine-secreting cells induce significantly more chemotaxis of cancer cells when the 3-D extracellular matrix (ECM) includes components that bind the secreted CXCL12 chemokines. Experimental observations using cells that secrete CXCL12 isoforms with different matrix affinities together with computational simulations show that stronger ligand-matrix interactions sharpen chemoattractant gradients, leading to increased chemotaxis of the CXCL12 gradient-sensing CXCR4 receptor-expressing (CXCR4+) cells patterned in the hydrogel. These results extend our recent report on CXCL12 isoform-dependent chemotaxis studies from 2-D to 3-D environments and additionally reveal the important role of ECM composition. The developed technology is simple, versatile and robust; and as chemoattractant-matrix interactions are common, the methods described here should be broadly applicable for study of physiological migration of many different cell types in response to a variety of chemoattractants.