Isolation of nucleic acids using liquid-liquid phase separation of pH-sensitive elastin-like polypeptides.

Extraction of nucleic acids (NAs) is critical for many methods in molecular biology and bioanalytical chemistry. NA extraction has been extensively studied and optimized for a wide range of applications and its importance to society has significantly increased. The COVID-19 pandemic highlighted the importance of early and efficient NA testing, for which NA extraction is a critical analytical step prior to the detection by methods like polymerase chain reaction. This study explores simple, new approaches to extraction using engineered smart nanomaterials, namely NA-binding, intrinsically disordered proteins (IDPs), that undergo triggered liquid-liquid phase separation (LLPS). Two types of NA-binding IDPs are studied, both based on genetically engineered elastin-like polypeptides (ELPs), model IDPs that exhibit a lower critical solution temperature in water and can be designed to exhibit LLPS at desired temperatures in a variety of biological solutions. We show that ELP fusion proteins with natural NA-binding domains can be used to extract DNA and RNA from physiologically relevant solutions. We further show that LLPS of pH responsive ELPs that incorporate histidine in their sequences can be used for both binding, extraction and release of NAs from biological solutions, and can be used to detect SARS-CoV-2 RNA in samples from COVID-positive patients.

Microfluidic Fabrication of Silica Microspheres Infused with Positron Emission Tomography Imaging Agents.

Selective internal radiation therapy (SIRT) is a treatment which delivers radioactive therapeutic microspheres via the hepatic artery to destroy tumorigenic tissue of the liver. However, the dose required varies significantly from patient to patient due to nuances in individual biology. Therefore, a positron emission tomography (PET) imaging surrogate, or radiotracer, is used to predict in vivo behavior of therapeutic Y-90 spheres. The ideal surrogate should closely resemble Y-90 microspheres in morphology for highest predictive accuracy. This work presents the fabrication of positron-emitting silica microspheres infused with PET radiotracers copper, fluorine, and gallium. A quick one-pot synthesis is used to create precursor sol, followed by droplet formation with flow-focusing microfluidics, and finally thermal treatment to yield 10-50 µm microspheres with narrow size distribution. Loading of the infused element is controllable in the sol synthesis, while the final sphere size is tunable based on microfluidic flow rates and device channel width. The system is then employed to make radioactive Ga-68 microspheres, which are tested for radioactivity and stability. The fabrication method can be completed within a few hours, depending on the desired microsphere quantity. A microfluidic system is applied to fabricate silica particles loaded with diverse elemental infusions, including radioactive Ga-68.

Rapid capture of biomolecules from blood via stimuli-responsive elastomeric particles for acoustofluidic separation.

The detection of biomarkers in blood often requires extensive and time-consuming sample preparation to remove blood cells and concentrate the biomarker(s) of interest. We demonstrate proof-of-concept for a chip-based, acoustofluidic method that enables the rapid capture and isolation of a model protein biomarker (i.e., streptavidin) from blood for off-chip quantification. Our approach makes use of two key components - namely, soluble, thermally responsive polypeptides fused to ligands for the homogeneous capture of biomarkers from whole blood and silicone microparticles functionalized with similar, tethered, thermally responsive polypeptides. When the two components are mixed together and subjected to a mild thermal trigger, the thermally responsive moieties undergo a phase transition, causing the untethered (soluble) polypeptides to co-aggregate with the particle-bound polypeptides. The mixture is then diluted with warm buffer and injected into a microfluidic channel supporting a bulk acoustic standing wave. The biomarker-bearing particles migrate to the pressure antinodes, whereas blood cells migrate to the pressure node, leading to rapid separation with efficiencies exceeding 90% in a single pass. The biomarker-bearing particles can then be analyzed via flow cytometry, with a limit of detection of 0.75 nM for streptavidin spiked in blood plasma. Finally, by cooling the solution below the solubility temperature of the polypeptides, greater than 75% of the streptavidin is released from the microparticles, offering a unique approach for downstream analysis (e.g., sequencing or structural analysis). Overall, this methodology has promise for the detection, enrichment and analysis of some biomarkers from blood and other complex biological samples.

Migration of Microparticle-Containing Amoeba through Constricted Environments.

In many situations, cells migrate through tiny orifices. Examples include the extravasation of immune cells from the bloodstream for fighting infections, the infiltration of cancer cells during metastasis, and the migration of human pathogens. An extremely motile and medically relevant type of human pathogen is Acanthamoeba castellanii. In the study presented here, we investigated how a combination of microparticles and microstructured interfaces controls the migration of A. castellanii trophozoites. The microinterfaces comprised well-defined micropillar arrays, and the trophozoites easily migrated through the given constrictions by adapting the shape and size of their intracellular vacuoles and by adapting intracellular motion. After feeding the trophozoite cells in microinterfaces with synthetic, stiff microparticles of various sizes and shapes, their behavior changed drastically: if the particles were smaller than the micropillar gap, migration was still possible. If the cells incorporated particles larger than the pillar gap, they could become immobilized but could also display remarkable problem-solving capabilities. For example, they turned rod-shaped microparticles such that their short axis fit through the pillar gap or they transported the particles above the structure. As migration is a crucial contribution to A. castellanii pathogenicity and is also relevant to other biological processes in microenvironments, such as cancer metastasis, our results provide an interesting strategy for controlling the migration of cells containing intracellular particles by microstructured interfaces that serve as migration-limiting environments.

Oil-Free Acoustofluidic Droplet Generation for Multicellular Tumor Spheroid Culture.

We present an easy-to-assemble microfluidic system for synthesizing cell-loaded dextran/alginate (DEX/ALG) hydrogel spheres using an aqueous two-phase system (ATPS) for templated fabrication of multicellular tumor spheroids (MTSs). An audio speaker driven by an amplified output of a waveform generator or smartphone provides acoustic modulation to drive the breakup of an ATPS into MTS template droplets within microcapillary fluidic devices. We apply extensions of Plateau-Rayleigh theory to help define the flow and frequency parameter space necessary for acoustofluidic ATPS droplet formation in these devices. This method provides a simple droplet microfluidic approach using off-the-shelf acoustic components for quickly initiating MTSs and subsequent 3D cell culture.

Functional Modification of Silica through Enhanced Adsorption of Elastin-Like Polypeptide Block Copolymers.

A powerful tool for controlling interfacial properties and molecular architecture relies on the tailored adsorption of stimuli-responsive block copolymers onto surfaces. Here, we use computational and experimental approaches to investigate the adsorption behavior of thermally responsive polypeptide block copolymers (elastin-like polypeptides, ELPs) onto silica surfaces, and to explore the effects of surface affinity and micellization on the adsorption kinetics and the resultant polypeptide layers. We demonstrate that genetic incorporation of a silica-binding peptide (silaffin R5) results in enhanced adsorption of these block copolymers onto silica surfaces as measured by quartz crystal microbalance and ellipsometry. We find that the silaffin peptide can also direct micelle adsorption, leading to close-packed micellar arrangements that are distinct from the sparse, patchy arrangements observed for ELP micelles lacking a silaffin tag, as evidenced by atomic force microscopy measurements. These experimental findings are consistent with results of dissipative particle dynamics simulations. Wettability measurements suggest that surface immobilization hampers the temperature-dependent conformational change of ELP micelles, while adsorbed ELP unimers (i.e., unmicellized block copolymers) retain their thermally responsive property at interfaces. These observations provide guidance on the use of ELP block copolymers as building blocks for fabricating smart surfaces and interfaces with programmable architecture and functionality.

Self-assembled hybrid elastin-like polypeptide/silica nanoparticles enable triggered drug release.

The discovery of biomimetic polypeptides that enable the biomineralization of synthetic and biosynthetic materials has resulted in the development of hybrid materials that incorporate inorganic components for potential application in drug delivery, enzyme immobilization, and surface modification. Here, we describe an approach that uses micellar assemblies of an elastin-like polypeptide (ELP) modified with silica-promoting sequences and drug conjugates that are subsequently encapsulated within a silica matrix. Incorporation of a lysine-rich tag derived from the silaffin R5 peptide into the N-terminus of a hydrophilic ELP that self-assembles upon conjugation of hydrophobic molecules at the C-terminus results in the formation of spherical micelles with a conjugated drug embedded in the core and a corona that is decorated with the silaffin peptide. These micelles serve as the building blocks for the polycondensation of silica into uniform, hybrid polypeptide-silica nanoparticles. We demonstrate proof-of-concept examples using a model hydrophobic small molecule and doxorobucin, a small molecule chemotherapeutic, and further show pH-dependent doxorubicin release from the hybrid nanoparticles.

Strong, Tough, Stretchable, and Self-Adhesive Hydrogels from Intrinsically Unstructured Proteins.

Strong, tough, stretchable, and self-adhesive hydrogels are designed with intrinsically unstructured proteins. The extraordinary mechanical properties exhibited by these materials are enabled by an integration of toughening mechanisms that maintain high elasticity and dissipate mechanical energy within the protein networks.

Magnetic separation of acoustically focused cancer cells from blood for magnetographic templating and analysis.

Liquid biopsies hold enormous promise for the next generation of medical diagnoses. At the forefront of this effort, many are seeking to capture, enumerate and analyze circulating tumor cells (CTCs) as a means to prognosticate and develop individualized treatments for cancer. Capturing these rare cells, however, represents a major engineering challenge due to their low abundance, morphology and heterogeneity. A variety of microfluidic tools have been developed to isolate CTCs from drawn blood samples; however, few of these approaches offer a means to separate and analyze cells in an integrated system. We have developed a microfluidic platform comprised of three modules that offers high throughput separation of cancer cells from blood and on-chip organization of those cells for streamlined analyses. The first module uses an acoustic standing wave to rapidly align cells in a contact-free manner. The second module then separates magnetically labeled cells from unlabeled cells, offering purities exceeding 85% for cells and 90% for binary mixtures of synthetic particles. Finally, the third module contains a spatially periodic array of microwells with underlying micromagnets to capture individual cells for on-chip analyses (e.g., staining, imaging and quantification). This array is capable of capturing with accuracies exceeding 80% for magnetically labeled cells and 95% for magnetic particles. Overall, by virtue of its holistic processing of complex biological samples, this system has promise for the isolation and evaluation of rare cancer cells and can be readily extended to address a variety of applications across single cell biology and immunology.

Incorporation of silicone oil into elastomers enhances barnacle detachment by active surface strain.

Silicone-oil additives are often used in fouling-release silicone coatings to reduce the adhesion strength of barnacles and other biofouling organisms. This study follows on from a recently reported active approach to detach barnacles, which was based on the surface strain of elastomeric materials, by investigating a new, dual-action approach to barnacle detachment using Ecoflex®-based elastomers incorporated with poly(dimethylsiloxane)-based oil additives. The experimental results support the hypothesis that silicone-oil additives reduce the amount of substratum strain required to detach barnacles. The study also de-coupled the two effects of silicone oils (ie surface-activity and alteration of the bulk modulus) and examined their contributions in reducing barnacle adhesion strength. Further, a finite element model based on fracture mechanics was employed to qualitatively understand the effects of surface strain and substratum modulus on barnacle adhesion strength. The study demonstrates that dynamic substratum deformation of elastomers with silicone-oil additives provides a bifunctional approach towards management of biofouling by barnacles.

Experimental verification of theoretical equations for acoustic radiation force on compressible spherical particles in traveling waves.

Acoustophoresis uses acoustic radiation force to remotely manipulate particles suspended in a host fluid for many scientific, technological, and medical applications, such as acoustic levitation, acoustic coagulation, contrast ultrasound imaging, ultrasound-assisted drug delivery, etc. To estimate the magnitude of acoustic radiation forces, equations derived for an inviscid host fluid are commonly used. However, there are theoretical predictions that, in the case of a traveling wave, viscous effects can dramatically change the magnitude of acoustic radiation forces, which make the equations obtained for an inviscid host fluid invalid for proper estimation of acoustic radiation forces. To date, experimental verification of these predictions has not been published. Experimental measurements of viscous effects on acoustic radiation forces in a traveling wave were conducted using a confocal optical and acoustic system and values were compared with available theories. Our results show that, even in a low-viscosity fluid such as water, the magnitude of acoustic radiation forces is increased manyfold by viscous effects in comparison with what follows from the equations derived for an inviscid fluid.

Creating cellular patterns using genetically engineered, gold- and cell-binding polypeptides.

Patterning cells on material surfaces is an important tool for the study of fundamental cell biology, tissue engineering, and cell-based bioassays. Here, the authors report a simple approach to pattern cells on gold patterned silicon substrates with high precision, fidelity, and stability. Cell patterning is achieved by exploiting adsorbed biopolymer orientation to either enhance (gold regions) or impede (silicon oxide regions) cell adhesion at particular locations on the patterned surface. Genetic incorporation of gold binding domains enables C-terminal chemisorption of polypeptides onto gold regions with enhanced accessibility of N-terminal cell binding domains. In contrast, the orientation of polypeptides adsorbed on the silicon oxide regions limit the accessibility of the cell binding domains. The dissimilar accessibility of cell binding domains on the gold and silicon oxide regions directs the cell adhesion in a spatially controlled manner in serum-free medium, leading to the formation of well-defined cellular patterns. The cells are confined within the polypeptide-modified gold regions and are viable for eight weeks, suggesting that bioactive polypeptide modified surfaces are suitable for long-term maintenance of patterned cells. This study demonstrates an innovative surface-engineering approach for cell patterning by exploiting distinct ligand accessibility on heterogeneous surfaces.

3D Printing of Highly Stretchable and Tough Hydrogels into Complex, Cellularized Structures.

A 3D printable and highly stretchable tough hydrogel is developed by combining poly(ethylene glycol) and sodium alginate, which synergize to form a hydrogel tougher than natural cartilage. Encapsulated cells maintain high viability over a 7 d culture period and are highly deformed together with the hydrogel. By adding biocompatible nanoclay, the tough hydrogel is 3D printed in various shapes without requiring support material.

Bio-inspired synthesis of hybrid silica nanoparticles templated from elastin-like polypeptide micelles.

The programmed self-assembly of block copolymers into higher order nanoscale structures offers many attractive attributes for the development of new nanomaterials for numerous applications including drug delivery and biosensing. The incorporation of biomimetic silaffin peptides in these block copolymers enables the formation of hybrid organic-inorganic materials, which can potentially enhance the utility and stability of self-assembled nanostructures. We demonstrate the design, synthesis and characterization of amphiphilic elastin-like polypeptide (ELP) diblock copolymers that undergo temperature-triggered self-assembly into well-defined spherical micelles. Genetically encoded incorporation of the silaffin R5 peptide at the hydrophilic terminus of the diblock ELP leads to presentation of the silaffin R5 peptide on the coronae of the micelles, which results in localized condensation of silica and the formation of near-monodisperse, discrete, sub-100 nm diameter hybrid ELP-silica particles. This synthesis method, can be carried out under mild reaction conditions suitable for bioactive materials, and will serve as the basis for the development and application of functional nanomaterials. Beyond silicification, the general strategies described herein may also be adapted for the synthesis of other biohybrid nanomaterials as well.

Microfluidic cell sorting: a review of the advances in the separation of cells from debulking to rare cell isolation.

Accurate and high throughput cell sorting is a critical enabling technology in molecular and cellular biology, biotechnology, and medicine. While conventional methods can provide high efficiency sorting in short timescales, advances in microfluidics have enabled the realization of miniaturized devices offering similar capabilities that exploit a variety of physical principles. We classify these technologies as either active or passive. Active systems generally use external fields (e.g., acoustic, electric, magnetic, and optical) to impose forces to displace cells for sorting, whereas passive systems use inertial forces, filters, and adhesion mechanisms to purify cell populations. Cell sorting on microchips provides numerous advantages over conventional methods by reducing the size of necessary equipment, eliminating potentially biohazardous aerosols, and simplifying the complex protocols commonly associated with cell sorting. Additionally, microchip devices are well suited for parallelization, enabling complete lab-on-a-chip devices for cellular isolation, analysis, and experimental processing. In this review, we examine the breadth of microfluidic cell sorting technologies, while focusing on those that offer the greatest potential for translation into clinical and industrial practice and that offer multiple, useful functions. We organize these sorting technologies by the type of cell preparation required (i.e., fluorescent label-based sorting, bead-based sorting, and label-free sorting) as well as by the physical principles underlying each sorting mechanism.

Simple assay for proteases based on aggregation of stimulus-responsive polypeptides.

Unregulated changes in protease activity are linked to many diseases including cancer. Fast, accurate, and low-cost assays for detection of these changes are being explored for early diagnosis and monitoring of these diseases and can also be used as platforms for the discovery of new drugs. We report a new methodology for the simple detection and quantification of protease activity in buffer and human serum. The assay is based on recombinant diblock polypeptides that undergo temperature- or salt-triggered micellization in water. The coronae of the micelles are linked to the water-insoluble cores by a peptide substrate that is cleaved in the presence of the target protease. Protease cleavage of the diblock polypeptide triggers the aggregation of the core-forming segment, leading to a change in solution optical density, which can be used to detect the presence of, and to quantify the concentration of, protease. We used matrix metalloproteinase-1 (MMP-1) as a model protease and found peptide aggregation time to be proportional to enzyme concentration over a range from endogenous MMP-1 level in human serum (∼3 ng/mL) to 100 ng/mL (0.15-5 nM) in 40% human serum and 1-100 ng/mL in buffer. The assay does not require any intermediate steps or sophisticated data analysis, and the modular design of the assay system is amenable to straightforward adaptation for the detection of a wide range of proteases.

Elastomeric negative acoustic contrast particles for capture, acoustophoretic transport, and confinement of cells in microfluidic systems.

We present a particle-based method for the immunospecific capture and confinement of cells using acoustic radiation forces. Ultrasonic standing waves in microfluidic systems have previously been used for the continuous focusing of cells in rapid screening and sorting applications. In aqueous fluids, cells typically exhibit positive acoustic contrast and are thus forced toward the pressure nodes of a standing wave. Conversely, elastomeric particles exhibit negative acoustic contrast and travel toward the pressure antinodes. We have developed a class of elastomeric particles that are synthesized in bulk using a simple nucleation and growth process, providing precise control over their size and functional properties. We demonstrate that the biofunctionalization of these particles can allow the capture and transport of cells to the pressure antinodes solely via acoustic radiation forces, which may enable new acoustics-based cell handling techniques such as the washing, labeling, and sorting of cells with minimal preparatory steps.

Membrane-mediated neuroprotection by curcumin from amyloid-ß-peptide-induced toxicity.

Amyloid-ß peptide (Aß)-membrane interactions have been implicated in the formation of toxic oligomers that permeabilize membranes, allowing an influx of calcium ions and triggering cell death in the pathogenesis of Alzheimer's disease (AD). Curcumin, a small dietary polyphenolic molecule, has been shown to reduce Aß-induced toxicity and AD pathology. We investigate here the effect of curcumin on Aß40-induced toxicity in cultured human neuroblastoma SH-SY5Y cells and test a novel neuroprotection mechanism in which curcumin reduces Aß-membrane interactions and attenuates Aß-induced membrane disruptions. Predominantly monomeric Aß40 exerts toxicity toward SH-SY5Y cells and has been shown to insert spontaneously into anionic lipid monolayers at the air/water interface, resulting in the misfolding and assembly of Aß into ß-sheet-enriched oligomers. Concomitantly, membrane morphology and lipid packing are disrupted. Curcumin dose-dependently ameliorates Aß-induced neurotoxicity and reduces either the rate or extent of Aß insertion into anionic lipid monolayers. Moreover, curcumin reduces Aß-induced dye leakage from lipid-bilayer-covered, dye-loaded, porous silica microspheres. Because curcumin neither affects the inherent surface activity of Aß nor modifies the membrane properties, it reduces Aß insertion by directly attenuating Aß-membrane interactions and reducing Aß-induced membrane disruption. Although the exact molecular mechanism of curcumin's membrane protective effect remains unclear, this effect could in part contribute to curcumin's neuroprotective effect with respect to Aß-induced toxicity. Our work reveals a novel molecular mechanism by which curcumin reduces Aß-related pathology and toxicity and suggests a therapeutic strategy for preventing or treating AD by targeting the inhibition of Aß-induced membrane disruption.

Elastomeric negative acoustic contrast particles for affinity capture assays.

This report describes the development of elastomeric capture microparticles (ECµPs) and their use with acoustophoretic separation to perform microparticle assays via flow cytometry.We have developed simple methods to form ECµPs by cross-linking droplets of common commercially available silicone precursors in suspension followed by surface functionalization with biomolecular recognition reagents. The ECµPs are compressible particles that exhibit negative acoustic contrast in ultrasound when suspended in aqueous media, blood serum, or diluted blood. In this study, these particles have been functionalized with antibodies to bind prostate specific antigen and immunoglobulin (IgG). Specific separation of the ECµPs from blood cells is achieved by flowing them through a microfluidic acoustophoretic device that uses an ultrasonic standing wave to align the blood cells, which exhibit positive acoustic contrast, at a node in the acoustic pressure distribution while aligning the negative acoustic contrast ECµPs at the antinodes. Laminar flow of the separated particles to downstream collection ports allows for collection of the separated negative contrast (ECµPs) and positive contrast particles (cells). Separated ECµPs were analyzed via flow cytometry to demonstrate nanomolar detection for prostate specific antigen in aqueous buffer and picomolar detection for IgG in plasma and diluted blood samples. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types.

Effect of detergents on the thermal behavior of elastin-like polypeptides.

Elastin-like polypeptide (ELP) fusions have been designed to allow large-scale, nonchromatographic purification of many soluble proteins by using the inverse transition cycling (ITC) method; however, the sensitivity of the aqueous lower critical solubility phase transition temperature (T(t)) of ELPs to the addition of cosolutes, including detergents, may be a potential hindrance in purification of proteins with surface hydrophobicity in such a manner. To identify detergents that are known to solubilize such proteins (e.g., membrane proteins) and that have little effect on the T(t) of the ELP, we screened a number of detergents with respect to their effects on the T(t) and secondary structures of a model ELP (denoted here as ELP180). We found that mild detergents (e.g., n-dodecyl-ß-D-maltoside, Triton-X100, and 3-[(3-cholamidopropyl) dimethylamino]-1-propanesulfonate) do not alter the phase transition behavior or structure (as probed by circular dichroism) of ELP180. This result is in contrast to previous studies that showed a strong effect of other detergents (e.g., sodium dodecylsulfate) on the T(t) of ELPs. Our results clearly indicate that mild detergents do not preclude ITC-based separation of ELPs, and thus that ELP fusions may prove to be useful in the purification of detergent-solubilized recombinant hydrophobic proteins, including membrane proteins, which are otherwise notoriously difficult to extract and purify by conventional separation methods (e.g., chromatography).