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.

Developing a Graduate Class on Synthetic Cells at a Minority Serving Institution: Lessons from the University of New Mexico.

This article describes the development, methodology, enrollment, and outcomes of a graduate technical elective course on synthetic cells and organelles offered at the University of New Mexico, a minority-majority institution, in Fall 2022. The course had a significant ethics component and took advantage of readily available, low cost, and no-cost teaching materials that are available online. The course was effective in attracting a diverse enrollment of graduate students and senior undergraduates, some of whom participated in a survey of their backgrounds and motivations after the course was over. The article also provides results from this survey. Courses such as the one described have the potential to increase access and participation in emerging fields of research and technology such as synthetic cells.

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.

Building a community to engineer synthetic cells and organelles from the bottom-up.

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives.

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.

On-demand release of Candida albicans biofilms from urinary catheters by mechanical surface deformation.

Candida albicans is a leading cause of catheter-associated urinary tract infections and elimination of these biofilm-based infections without antifungal agents would constitute a significant medical advance. A novel urinary catheter prototype that utilizes on-demand surface deformation is effective at eliminating bacterial biofilms and here the broader applicability of this prototype to remove fungal biofilms has been demonstrated. C. albicans biofilms were debonded from prototypes by selectively inflating four additional intralumens surrounding the main lumen of the catheters to provide the necessary surface strain to remove the adhered biofilm. Deformable catheters eliminated significantly more biofilm than the controls (>90% eliminated vs 10% control; p < 0.001). Mechanical testing revealed that fungal biofilms have an elastic modulus of 45 ± 6.7 kPa with a fracture energy of 0.4-2 J m-2. This study underscores the potential of mechanical disruption as a materials design strategy to combat fungal device-associated infections.

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.

Sequence-encoded colloidal origami and microbot assemblies from patchy magnetic cubes.

Colloidal-scale assemblies that reconfigure on demand may serve as the next generation of soft "microbots," artificial muscles, and other biomimetic devices. This requires the precise arrangement of particles into structures that are preprogrammed to reversibly change shape when actuated by external fields. The design and making of colloidal-scale assemblies with encoded directional particle-particle interactions remain a major challenge. We show how assemblies of metallodielectric patchy microcubes can be engineered to store energy through magnetic polarization and release it on demand by microscale reconfiguration. The dynamic pattern of folding and reconfiguration of the chain-like assemblies can be encoded in the sequence of the cube orientation. The residual polarization of the metallic facets on the microcubes leads to local interactions between the neighboring particles, which is directed by the conformational restrictions of their shape after harvesting energy from external magnetic fields. These structures can also be directionally moved, steered, and maneuvered by global forces from external magnetic fields. We illustrate these capabilities by examples of assemblies of specific sequences that can be actuated, reoriented, and spatially maneuvered to perform microscale operations such as capturing and transporting live cells, acting as prototypes of microbots, micromixers, and other active microstructures.

Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity.

Dynamic protein-rich intracellular structures that contain phase-separated intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase separate into diverse assemblies within droplet microenvironments. Using theoretical analyses, we interpret the phase behaviour of archetypal IDP sequences and demonstrate the rational design of a vast library of multicomponent protein-rich structures that ranges from uniform nano-, meso- and microscale puncta (distinct protein droplets) to multilayered orthogonally phase-separated granular structures. The ability to predict and program IDP-rich assemblies in this fashion offers new insights into (1) genetic-to-molecular-to-macroscale relationships that encode hierarchical IDP assemblies, (2) design rules of such assemblies in cell biology and (3) molecular-level engineering of self-assembled recombinant IDP-rich materials.

Assembly of hard spheres in a cylinder: a computational and experimental study.

Hard spheres are an important benchmark of our understanding of natural and synthetic systems. In this work, colloidal experiments and Monte Carlo simulations examine the equilibrium and out-of-equilibrium assembly of hard spheres of diameter σ within cylinders of diameter σ≤D≤ 2.82σ. Although phase transitions formally do not exist in such systems, marked structural crossovers can nonetheless be observed. Over this range of D, we find in simulations that structural crossovers echo the structural changes in the sequence of densest packings. We also observe that the out-of-equilibrium self-assembly depends on the compression rate. Slow compression approximates equilibrium results, while fast compression can skip intermediate structures. Crossovers for which no continuous line-slip exists are found to be dynamically unfavorable, which is the main source of this difference. Results from colloidal sedimentation experiments at low diffusion rate are found to be consistent with the results of fast compressions, as long as appropriate boundary conditions are used.

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.

Translating microfluidics: Cell separation technologies and their barriers to commercialization.

Advances in microfluidic cell sorting have revolutionized the ways in which cell-containing fluids are processed, now providing performances comparable to, or exceeding, traditional systems, but in a vastly miniaturized format. These technologies exploit a wide variety of physical phenomena to manipulate cells and fluid flow, such as magnetic traps, sound waves and flow-altering micropatterns, and they can evaluate single cells by immobilizing them onto surfaces for chemotherapeutic assessment, encapsulate cells into picoliter droplets for toxicity screenings and examine the interactions between pairs of cells in response to new, experimental drugs. However, despite the massive surge of innovation in these high-performance lab-on-a-chip devices, few have undergone successful commercialization, and no device has been translated to a widely distributed clinical commodity to date. Persistent challenges such as an increasingly saturated patent landscape as well as complex user interfaces are among several factors that may contribute to their slowed progress. In this article, we identify several of the leading microfluidic technologies for sorting cells that are poised for clinical translation; we examine the principal barriers preventing their routine clinical use; finally, we provide a prospectus to elucidate the key criteria that must be met to overcome those barriers. Once established, these tools may soon transform how clinical labs study various ailments and diseases by separating cells for downstream sequencing and enabling other forms of advanced cellular or sub-cellular analysis. © 2016 International Clinical Cytometry Society.

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.

Hollow silica microspheres for buoyancy-assisted separation of infectious pathogens from stool.

Separation of cells and microorganisms from complex biological mixtures is a critical first step in many analytical applications ranging from clinical diagnostics to environmental monitoring for food and waterborne contaminants. Yet, existing techniques for cell separation are plagued by high reagent and/or instrumentation costs that limit their use in many remote or resource-poor settings, such as field clinics or developing countries. We developed an innovative approach to isolate infectious pathogens from biological fluids using buoyant hollow silica microspheres that function as "molecular buoys" for affinity-based target capture and separation by floatation. In this process, antibody functionalized glass microspheres are mixed with a complex biological sample, such as stool. When mixing is stopped, the target-bound, low-density microspheres float to the air/liquid surface, which simultaneously isolates and concentrates the target analytes from the sample matrix. The microspheres are highly tunable in terms of size, density, and surface functionality for targeting diverse analytes with separation times of ≤2min in viscous solutions. We have applied the molecular buoy technique for isolation of a protozoan parasite that causes diarrheal illness, Cryptosporidium, directly from stool with separation efficiencies over 90% and low non-specific binding. This low-cost method for phenotypic cell/pathogen separation from complex mixtures is expected to have widespread use in clinical diagnostics as well as basic research.

Magnetically Responsive Negative Acoustic Contrast Microparticles for Bioanalytical Applications.

Smart colloidal particles are routinely used as carriers for biological molecules, fluorescent reporters, cells, and other analytes for the purposes of sample preparation and detection. However, such particles are typically engineered to respond to a single type of stimulus (e.g., commercial magnetic beads to magnetic fields). Here, we demonstrate a unique class of particles that display both positive magnetic contrast and negative acoustic contrast in water. This dual functionality allows for fine spatiotemporal control, enabling multiple separation modalities and increasing the utility of the particles in various chemical and biological assays.

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.