Localized Plasmonic Heating for Single-Molecule DNA Rupture Measurements in Optical Tweezers.

To date, studies on the thermodynamic and kinetic processes that underlie biological function and nanomachine actuation in biological- and biology-inspired molecular constructs have primarily focused on photothermal heating of ensemble systems, highlighting the need for probes that are localized within the molecular construct and capable of resolving single-molecule response. Here we present an experimental demonstration of wavelength-selective, localized heating at the single-molecule level using the surface plasmon resonance of a 15 nm gold nanoparticle (AuNP). Our approach is compatible with force-spectroscopy measurements and can be applied to studies of the single-molecule thermodynamic properties of DNA origami nanomachines as well as biomolecular complexes. We further demonstrate wavelength selectivity and establish the temperature dependence of the reaction coordinate for base-pair disruption in the shear-rupture geometry, demonstrating the utility and flexibility of this approach for both fundamental studies of local (nanometer-scale) temperature gradients and rapid and multiplexed nanomachine actuation.

Structural interactions in polymer-stabilized magnetic nanocomposites.

Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted significant attention because of their nanoscale magnetic properties. SPION aggregates may afford emergent properties, resulting from dipole-dipole interactions between neighbors. Such aggregates can display internal order, with high packing fractions (>20%), and can be stabilized with block co-polymers (BCPs), permitting design of tunable composites for potential nanomedicine, data storage, and electronic sensing applications. Despite the routine use of magnetic fields for aggregate actuation, the impact of those fields on polymer structure, SPION ordering, and magnetic properties is not fully understood. Here, we report that external magnetic fields can induce ordering in SPION aggregates that affect their structure, inter-SPION distance, magnetic properties, and composite Tg. SPION aggregates were synthesized in the presence or absence of magnetic fields or exposed to magnetic fields post-synthesis. They were characterized using transmission electron microscopy (TEM), small angle X-ray scattering (SAXS), superconducting quantum interference device (SQUID) analysis, and differential scanning calorimetry (DSC). SPION aggregate properties depended on the timing of field application. Magnetic field application during synthesis encouraged preservation of SPION chain aggregates stabilized by polymer coatings even after removal of the field, whereas post synthesis application triggered subtle internal reordering, as indicated by increased blocking temperature (TB), that was not observed via SAXS or TEM. These results suggest that magnetic fields are a simple, yet powerful tool to tailor the structure, ordering, and magnetic properties of polymer-stabilized SPION nanocomposites.

Self-Assembly and sedimentation of 5 nm SPIONs using horizontal, high magnetic fields and gradients.

Superparamagnetic iron oxide nanoparticles (SPIONs) are employed in multiple applications, especially within medical and chemical engineering fields. However, their magnetic separation is very challenging as the magnetophoretic motion is hindered by thermal energy and viscous drag. Recent studies have addressed the recovery of SPIONs by a combination of cooperative magnetophoresis and sedimentation. Nevertheless, the effect of horizontal, high fields and gradients on the vertical sedimentation of SPIONs has not been described. In this work, we report, for the first time, the magnetically facilitated sedimentation of 5 nm particles by applying fields and gradients perpendicular to gravity. The magnetic field was generated by quadrupole magnetic sorters and the process was measured with time by tracking the concentration along the length of a channel contacting the 5 nm SPIONs within the quadrupole field. Our experimental data suggest that aggregates of 60-90 particles are formed in the system; thus, particle agglomeration by dipole-dipole interactions was promoted, and these clusters settled down as a result of gravitational forces. Multiple variables and parameters were evaluated, including the initial SPION concentration, the temperature, the magnetic field and gradient and operation time. It was found that the process was improved by decreasing the initial concentration and the temperature, but the magnitude of the magnetic field and gradient did not significantly affect the sedimentation. Finally, the separation process was rapid, with the systems reaching the equilibrium in approximately 20 minutes, which is a significant advantage in comparison to other systems that require longer times and larger particle sizes.

Reciprocal Control of Hierarchical DNA Origami-Nanoparticle Assemblies.

DNA origami mechanisms offer promising tools for precision nanomanipulation of molecules or nanomaterials. Recent advances have extended the function of individual DNA origami devices to material scales via hierarchical assemblies. However, achieving rapid and precise control of large conformational changes in hierarchical assemblies remains a critical challenge. Here, we demonstrate a method for controlling DNA origami-nanoparticle assemblies through a multiscale approach, in which nanoparticles impart control on the conformation of individual DNA origami mechanisms, whereas DNA origami assemblies control the conformation of nanoparticle arrays. Specifically, we show that the angular distributions of DNA origami hinge mechanisms are tunable as a function of nanoparticle size and distance from the hinge vertex. We selectively adjust the affinity of nanoparticle binding sites, resulting in hinge actuation via DNA melting without releasing the nanoparticle, thereby enabling rapid and reversible temperature-based actuation. Finally, we demonstrate this rapid actuation in DNA origami-nanoparticle arrays of length scales extending over a micron. These results provide guiding principles toward the design of dynamic, DNA-origami hierarchical materials capable of storing and releasing mechanical energy.

Compact quantum dot surface modification to enable emergent behaviors in quantum dot-DNA composites.

Quantum dot (QD) biological imaging and sensing applications often require surface modification with single-stranded deoxyribonucleic acid (ssDNA) oligonucleotides. Furthermore, ssDNA conjugation can be leveraged for precision QD templating via higher-order DNA nanostructures to exploit emergent behaviors in photonic applications. Use of ssDNA-QDs across these platforms requires compact, controlled conjugation that engenders QD stability over a wide pH range and in solutions of high ionic strength. However, current ssDNA-QD conjugation approaches suffer from limitations, such as the requirement for thick coatings, low control over ssDNA labeling density, requirement of large amounts of ssDNA, or low colloidal or photostability, restraining implementation in many applications. Here, we combine thin, multidentate, phytochelatin-3 (PC3) QD passivation techniques with strain-promoted copper-free alkyne-azide click chemistry to yield functional ssDNA-QDs with high stability. This process was broadly applicable across QD sizes (i.e., λem = 540, 560, 600 nm), ssDNA lengths (i.e., 10-16 base pairs, bps), and sequences (poly thymine, mixed bps). The resulting compact ssDNA-QDs displayed a fluorescence quenching efficiency of up to 89% by hybridization with complementary ssDNA-AuNPs. Furthermore, ssDNA-QDs were successfully incorporated with higher-order DNA origami nanostructure templates. Thus, this approach, combining PC3 passivation with click chemistry, generates ssDNA-PC3-QDs that enable emergent QD properties in DNA-based devices and applications.

Nanoparticle packing within block copolymer micelles prepared by the interfacial instability method.

The interfacial instability method has emerged as a viable approach for encapsulating high concentrations of nanoparticles (NPs) within morphologically diverse micelles. In this method, transient interfacial instabilities at the surface of an emulsion droplet guide self-assembly of block co-polymers and NP encapsulants. Although used by many groups, there are no systematic investigations exploring the relationship between NP properties and micelle morphology. Here, the effect of quantum dot (QD) and superparamagnetic iron oxide NP (SPION) concentration on the shape, size, and surface deformation of initially spherical poly(styrene-b-ethylene oxide) (PS-b-PEO) micelles was examined. Multi-NP encapsulation and uniform dispersion within micelles was obtained even at low NP concentrations. Increasing NP concentration initially resulted in larger numbers of elongated micelles and cylinders with tightly-controlled diameters smaller than those of spherical micelles. Beyond a critical NP concentration, micelle formation was suppressed; the dominant morphology became densely-loaded NP structures that were coated with polymer and exhibited increased polydispersity. Transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS) revealed that NPs in densely-loaded structures can be well-ordered, with packing volume fractions of up to 24%. These effects were enhanced in magnetic composites, possibly by dipole interactions. Mechanisms governing phase transitions triggered by NP loading in the interfacial instability process were proposed. The current study helps establish and elucidate the active role played by NPs in directing block copolymer assembly in the interfacial instability process, and provides important guiding principles for the use of this approach in generating NP-loaded block copolymer composites.

Scalable, semicontinuous production of micelles encapsulating nanoparticles via electrospray.

Nanoparticle encapsulation within micelles has been demonstrated as a versatile platform for creating water-soluble nanocomposites. However, in contrast to typical micelle encapsulants, such as small molecule drugs and proteins, nanoparticles are substantially larger, which creates significant challenges in micelle synthesis, especially at large scale. Here, we describe a new nanocomposite synthesis method that combines electrospray, a top-down, continuous manufacturing technology currently used for polymer microparticle fabrication, with bottom-up micellar self-assembly to yield a scalable, semicontinuous micelle synthesis method: i.e., micellar electrospray. Empty micelles and micellar nanocomposites containing quantum dots (QDs), superparamagnetic iron oxide nanoparticles (SPIONs), and their combination were produced using micellar electrospray with a 30-fold increase in yield by weight over batch methods. Particles were characterized using dynamic light scattering, transmission electron microscopy, and scanning mobility particle sizing, with remarkable agreement between methods, which indicated size distributions with variations of as little as ~5%. In addition, new methodologies for qualitatively evaluating nanoparticle loading in the resultant micelles are presented. Micellar electrospray is a broad, scalable nanomanufacturing approach that should be easily adapted to virtually any hydrophobic molecule or nanoparticle with a diameter smaller than the micelle core, potentially enabling synthesis of a vast array of nanocomposites and self-assembled nanostructures.

Micelle-templated composite quantum dots for super-resolution imaging.

Quantum dots (QDs) have tremendous potential for biomedical imaging, including super-resolution techniques that permit imaging below the diffraction limit. However, most QDs are produced via organic methods, and hence require surface treatment to render them water-soluble for biological applications. Previously, we reported a micelle-templating method that yields nanocomposites containing multiple core/shell ZnS-CdSe QDs within the same nanocarrier, increasing overall particle brightness and virtually eliminating QD blinking. Here, this technique is extended to the encapsulation of Mn-doped ZnSe QDs (Mn-ZnSe QDs), which have potential applications in super-resolution imaging as a result of the introduction of Mn(2+) dopant energy levels. The size, shape and fluorescence characteristics of these doped QD-micelles were compared to those of micelles created using core/shell ZnS-CdSe QDs (ZnS-CdSe QD-micelles). Additionally, the stability of both types of particles to photo-oxidation was investigated. Compared to commercial QDs, micelle-templated QDs demonstrated superior fluorescence intensity, higher signal-to-noise ratios, and greater stability against photo-oxidization,while reducing blinking. Additionally, the fluorescence of doped QD-micelles could be modulated from a bright 'on' state to a dark 'off' state, with a modulation depth of up to 76%, suggesting the potential of doped QD-micelles for applications in super-resolution imaging.

Cooperative control of a DNA origami force sensor.

Biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces, but they are not optimized for studies in constrained environments as they require attachment to micron-scale probes such as beads or cantilevers. Nanomechanical devices are a promising alternative, but this requires versatile designs that can be tuned to respond to a wide range of forces. We investigate the properties of a nanoscale force sensitive DNA origami device which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, has a binary (open or closed) response to an applied force by undergoing a reversible structural transition. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). The DNA oligonucleotide design parameters also strongly influence the efficiency of resetting the initial state, with higher stability devices (≳10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show the opening force is tunable in real time by adding a single DNA oligonucleotide. These results establish the potential of the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.

Cooperative control of a DNA origami force sensor.

Most biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces. These techniques, however, are not optimized for studies in constrained or crowded environments as they typically require micron-scale beads in the case of magnetic or optical tweezers, or direct attachment to a cantilever in the case of atomic force microscopy. We implement a nanoscale force-sensing device using a DNA origami which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, functions as a binary (open or closed) force sensor that undergoes a structural transition under an external force. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). This actuation of the NanoDyn is reversible and the design parameters strongly influence the efficiency of resetting the initial state, with higher stability devices (≳10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show that the opening force can be adjusted in real time by the addition of a single DNA oligonucleotide. These results establish the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.

Evaluating glioblastoma tumour sphere growth and migration in interaction with astrocytes using 3D collagen-hyaluronic acid hydrogels.

Glioblastoma (GB) is an astrocytic brain tumour with a low survival rate, partly because of its highly invasive nature. The GB tumour microenvironment (TME) includes its extracellular matrix (ECM), a variety of brain cell types, unique anatomical structures, and local mechanical cues. As such, researchers have attempted to create biomaterials and culture models that mimic features of TME complexity. Hydrogel materials have been particularly popular because they enable 3D cell culture and mimic TME mechanical properites and chemical composition. Here, we used a 3D collagen I-hyaluronic acid hydrogel material to explore interactions between GB cells and astrocytes, the normal cell type from which GB likely derives. We demonstrate three different spheroid culture configurations, including GB multi-spheres (i.e., GB and astrocyte cells in spheroid co-culture), GB-only mono-spheres cultured with astrocyte-conditioned media, and GB-only mono-spheres cultured with dispersed live or fixed astrocytes. Using U87 and LN229 GB cell lines and primary human astrocytes, we investigated material and experiment variability. We then used time-lapse fluorescence microscopy to measure invasive potential by characterizing the sphere size, migration capacity, and weight-averaged migration distance in these hydrogels. Finally, we developed methods to extract RNA for gene expression analysis from cells cultured in hydrogels. U87 and LN229 cells displayed different migration behaviors. U87 migration occurred primarily as single cells and was reduced with higher numbers of astrocytes in both multi-sphere and mono-sphere plus dispersed astrocyte cultures. In contrast, LN229 migration exhibited features of collective migration and was increased in monosphere plus dispersed astrocyte cultures. Gene expression studies indicated that the most differentially expressed genes in these co-cultures were CA9, HLA-DQA1, TMPRSS2, FPR1, OAS2, and KLRD1. Most differentially expressed genes were related to immune response, inflammation, and cytokine signalling, with greater influence on U87 than LN229. These data show that 3D in vitro hydrogel co-culture models can be used to reveal cell line specific differences in migration and to study differential GB-astrocyte crosstalk.

DNA-caged nanoparticles via electrostatic self-assembly.

DNA-modified nanoparticles enable DNA sensing and therapeutics in nanomedicine and are also crucial for nanoparticle self-assembly with DNA-based materials. However, methods to conjugate DNA to nanoparticle surfaces are limited, inefficient, and lack control. Inspired by DNA tile nanotechnology, we demonstrate a new approach to nanoparticle modification based on electrostatic attraction between negatively charged DNA tiles and positively charged nanoparticles. This approach does not disrupt nanoparticle surfaces and leverages the programmability of DNA nanotechnology to control DNA presentation. We demonstrated this approach using a vareity of nanoparticles, including polymeric micelles, polystyrene beads, gold nanoparticles, and superparamagnetic iron oxide nanoparticles with sizes ranging from 5-20 nm in diameter. DNA cage formation was confirmed through transmission electron microscopy (TEM), neutralization of zeta potential, and a series of fluorescence experiments. DNA cages present "handle" sequences that can be used for reversible target attachment or self-assembly. Handle functionality was verified in solution, at the solid-liquid interface, and inside fixed cells, corresponding to applications in biosensing, DNA microarrays, and erasable immunocytochemistry. These experiments demonstrate the versatility of the electrostatic DNA caging approach and provide a new pathway to nanoparticle modification with DNA that will empower further applications of these materials in medicine and materials science.

Simultaneous, single particle, magnetization and size measurements of micron sized, magnetic particles.

Single particle magnetization and size measurements of micron and nano sized, magnetic particles were made using a previously described device referred to as Cell Tracking Velocimetry, CTV. Three types of commercially available, and commonly used, magnetic particles were studied in this report. While the CTV instrument provides individual particles measurements, the average magnetization and size measurements were found to have reasonable agreements with reported values from instruments which measure bulk values. In addition, the CTV instrument, using electromagnets, can also determine magnetization curves, which also proved to have reasonable agreement with other published studies. Given that magnetic separation and analysis technology is dependent on the quality of the magnetic particles used, studies such as this one using CTV provide not only average data, but also provides information with respect to the distribution of the properties such as magnetization and size. For example, the spread of the data in magnetic and settling velocities were found to be predominately due to the size distribution of the analyzed particles.

Alternating-color quantum dot nanocomposites for particle tracking.

Because of their extraordinary brightness and photostability, quantum dots (QDs) have tremendous potential for long-term, particle tracking in heterogeneous systems (e.g., living cells, microfluidic flow). However, one of their major limitations is blinking, an intermittent loss of fluorescence, characteristic of individual and small clusters of QDs, that interrupts particle tracking. Recently, several research groups have reported "nonblinking QDs". However, blinking is the primary method used to confirm nanoparticle aggregation status in situ, and single or small clusters of nanoparticles with continuous fluorescence emission are difficult to discern from large aggregates. Here, we describe a new class of quantum dot-based composite nanoparticles that solve these two seemingly irreconcilable problems by exhibiting near-continuous, alternating-color fluorescence, which permits aggregation status discrimination by observable color changes even during motion across the focal plane. These materials will greatly enhance particle tracking in cell biology, biophysics, and fluid mechanics.

A MagDot-Nanoconveyor Assay Detects and Isolates Molecular Biomarkers.

The ability to quickly analyze, separate, and manipulate multiple types of biomarkers from small sample volumes is a significant step toward personalized medicine.

One step closer to cancer nanomedicine.

High-throughput tool uncovers links between cell signaling and nanomaterial uptake.

Nanoparticles caged with DNA nanostructures.

Nanoparticles (NPs) offer many benefits in biotechnology because of their small size and unique properties. However, many applications require precise positioning of the NPs or biological targeting molecules on their surfaces. DNA cages constructed from DNA tile, origami, or wireframe nanostructures offer a promising path forward because of their simplicity and programmability that can be used to generate complex, dynamic 2D and 3D geometries. Such materials can be used to pattern DNA on NP surfaces and organize NPs into specific supramolecular structures. DNA-caged NPs can be implemented in biosensing and drug delivery applications with cavities precisely designed to encapsulate-specific biomolecules. Ultimately, such approaches provide a springboard for future DNA robot designs that will enable controlled interactions with biological systems.

Oxidation of quantum dots encapsulated in block copolymer micelles as a function of polymer terminal charge.

Most high-quality quantum dots (QDs) are synthesized in the organic phase, and are often coated with polymers for use in aqueous biological environments. QDs can exhibit fluorescence losses during phase transfer, but evaluating underlying mechanisms (e.g., oxidation, surface etching, loss of colloidal stability) can be challenging because of variation in synthesis methods. Here, fluorescence stability of QDs encapsulated in block co-polymer (BCP) micelles was investigated as a function of BCP terminal functionalization (i.e., -OH, -COOH, and -NH2 groups) and synthesis method (i.e., electrohydrodynamic emulsification-mediated selfassembly (EE-SA), sonication, and manual shaking). Fluorescence losses, fluorescence intensity, energy spectra, and surface composition were assessed using spectrofluorometry and cathodoluminescence spectroscopy (CL) with integrated X-ray photoemission spectroscopy (XPS). QDs passivated using charged BCPs exhibited 50-80% lower fluorescence intensity than those displaying neutral groups (e.g., -OH), which CL/XPS revealed to result from oxidation of surface Cd to CdO. Fluorescence losses were higher for processes with slow formation speed, but minimized in the presence of poly(vinyl alcohol) (PVA) surfactant. These data suggest slower BCP aggregation kinetics rather than electrostatic chain repulsion facilitated QD oxidation. Thus, polymer coating method and BCP structure influence QD oxidation during phase transfer and should be selected to maximize fast aggregation kinetics.

Simultaneous magnetic manipulation and fluorescent tracking of multiple individual hybrid nanostructures.

Controlled transport of multiple individual nanostructures is crucial for nanoassembly and nanodelivery but is challenging because of small particle size. Although atomic force microscopy and optical and magnetic tweezers can control single particles, it is extremely difficult to scale these technologies for multiple structures. Here, we demonstrate a "nano-conveyer-belt" technology that permits simultaneous transport and tracking of multiple individual nanospecies in a selected direction. The technology consists of two components: nanocontainers, which encapsulate the nanomaterials transported, and nanoconveyer arrays, which use magnetic force to manipulate individual or aggregate nanocontainers. This technology is extremely versatile. For example, nanocontainers encapsulate quantum dots or rods and superparamagnetic iron oxide nanoparticles in <100 nm nanocontainers, the smallest magnetic composites to have been simultaneously moved and optically tracked. Similarly, the nanoconveyers consist of patterned microdisks or zigzag nanowires, whose dimensions can be controlled through micropatterning. The nanoconveyer belt technology could impact multiple fields, including nanoassembly, biomechanics, nanomedicine, and nanofluidics.