Integrated laser ablation-dropletProbe-mass spectrometry for absolute drug quantitation, metabolite detection, and distribution in tissue.

RATIONALE: Spatially resolved and accurate quantitation of drug-related compounds in tissue is a much-needed capability in drug discovery research. Here, application of an integrated laser ablation-dropletProbe-mass spectrometry surface sampling system (LADP-MS) is reported, which achieved absolute quantitation of propranolol measured from <500 × 500 µm thin tissue samples. METHODS: Mouse liver and kidney thin tissue sections were coated with parylene C and analyzed for propranolol by a laser ablation/liquid extraction workflow. Non-coated adjacent sections were microdissected for validation and processed using standard bulk tissue extraction protocols. High-performance liquid chromatography with positive ion mode electrospray ionization tandem mass spectrometry was applied to detect the drug and its metabolites. RESULTS: Absolute propranolol concentration in ~500 × 500 µm tissue regions measured by the two methods agreed within ±8% and had a relative standard deviation within ±17%. Quantitation down to ~400 × 400 µm tissue regions was shown, and this resolution was also used for automated mapping of propranolol and phase II hydroxypropranolol glucuronide metabolites in kidney tissue. CONCLUSIONS: This study exemplifies the capabilities of integrated laser ablation-dropletProbe-mass spectrometry (LADP-MS) for high resolution absolute drug quantitation analysis of thin tissue sections. This capability will be valuable for applications needing to quantitatively understand the spatial distribution of small molecules in tissue.

Absolute quantitation of propranolol from 200-µm regions of mouse brain and liver thin tissues using laser ablation-dropletProbe-mass spectrometry.

RATIONALE: The ability to quantify drugs and metabolites in tissue with sub-mm resolution is a challenging but much needed capability in pharmaceutical research. To fill this void, a novel surface sampling approach combining laser ablation with the commercial dropletProbe automated liquid surface sampling system (LA-dropletProbe) was developed and is presented here. METHODS: Parylene C-coated 200 × 200 µm tissue regions of mouse brain and kidney thin tissue sections were analyzed for propranolol by laser ablation of tissue directly into a preformed liquid junction. Propranolol was detected by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) in positive electrospray ionization mode. Quantitation was achieved via application of a stable-isotope-labeled internal standard and an external calibration curve. RESULTS: The absolute concentrations of propranolol determined from 200 × 200 µm tissue regions were compared with the propranolol concentrations obtained from 2.3-mm-diameter tissue punches of adjacent, non-coated sections using standard bulk tissue extraction protocols followed by regular HPLC/MS/MS analysis. The average concentration of propranolol in both organs determined by the two employed methods agreed to within ±12%. Furthermore, the relative abundances of phase II hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous results. CONCLUSIONS: This work illustrates that depositing a thin layer of parylene C onto thin tissue prior to analysis, which seals the surface and prevents direct liquid extraction of the drug from the tissue, coupled to the novel LA-dropletProbe surface sampling system is a viable approach for sub-mm resolution quantitative drug distribution analysis.

Atomic Force Microscopy Thermally-Assisted Microsampling with Atmospheric Pressure Temperature Ramped Thermal Desorption/Ionization-Mass Spectrometry Analysis.

The use of atomic force microscopy controlled nanothermal analysis probes for reproducible spatially resolved thermally assisted sampling of micrometer-sized areas (ca. 11 × 17 µm wide × 2.4 µm deep) from relatively low number-average molecular weight (Mn < 3000) polydisperse thin films of poly(2-vinylpyridine) (P2VP) is presented. Following sampling, the nanothermal analysis probes were moved up from the surface and the probe temperature ramped to liberate the sampled materials into the gas phase for atmospheric pressure chemical ionization and mass spectrometric analysis. The procedure and mechanism for material pickup, the sampling reproducibility and sampling size are discussed, and the oligomer distribution information available from slow temperature ramps versus ballistic temperature jumps is presented. For the Mn = 970 P2VP, the Mn and polydispersity index determined from the mass spectrometric data were in line with both the label values from the sample supplier and the value calculated from the simple infusion of a solution of polymer into the commercial atmospheric pressure chemical ionization source on this mass spectrometer. With a P2VP sample of higher Mn (Mn = 2070 and 2970), intact oligomers were still observed (as high as m/z 2793 corresponding to the 26-mer), but a significant abundance of thermolysis products were also observed. In addition, the capability for confident identification of the individual oligomers by slowly ramping the probe temperature and collecting data-dependent tandem mass spectra was also demonstrated. The material type limits to the current sampling and analysis approach as well as possible improvements in nanothermal analysis probe design to enable smaller area sampling and to enable controlled temperature ramps beyond the present upper limit of about 415 °C are also discussed.

Hidden Area and Mechanical Nonlinearities in Freestanding Graphene.

We investigated the effect of out-of-plane crumpling on the mechanical response of graphene membranes. In our experiments, stress was applied to graphene membranes using pressurized gas while the strain state was monitored through two complementary techniques: interferometric profilometry and Raman spectroscopy. By comparing the data obtained through these two techniques, we determined the geometric hidden area which quantifies the crumpling strength. While the devices with hidden area ∼0% obeyed linear mechanics with biaxial stiffness 428±10 N/m, specimens with hidden area in the range 0.5%-1.0% were found to obey an anomalous nonlinear Hooke's law with an exponent ∼0.1.

Polymeric spatial resolution test patterns for mass spectrometry imaging using nano-thermal analysis with atomic force microscopy.

RATIONALE: As the spatial resolution of mass spectrometry imaging technologies has begun to reach into the nanometer regime, finding readily available or easily made resolution reference materials has become particularly challenging for molecular imaging purposes. This paper describes the fabrication, characterization and use of vertical line array polymeric spatial resolution test patterns for nano-thermal analysis/atomic force microscopy/mass spectrometry chemical imaging. METHODS: Test patterns of varied line width (0.7 or 1.0 µm) and spacing (0.7 or 1.0 µm) were created in an ~1-µm-thick poly(methyl methacrylate) thin film using electron beam lithography. The patterns were characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, atomic force microscopy topography and nano-thermal analysis/mass spectrometry imaging. RESULTS: The efficacy of these polymeric test patterns for the advancement of chemical imaging techniques was illustrated by their use to judge the spatial resolution improvement achieved by heating the ionization interface of the current instrument platform. The spatial resolution of the mass spectral chemical images was estimated to be 1.4 µm, based on the ability to statistically distinguish 0.7-µm-wide lines separated by 0.7-µm-wide spacings in those images when the interface cross was heated to 200°C. CONCLUSIONS: This work illustrates that e-beam lithography is a viable method to create spatial resolution test patterns in a thin film of high molecular weight polymer to allow unbiased judgment of intra-laboratory advancement and/or inter-laboratory comparison of instrument advances in nano-thermal analysis/atomic force microscopy/mass spectrometry chemical imaging. Published in 2017. This article is a U.S. Government work and is in the public domain in the USA.

A Comparison of Single-Molecule Emission in Aluminum and Gold Zero-Mode Waveguides.

The effect of gold and aluminum zero-mode waveguides (ZMWs) on the brightness of immobilized single emitters was characterized by probing fluorophores that absorb in the green and red regions of the visible spectrum. Aluminum ZMWs enhance the emission of Atto565 fluorophores upon green excitation, but they do not enhance the emission of Atto647N fluorophores upon red excitation. Gold ZMWs increase emission of both fluorophores with Atto647N showing enhancement that is threefold higher than that observed for Atto565. This work indicates that 200 nm gold ZMWs are better suited for single-molecule fluorescence studies in the red region of the visible spectrum, while aluminum appears more suited for the green region of the visible spectrum.

Dynamic defrosting on nanostructured superhydrophobic surfaces.

Water suspended on chilled superhydrophobic surfaces exhibits delayed freezing; however, the interdrop growth of frost through subcooled condensate forming on the surface seems unavoidable in humid environments. It is therefore of great practical importance to determine whether facile defrosting is possible on superhydrophobic surfaces. Here, we report that nanostructured superhydrophobic surfaces promote the growth of frost in a suspended Cassie state, enabling its dynamic removal upon partial melting at low tilt angles (<15°). The dynamic removal of the melting frost occurred in two stages: spontaneous dewetting followed by gravitational mobilization. This dynamic defrosting phenomenon is driven by the low contact angle hysteresis of the defrosted meltwater relative to frost on microstructured superhydrophobic surfaces, which forms in the impaled Wenzel state. Dynamic defrosting on nanostructured superhydrophobic surfaces minimizes the time, heat, and gravitational energy required to remove frost from the surface, and is of interest for a variety of systems in cold and humid environments.

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants.

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to plants. As exciting as these new tools are, they have yet to be widely applied. Nanomaterials fabricated on rigid substrate (backing) are particularly difficult to successfully apply to curved plant structures. This study describes the process for microfabricating vertically aligned carbon nanofiber arrays and transferring them from a rigid to a flexible substrate. We detail and demonstrate how these fibers (on either rigid or flexible substrates) can be used for transient transformation or dye (e.g., fluorescein) delivery to plants. We show how VACNFs can be transferred from rigid silicon substrate to a flexible SU-8 epoxy substrate to form flexible VACNF arrays. To overcome the hydrophobic nature of SU-8, fibers in the flexible film were coated with a thin silicon oxide layer (2-3 nm). To use these fibers for delivery to curved plant organs, we deposit a 1 µL droplet of dye or DNA solution on the fiber side of VACNF films, wait 10 min, place the films on the plant organ and employ a swab with a rolling motion to drive fibers into plant cells. With this method, we have achieved dye and DNA delivery in plant organs with curved surfaces.

Gold Ion Beam Milled Gold Zero-Mode Waveguides.

Zero-mode waveguides (ZMWs) are widely used in single molecule fluorescence microscopy for their enhancement of emitted light and the ability to study samples at physiological concentrations. ZMWs are typically produced using photo or electron beam lithography. We report a new method of ZMW production using focused ion beam (FIB) milling with gold ions. We demonstrate that ion-milled gold ZMWs with 200 nm apertures exhibit similar plasmon-enhanced fluorescence seen with ZMWs fabricated with traditional techniques such as electron beam lithography.

Photoluminescence Enhancement, Blinking Suppression, and Improved Biexciton Quantum Yield of Single Quantum Dots in Zero Mode Waveguides.

The capability of quantum dots to generate both single and multiexcitons can be harnessed for a wide variety of applications, including those that require high optical gain. Here, we use time-correlated photoluminescence (PL) spectroscopy to demonstrate that the isolation of single CdSeTe/ZnS core-shell, nanocrystal quantum dots (QDs) in Zero Mode Waveguides (ZMWs) leads to a significant modification in PL intensity, blinking dynamics, and biexciton behavior. QDs in aluminum ZMWs (AlZMWs) exhibited a 15-fold increase in biexciton emission, indicating a preferential enhancement of the biexciton radiative decay rate as compared to the single exciton rate. The increase in biexciton behavior was accompanied by a decrease in blinking events due to a shortening in the dark state residence time. These results indicate that plasmon mediated enhanced decay rates of QDs in AlZMWs lead to substantial changes in the photophysical properties of single quantum dots, including an increase in biexciton behavior.

Evaporation of squeezed water droplets between two parallel hydrophobic/superhydrophobic surfaces.

HYPOTHESIS: A liquid droplet is apt to be deformed within a compact space in various applications. The morphological change of a droplet and vapor accumulation in the confined space between two parallel surfaces with different gaps and surface wettability are expected to significantly affect the evaporation dynamics of the squeezed droplet therein. EXPERIMENTS: Here the evaporation dynamics of a squeezed droplet between two parallel hydrophobic/superhydrophobic surfaces are experimentally explored. By reducing the surface gap from 1000 µm to 400 µm, the evolution of contact angle, contact radius and volume of the evaporating droplet are measured. A diffusion-driven model based on a two-parameter ellipsoidal segment geometry is developed to predict the morphology and volume evolution of a squeezed droplet during evaporation. FINDINGS: Evaporation dynamics of a squeezed water droplet via the constant contact radius (CCR) mode, the constant contact angle (CCA) mode, or the mixed mode are experimentally observed. Confirmed by our ellipsoidal segment model, the evaporation of the squeezed droplet is significantly depressed with the decreasing surface gap, which is primarily attributed to vapor enrichment in a more confined geometry. A linear scaling law between droplet volume and evaporation time is unveiled, which is verified by a simplified cylindrical model.

Mixed metal zero-mode guides (ZMWs) for tunable fluorescence enhancement.

Zero-mode waveguides (ZMWs) are capable of modifying fluorescence emission through interactions with surface plasmon modes leading to either plasmon-enhanced fluorescence or quenching. Enhancement requires spectral overlap of the plasmon modes with the absorption or emission of the fluorophore. Thus, enhancement is limited to fluorophores in resonance with metals (e.g. Al, Au, Ag) used for ZMWs. The ability to tune interactions to match a wider range of fluorophores across the visible spectra would significantly extend the utility of ZMWs. We fabricated ZMWs composed of aluminum and gold individually and also in mixtures of three different ratios, (Al : Au; 75 : 25, 50 : 50, 25 : 75). We characterized the effect of mixed-metal ZMWs on single-molecule emission for a range fluorophores across the visible spectrum. Mixed metal ZMWs exhibited a shift in the spectral range where they exhibited the maximum fluorescence enhancement allowing us to match the emission of fluorophores that were nonresonant with single metal ZMWs. We also compared the effect of mixed-metal ZMWs on the photophysical properties of fluorescent molecules due to metal-molecule interactions. We quantified changes in fluorescence lifetimes and photostability that were dependent on the ratio of Au and Al. Tuning the enhancement properties of ZMWs by changing the ratio of Au and Al allowed us to match the fluorescence of fluorophores that emit in different regions of the visible spectrum.

Programmable Electrofluidics for Ionic Liquid Based Neuromorphic Platform.

Due to the limit in computing power arising from the Von Neumann bottleneck, computational devices are being developed that mimic neuro-biological processing in the brain by correlating the device characteristics with the synaptic weight of neurons. This platform combines ionic liquid gating and electrowetting for programmable placement/connectivity of the ionic liquid. In this platform, both short-term potentiation (STP) and long-term potentiation (LTP) are realized via electrostatic and electrochemical doping of the amorphous indium gallium zinc oxide (aIGZO), respectively, and pulsed bias measurements are demonstrated for lower power considerations. While compatible with resistive elements, we demonstrate a platform based on transitive amorphous indium gallium zinc oxide (aIGZO) pixel elements. Using a lithium based ionic liquid, we demonstrate both potentiation (decrease in device resistance) and depression (increase in device resistance), and propose a 2D platform array that would enable a much higher pixel count via Active Matrix electrowetting.

Self-Stabilizing Transpiration in Synthetic Leaves.

Over the past decade, synthetic trees have been engineered to mimic the transpiration cycle of natural plants, but the leaves are prone to dry out beneath a critical relative humidity. Here, we create large-area synthetic leaves whose transpiration process is remarkably stable over a wide range of humidities, even without synthetic stomatal chambers atop the nanopores of the leaf. While the water menisci cannot initially withstand the Kelvin stress of the subsaturated air, they self-stabilized by locally concentrating vapor within the top layers of nanopores that have dried up. Transpiration rates were found to vary nonmonotonically with the ambient humidity because of the tradeoff of dry air increasing the retreat length of the menisci. It is our hope that these findings will encourage the development of large-area synthetic trees that exhibit excellent stability and high throughput for water-harvesting applications.

Strain tolerance of two-dimensional crystal growth on curved surfaces.

Two-dimensional (2D) crystal growth over substrate features is fundamentally guided by the Gauss-Bonnet theorem, which mandates that rigid, planar crystals cannot conform to surfaces with nonzero Gaussian curvature. Here, we reveal how topographic curvature of lithographically designed substrate features govern the strain and growth dynamics of triangular WS2 monolayer single crystals. Single crystals grow conformally without strain over deep trenches and other features with zero Gaussian curvature; however, features with nonzero Gaussian curvature can easily impart sufficient strain to initiate grain boundaries and fractured growth in different directions. Within a strain-tolerant regime, however, triangular single crystals can accommodate considerable (<1.1%) localized strain exerted by surface features that shift the bandgap up to 150 meV. Within this regime, the crystal growth accelerates in specific directions, which we describe using a growth model. These results present a previously unexplored strategy to strain-engineer the growth directions and optoelectronic properties of 2D crystals.

Passive Antifrosting Surfaces Using Microscopic Ice Patterns.

Despite exceptional recent advances in tailoring the wettability of surfaces, to date, no engineered surface can passively suppress the in-plane growth of frost that invariably occurs in humid, subfreezing environments. Here, we show that up to 90% of a surface can exhibit passive antifrosting by using chemical or physical wettability patterns to template "ice stripes" across the surface. As ice exhibits a depressed vapor pressure relative to liquid water, these sacrificial ice stripes siphon the supersaturated water vapor to keep the intermediate surface areas dry from dew and frost. Further, we show that when these sacrificial ice stripes are elevated atop microfins, they diffusively coarsen in a suspended state above the surface. The suspended state of the coarsening ice results in a diffusive growth rate an order of magnitude slower than frost coarsening directly on a solid substrate and should also minimize its adhesive strength to the surface.

Tuning Superhydrophobic Nanostructures To Enhance Jumping-Droplet Condensation.

It was recently discovered that condensation growing on a nanostructured superhydrophobic surface can spontaneously jump off the surface, triggered by naturally occurring coalescence events. Many reports have observed that droplets must grow to a size of order 10 µm before jumping is enabled upon coalescence; however, it remains unknown how the critical jumping size relates to the topography of the underlying nanostructure. Here, we characterize the dynamic behavior of condensation growing on six different superhydrophobic nanostructures, where the topography of the nanopillars was systematically varied. The critical jumping diameter was observed to be highly dependent upon the height, diameter, and pitch of the nanopillars: tall and slender nanopillars promoted 2 µm jumping droplets, whereas short and stout nanopillars increased the critical size to over 20 µm. The topology of each surface is successfully correlated to the critical jumping diameter by constructing an energetic model that predicts how large a nucleating embryo needs to grow before it can inflate into the air with an apparent contact angle large enough for jumping. By extending our model to consider any possible surface, it is revealed that properly designed nanostructures should enable nanometric jumping droplets, which would further enhance jumping-droplet condensers for heat transfer, antifogging, and antifrosting applications.

Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices.

The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor. We observed single ligands binding to EGFRs, allowing us to quantify the ligand turnover in real time. These results demonstrate that this nanoaperture-coupled microfluidic device allows for the spatial isolation of individual membrane proteins while maintaining them in their cellular environment, providing the capability to monitor single-ligand binding events while maintaining receptors in their physiological environment. These methods should be applicable to a wide range of membrane proteins.

Tilt Grain Boundary Topology Induced by Substrate Topography.

Synthesis of two-dimensional (2D) crystals is a topic of great current interest, since their chemical makeup, electronic, mechanical, catalytic, and optical properties are so diverse. A universal challenge, however, is the generally random formation of defects caused by various growth factors on flat surfaces. Here we show through theoretical analysis and experimental demonstration that nonplanar, curved-topography substrates permit the intentional and controllable creation of topological defects within 2D materials. We augment a common phase-field method by adding a geometric phase to track the crystal misorientation on a curved surface and to detect the formation of grain boundaries, especially when a growing monocrystal "catches its own tail" on a nontrivial topographical feature. It is specifically illustrated by simulated growth of a trigonal symmetry crystal on a conical-planar substrate, to match the experimental synthesis of WS2 on silicon template, with satisfactory and in some cases remarkable agreement of theory predictions and experimental evidence.

Transport and dispersion of nanoparticles in periodic nanopost arrays.

Nanoparticles transported through highly confined porous media exhibit faster breakthrough than small molecule tracers. Despite important technological applications in advanced materials, human health, energy, and environment, the microscale mechanisms leading to early breakthrough have not been identified. Here, we measure dispersion of nanoparticles at the single-particle scale in regular arrays of nanoposts and show that for highly confined flows of dilute suspensions of nanoparticles the longitudinal and transverse velocities exhibit distinct scaling behaviors. The distributions of transverse particle velocities become narrower and more non-Gaussian when the particles are strongly confined. As a result, the transverse dispersion of highly confined nanoparticles at low Péclet numbers is significantly less important than longitudinal dispersion, leading to early breakthrough. This finding suggests a fundamental mechanism by which to control dispersion and thereby improve efficacy of nanoparticles applied for advanced polymer nanocomposites, drug delivery, hydrocarbon production, and environmental remediation.