Conformational Changes of Immobilized Polythymine due to External Stressors Studied with Temperature-Controlled Electrochemical Microdevices.

Conformational changes of single-stranded DNA (ssDNA) play an important role in a DNA strand's ability to bind to target ligands. A variety of factors can influence conformation, including temperature, ionic strength, pH, buffer cation valency, strand length, and sequence. To better understand the effects of these factors on immobilized DNA structures, we employ temperature-controlled electrochemical microsensors to study the effects of salt concentration and temperature variation on the conformation and motion of polythymine (polyT) strands of varying lengths (10, 20, 50 nucleotides). PolyT strands were tethered to a gold working electrode at the proximal end through a thiol linker via covalent bonding between the Au electrode and sulfur link, which can tend to decompose between a temperature range of 60 and 90 °C. The strands were also modified with an electrochemically active methylene blue (MB) moiety at the distal end. Electron transfer (eT) was measured by square wave voltammetry (SWV) and used to infer information pertaining to the average distance between the MB and the working electrode. We observe changes in DNA flexibility due to varying ionic strength, while the effects of increased DNA thermal motion are tracked for elevated temperatures. This work elucidates the behavior of ssDNA in the presence of a phosphate-buffered saline at NaCl concentrations ranging from 20 to 1000 mmol/L through a temperature range of 10-50 °C in 1° increments, well below the decomposition temperature range. The results lay the groundwork for studies on more complex DNA strands in conjunction with different chemical and physical conditions.

Miniaturized nanohole array based plasmonic sensor for the detection of acetone and ethanol with insights into the kinetics of adsorptive plasmonic sensing.

The present work demonstrates development of a miniaturized plasmonic platform comprised of a Au nanohole array (NHA) on a Si/Si3N4 substrate. Plasmonic responses of the NHA platform, which is coated with Cu-benzenetricarboxylate metal organic framework (MOF), are found to be promising even towards 500 nmol mol-1 (ppb) of acetone or ethanol vapors at room temperature. The sensing characteristics are further investigated by varying the operating temperature (296 K to 318 K) of the sensor and the concentrations of vapors (500 nmol mol-1 to 320 µmol mol-1). The plasmonic responses for the sensors are correlated with the adsorption of vapors on the MOF surface and modeled in accordance to Langmuir-type adsorption. Kinetic parameters are estimated for the adsorption of fixed concentrations of acetone and ethanol vapors within the studied operating temperature range. The linear variation of characteristic response time constants with the operating temperature provides Arrhenius activation energies for the adsorption of acetone and ethanol vapors. The comparatively lower activation energy estimated for the adsorption of ethanol results in faster and more sensitive response of the sensor towards that analyte. The plasmonic sensor for the detection of nmol mol-1 level acetone and ethanol vapors at room temperature along with the kinetic correlation on plasmonic response with the adsorption of the analytes described herein offer new insights to existing reports on surface modification and plasmonic detection.

Ligand-Based Stability Changes in Duplex DNA Measured with a Microscale Electrochemical Platform.

Development of technologies for rapid screening of DNA secondary structure thermal stability and the effects on stability for binding of small molecule drugs is important to the drug discovery process. In this report, we describe the capabilities of an electrochemical, microdevice-based approach for determining the melting temperatures (Tm) of electrode-bound duplex DNA structures. We also highlight new features of the technology that are compatible with array development and adaptation for high-throughput screening. As a foundational study to exhibit device performance and capabilities, melting-curve analyses were performed on 12-mer DNA duplexes in the presence/absence of two binding ligands: diminazene aceturate (DMZ) and proflavine. By measuring electrochemical current as a function of temperature, our measurement platform has the ability to determine the effect of binding ligands on Tm values with high signal-to-noise ratios and good reproducibility. We also demonstrate that heating our three-electrode cell with either an embedded microheater or a thermoelectric module produces similar results. The ΔTm values we report show the stabilizing ability of DMZ and proflavine when bound to duplex DNA structures. These initial proof-of-concept studies highlight the operating characteristics of the microdevice platform and the potential for future application toward other immobilized samples.

An interlaboratory comparison of sizing and counting of subvisible particles mimicking protein aggregates.

Accurate counting and sizing of protein particles has been limited by discrepancies of counts obtained by different methods. To understand the bias and repeatability of techniques in common use in the biopharmaceutical community, the National Institute of Standards and Technology has conducted an interlaboratory comparison for sizing and counting subvisible particles from 1 to 25 µm. Twenty-three laboratories from industry, government, and academic institutions participated. The circulated samples consisted of a polydisperse suspension of abraded ethylene tetrafluoroethylene particles, which closely mimic the optical contrast and morphology of protein particles. For restricted data sets, agreement between data sets was reasonably good: relative standard deviations (RSDs) of approximately 25% for light obscuration counts with lower diameter limits from 1 to 5 µm, and approximately 30% for flow imaging with specified manufacturer and instrument setting. RSDs of the reported counts for unrestricted data sets were approximately 50% for both light obscuration and flow imaging. Differences between instrument manufacturers were not statistically significant for light obscuration but were significant for flow imaging. We also report a method for accounting for differences in the reported diameter for flow imaging and electrical sensing zone techniques; the method worked well for diameters greater than 15 µm.

Particle shape effects on subvisible particle sizing measurements.

Particle analysis tools for the subvisible (<100 µm) size range, such as light obscuration, flow imaging (FI), and electrical sensing zone (ESZ), often produce results that do not agree with one another, despite their general agreement when characterizing polystyrene latex spheres of different sizes. To include the effect of shape in comparison studies, we have used the methods of photolithography to create rods and disks. Although the rods are highly monodisperse, the instruments produce broadened peaks and report mean size parameters that are different for different instruments. We have fabricated a microfluidic device that simultaneously performs ESZ and FI measurements on each particle to elucidate the causes of discrepancies and broadening. Alignment of the rods with flow causes an oversizing by FI and undersizing by ESZ. FI also oversizes rods because of the incorrect edge definition that results from diffraction and imperfect focus. We present an improved correction algorithm for this effect that reduces discrepancies for rod-shaped particles. Tumbling of particles is observed in the microfluidic ESZ/FI and results in particle oversizing and breadth of size distribution for the monodisperse rods.

Demonstration of fast and accurate discrimination and quantification of chemically similar species utilizing a single cross-selective chemiresistor.

Performance characteristics of gas-phase microsensors will determine the ultimate utility of these devices for a wide range of chemical monitoring applications. Commonly employed chemiresistor elements are quite sensitive to selected analytes, and relatively new methods have increased the selectivity to specific compounds, even in the presence of interfering species. Here, we have focused on determining whether purposefully driven temperature modulation can produce faster sensor-response characteristics, which could enable measurements for a broader range of applications involving dynamic compositional analysis. We investigated the response speed of a single chemiresitive In2O3 microhotplate sensor to four analytes (methanol, ethanol, acetone, 2-butanone) by systematically varying the oscillating frequency (semicycle periods of 20-120 ms) of a bilevel temperature cycle applied to the sensing element. It was determined that the fastest response (≈ 9 s), as indicated by a 98% signal-change metric, occurred for a period of 30 ms and that responses under such modulation were dramatically faster than for isothermal operation of the same device (>300 s). Rapid modulation between 150 and 450 °C exerts kinetic control over transient processes, including adsorption, desorption, diffusion, and reaction phenomena, which are important for charge transfer occurring in transduction processes and the observed response times. We also demonstrate that the fastest operation is accompanied by excellent discrimination within a challenging 16-category recognition problem (consisting of the four analytes at four separate concentrations). This critical finding demonstrates that both speed and high discriminatory capabilities can be realized through temperature modulation.

Porous tin oxide nanostructured microspheres for sensor applications.

We have sought to enhance the sensitivity of conductometric gas microsensors through the design and fabrication of porous, three-dimensional tin oxide nanoparticle structures. Electrostatically controlled layer-by-layer processing in aqueous solutions was used to decorate sacrificial latex microspheres with Sb:SnO2 nanoparticles. To evaluate their sensing performance, these structures were then deposited as films, via micropipetting, on MEMS micro-hot-plate platforms with interdigitated electrodes. Prior to gas testing, rapid heating of the micro-hot-plates was used to remove the sacrificial latex templates, thereby revealing a 3-D structure composed of interconnected spherical tin oxide nanoparticle shells with porous ultrathin walls. Changes in film conductance, caused by exposure to test gases (methanol, carbon monoxide, benzene, water) in a dry air background, were measured at different temperatures. Hollow nanoparticle microsphere films exhibited partial selectivity for these different gases, good dynamic range at different temperatures and gas concentrations, and good repeatability and stability over long runs. These films also yielded approximately 3-fold and 5-fold increases in sensitivity to methanol when compared to SnO2 polycrystalline chemical vapor deposition films and Sb:SnO2 microporous nanoparticle films, respectively. Gains in sensitivity are attributed to the multiscale porous architecture of the hollow microsphere films. This architecture promotes gas diffusion and increases the active surface area.