Electrically Guided DNA Immobilization and Multiplexed DNA Detection with Nanoporous Gold Electrodes.

Molecular diagnostics have significantly advanced the early detection of diseases, where the electrochemical sensing of biomarkers (e.g., DNA, RNA, proteins) using multiple electrode arrays (MEAs) has shown considerable promise. Nanostructuring the electrode surface results in higher surface coverage of capture probes and more favorable orientation, as well as transport phenomena unique to nanoscale, ultimately leading to enhanced sensor performance. The central goal of this study is to investigate the influence of electrode nanostructure on electrically-guided immobilization of DNA probes for nucleic acid detection in a multiplexed format. To that end, we used nanoporous gold (np-Au) electrodes that reduced the limit of detection (LOD) for DNA targets by two orders of magnitude compared to their planar counterparts, where the LOD was further improved by an additional order of magnitude after reducing the electrode diameter. The reduced electrode diameter also made it possible to create a np-Au MEA encapsulated in a microfluidic channel. The electro-grafting reduced the necessary incubation time to immobilize DNA probes into the porous electrodes down to 10 min (25-fold reduction compared to passive immobilization) and allowed for grafting a different DNA probe sequence onto each electrode in the array. The resulting platform was successfully used for the multiplexed detection of three different biomarker genes relevant to breast cancer diagnosis

A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation.

BackgroundInteractions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system. In vitro astrocyte and microglia cultures are powerful tools to study specific molecular pathways involved in neuroinflammation; however, in order to better understand the influence of cellular crosstalk on neuroinflammation, new multicellular culture models are required.MethodsPrimary cortical cells taken from neonatal rats were cultured in a serum-free "tri-culture" medium formulated to support neurons, astrocytes, and microglia, or a "co-culture" medium formulated to support only neurons and astrocytes. Caspase 3/7 activity and morphological changes were used to quantify the response of the two culture types to different neuroinflammatory stimuli mimicking sterile bacterial infection (lipopolysaccharide (LPS) exposure), mechanical injury (scratch), and seizure activity (glutamate-induced excitotoxicity). The secreted cytokine profile of control and LPS-exposed co- and tri-cultures were also compared.ResultsThe tri-culture maintained a physiologically relevant representation of neurons, astrocytes, and microglia for 14 days in vitro, while the co-cultures maintained a similar population of neurons and astrocytes, but lacked microglia. The continuous presence of microglia did not negatively impact the overall health of the neurons in the tri-culture, which showed reduced caspase 3/7 activity and similar neurite outgrowth as the co-cultures, along with an increase in the microglia-secreted neurotrophic factor IGF-1 and a significantly reduced concentration of CX3CL1 in the conditioned media. LPS-exposed tri-cultures showed significant astrocyte hypertrophy, increase in caspase 3/7 activity, and the secretion of a number of pro-inflammatory cytokines (e.g., TNF, IL-1α, IL-1β, and IL-6), none of which were observed in LPS-exposed co-cultures. Following mechanical trauma, the tri-culture showed increased caspase 3/7 activity, as compared to the co-culture, along with increased astrocyte migration towards the source of injury. Finally, the microglia in the tri-culture played a significant neuroprotective role during glutamate-induced excitotoxicity, with significantly reduced neuron loss and astrocyte hypertrophy in the tri-culture.ConclusionsThe tri-culture consisting of neurons, astrocytes, and microglia more faithfully mimics in vivo neuroinflammatory responses than standard mono- and co-cultures. This tri-culture can be a useful tool to study neuroinflammation in vitro with improved accuracy in predicting in vivo neuroinflammatory phenomena

Simulated surface diffusion in nanoporous gold and its dependence on surface curvature

The morphological evolution of nanoporous gold is generally believed to be governed by surface diffusion. This work specifically explores the dependence of mass transport by surface diffusion on the curvature of a gold surface. The surface diffusivity is estimated by molecular dynamics simulations for a variety of surfaces of constant mean curvature, eliminating any chemical potential gradients and allowing the possible dependence of the surface diffusivity on mean curvature to be isolated. The apparent surface diffusivity is found to have an activation energy of ~0.74 eV with a weak dependence on curvature, but is consistent with the values reported in the literature. The apparent concentration of mobile surface atoms is found to be highly variable, having an Arrhenius dependence on temperature with an activation energy that also has a weak curvature dependence. These activation energies depend on curvature in such a way that the rate of mass transport by surface diffusion is nearly independent of curvature, but with a higher activation energy of ~1.01 eV. The curvature dependencies of the apparent surface diffusivity and concentration of mobile surface atoms is believed to be related to the expected lifetime of a mobile surface atom, and has the practical consequence that a simulation study that does not account for this finite lifetime could underestimate the activation energy for mass transport via surface diffusion by ~0.27 eV.Comment: 14 pages, 12 figure

Configurable microfluidic platform for investigating therapeutic delivery from biomedical device coatings.

Advanced biomedical device coatings have shown significant promise in delivery of therapeutics (e.g., small-molecule drugs, proteins) for a wide range of medical interventions ranging from targeted cancer therapy to management of atherosclerosis. In order to accelerate the development of such coatings, there is a need for tools to investigate the loading capacity and release kinetics with high temporal resolution and in a variety of physiological conditions. To address this need, we report a microfluidic platform, where the coating on a substrate can be mounted onto the microchannel and the device can be configured in two physiologically-relevant modes: (i) flow-mode allows for monitoring the release from the coating in contact with a liquid flowing at a specific rate, modeling the case of a drug-eluting stent. (ii) Static-mode, where the channel is filled with a stationary gel, mimics the case of drug-eluting brain implant. We demonstrate the utility of the platform with a fluorescein-loaded nanoporous gold coating and monitor in real-time the release kinetics both under deionized water infusion and an agarose gel-filled channel via fluorescence microscopy coupled to a LabVIEW-based interface

In situ electrical modulation and monitoring of nanoporous gold morphology.

The ability to fine-tune feature size in nanostructured thin films is critical, as many desirable properties of these materials are dictated by their nanostructure. Accordingly, there is a need for techniques that allow for modifying nanostructure while monitoring the morphological changes in situ. Here, we demonstrate a closed-loop electro-annealing system which enables in situ monitoring of morphology evolution in sub-micron nanoporous gold (np-Au) thin films. Np-Au is produced by a microfabrication-compatible self-assembly process that produces a network of interconnected ligaments with tunable diameter (10 s to 100 s of nanometers), making it a desirable material for numerous applications and fundamental studies alike. We specifically investigate the relationship between np-Au morphology (i.e., ligament diameter) and electrical resistance of the thin film. A strong correlation emerges between ligament size and electrical resistance, which puts forward resistance as an effective parameter for monitoring morphology evolution. Surprisingly, np-Au films with thicker ligaments lead to an increase in electrical resistance, which is unexpected since the extent of charge carrier scattering at the ligament surface should decrease with increasing ligament size. Further examination of np-Au morphology with high-resolution electron microscopy revealed grain growth on the ligaments in highly-annealed np-Au thin films. This suggests that grains act as scattering centers for charge carriers and this becomes the dominant mechanism in dictating electrical resistance in a percolated network of thin conductive ligaments

Chemically-Gated and Sustained Molecular Transport through Nanoporous Gold Thin Films in Biofouling Conditions.

Sustained release and replenishment of the drug depot are essential for the long-term functionality of implantable drug-delivery devices. This study demonstrates the use nanoporous gold (np-Au) thin films for in-plane transport of fluorescein (a small-molecule drug surrogate) over large (mm-scale) distances from a distal reservoir to the site of delivery, thereby establishing a constant flux of molecular release. In the absence of halides, the fluorescein transport is negligible due to a strong non-specific interaction of fluorescein with the pore walls. However, in the presence of physiologically relevant concentration of ions, halides preferentially adsorb onto the gold surface, minimizing the fluorescein-gold interactions and thus enabling in-plane fluorescein transport. In addition, the nanoporous film serves as an intrinsic size-exclusion matrix and allows for sustained release in biofouling conditions (dilute serum). The molecular release is reproducibly controlled by gating it in response to the presence of halides at the reservoir (source) and the release site (sink) without external triggers (e.g., electrical and mechanical)