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.

Multimodal in vivo recording using transparent graphene microelectrodes illuminates spatiotemporal seizure dynamics at the microscale.

Neurological disorders such as epilepsy arise from disrupted brain networks. Our capacity to treat these disorders is limited by our inability to map these networks at sufficient temporal and spatial scales to target interventions. Current best techniques either sample broad areas at low temporal resolution (e.g. calcium imaging) or record from discrete regions at high temporal resolution (e.g. electrophysiology). This limitation hampers our ability to understand and intervene in aberrations of network dynamics. Here we present a technique to map the onset and spatiotemporal spread of acute epileptic seizures in vivo by simultaneously recording high bandwidth microelectrocorticography and calcium fluorescence using transparent graphene microelectrode arrays. We integrate dynamic data features from both modalities using non-negative matrix factorization to identify sequential spatiotemporal patterns of seizure onset and evolution, revealing how the temporal progression of ictal electrophysiology is linked to the spatial evolution of the recruited seizure core. This integrated analysis of multimodal data reveals otherwise hidden state transitions in the spatial and temporal progression of acute seizures. The techniques demonstrated here may enable future targeted therapeutic interventions and novel spatially embedded models of local circuit dynamics during seizure onset and evolution.

All-Electronic Quantification of Neuropeptide-Receptor Interaction Using a Bias-Free Functionalized Graphene Microelectrode.

Opioid neuropeptides play a significant role in pain perception, appetite regulation, sleep, memory, and learning. Advances in understanding of opioid peptide physiology are held back by the lack of methodologies for real-time quantification of affinities and kinetics of the opioid neuropeptide-receptor interaction at levels typical of endogenous secretion (<50 pM) in biosolutions with physiological ionic strength. To address this challenge, we developed all-electronic opioid-neuropeptide biosensors based on graphene microelectrodes functionalized with a computationally redesigned water-soluble µ-opioid receptor. We used the functionalized microelectrode in a bias-free charge measurement configuration to measure the binding kinetics and equilibrium binding properties of the engineered receptor with [d-Ala2, N-MePhe4, Gly-ol]-enkephalin and ß-endorphin at picomolar levels in real time.

Structural-functional analysis of engineered protein-nanoparticle assemblies using graphene microelectrodes.

The characterization of protein-nanoparticle assemblies in solution remains a challenge. We demonstrate a technique based on a graphene microelectrode for structural-functional analysis of model systems composed of nanoparticles enclosed in open-pore and closed-pore ferritin molecules. The method readily resolves the difference in accessibility of the enclosed nanoparticle for charge transfer and offers the prospect for quantitative analysis of pore-mediated transport, while shedding light on the spatial orientation of the protein subunits on the nanoparticle surface, faster and with higher sensitivity than conventional catalysis methods.

An Aptamer-Based Biosensor for the Azole Class of Antifungal Drugs.

This technical report describes the development of an aptamer for sensing azole antifungal drugs during therapeutic drug monitoring. Modified synthetic evolution of ligands through exponential enrichment (SELEX) was used to discover a DNA aptamer recognizing azole class antifungal drugs. This aptamer undergoes a secondary structural change upon binding to its target molecule, as shown through fluorescence anisotropy-based binding measurements. Experiments using circular dichroism spectroscopy revealed a unique G-quadruplex structure that was essential and specific for binding to the azole antifungal target. Aptamer-functionalized graphene field effect transistor (GFET) devices were created and used to measure the strength of binding of azole antifungals to this surface. In total, this aptamer and the supporting sensing platform provide a valuable tool for therapeutic drug monitoring of patients with invasive fungal infections. IMPORTANCE We have developed the first aptamer directed toward the azole class of antifungal drugs and a functional biosensor for these drugs. This aptamer has a unique secondary structure that allows it to bind to highly hydrophobic drugs. The aptamer works as a capture component of a graphene field effect transistor device. These devices can provide a quick and easy assay for determining drug concentrations. These will be useful for therapeutic drug monitoring of azole antifungal drugs, which is necessary to deal with the complex drug dosage profiles.

pH Sensing Properties of Flexible, Bias-Free Graphene Microelectrodes in Complex Fluids: From Phosphate Buffer Solution to Human Serum.

Advances in techniques for monitoring pH in complex fluids can have a significant impact on analytical and biomedical applications. This study develops flexible graphene microelectrodes (GEs) for rapid (<5 s), very-low-power (femtowatt) detection of the pH of complex biofluids by measuring real-time Faradaic charge transfer between the GE and a solution at zero electrical bias. For an idealized sample of phosphate buffer solution (PBS), the Faradaic current is varied monotonically and systematically with the pH, with a resolution of ≈0.2 pH unit. The current-pH dependence is well described by a hybrid analytical-computational model, where the electric double layer derives from an intrinsic, pH-independent (positive) charge associated with the graphene-water interface and ionizable (negative) charged groups. For ferritin solution, the relative Faradaic current, defined as the difference between the measured current response and a baseline response due to PBS, shows a strong signal associated with ferritin disassembly and the release of ferric ions at pH ≈2.0. For samples of human serum, the Faradaic current shows a reproducible rapid (<20 s) response to pH. By combining the Faradaic current and real-time current variation, the methodology is potentially suitable for use to detect tumor-induced changes in extracellular pH.

Scalable Production of High-Sensitivity, Label-Free DNA Biosensors Based on Back-Gated Graphene Field Effect Transistors.

Scalable production of all-electronic DNA biosensors with high sensitivity and selectivity is a critical enabling step for research and applications associated with detection of DNA hybridization. We have developed a scalable and very reproducible (>90% yield) fabrication process for label-free DNA biosensors based upon graphene field effect transistors (GFETs) functionalized with single-stranded probe DNA. The shift of the GFET sensor Dirac point voltage varied systematically with the concentration of target DNA. The biosensors demonstrated a broad analytical range and limit of detection of 1 fM for 60-mer DNA oligonucleotide. In control experiments with mismatched DNA oligomers, the impact of the mismatch position on the DNA hybridization strength was confirmed. This class of highly sensitive DNA biosensors offers the prospect of detection of DNA hybridization and sequencing in a rapid, inexpensive, and accurate way.