PFAS Electroanalysis in Low-Oxygen River Water Using Electrogenerated Dioxygen.

Per- and polyfluoroalkyl substances (PFAS), nicknamed "forever chemicals" due to the strength of their carbon-fluorine bonds, are a class of potent micropollutants that cause deleterious health effects in mammals. The current state-of-the-art detection method requires the collection and transport of water samples to a centralized facility where chromatography and mass spectrometry are performed for the separation, identification, and quantification of PFAS. However, for efficient remediation efforts to be properly informed, a more rapid in-field testing method is required. We previously demonstrated the development and use of dioxygen as the mediator molecule. The use of dioxygen is predicated on the assumption that there will be consistent ambient dioxygen levels in natural waters. This is not always the case in hypoxic groundwater and at high altitudes. To overcome this challenge and further advance the strategies that will enable in-field electroanalysis of PFAS, we demonstrate, as a proof of concept, that dioxygen can be generated in solution through the hydrolysis of water. The electrogenerated dioxygen can then be used as a mediator molecule for the indirect detection of PFOS via molecularly imprinted polymer (MIP)-based electroanalysis. We demonstrate that calibration curves can be constructed with high precision and sensitivity (LOD < 1 ppt or 1 ng/L). Our results provide a foundation for enabling in-field hypoxic PFAS electroanalysis.

Non-Enzymatic Detection of Glucose in Neutral Solution Using PBS-Treated Electrodeposited Copper-Nickel Electrodes.

Transition metals have been explored extensively for non-enzymatic electrochemical detection of glucose. However, to enable glucose oxidation, the majority of reports require highly alkaline electrolytes which can be damaging to the sensors and hazardous to handle. In this work, we developed a non-enzymatic sensor for detection of glucose in near-neutral solution based on copper-nickel electrodes which are electrochemically modified in phosphate-buffered saline (PBS). Nickel and copper were deposited using chronopotentiometry, followed by a two-step annealing process in air (Step 1: at room temperature and Step 2: at 150 °C) and electrochemical stabilization in PBS. Morphology and chemical composition of the electrodes were characterized using scanning electron microscopy and energy-dispersive X-ray spectroscopy. Cyclic voltammetry was used to measure oxidation reaction of glucose in sodium sulfate (100 mM, pH 6.4). The PBS-Cu-Ni working electrodes enabled detection of glucose with a limit of detection (LOD) of 4.2 nM, a dynamic response from 5 nM to 20 mM, and sensitivity of 5.47 ± 0.45 µA cm-2/log10(mole.L-1) at an applied potential of 0.2 V. In addition to the ultralow LOD, the sensors are selective toward glucose in the presence of physiologically relevant concentrations of ascorbic acid and uric acid spiked in artificial saliva. The optimized PBS-Cu-Ni electrodes demonstrate better stability after seven days storage in ambient compared to the Cu-Ni electrodes without PBS treatment.

Detection of bacterial metabolism in lag-phase using impedance spectroscopy of agar-integrated 3D microelectrodes.

Traditional methods for detection of metabolically-active bacterial cells, while effective, require several days to complete. Development of sensitive electrical biosensors is highly desirable for rapid detection and counting of pathogens in food, water, or clinical samples. Herein, we develop a highly-sensitive non-Faradaic impedance sensor which detects metabolic activity of E. coli cells in a mere 1 µl of sample volume and without any sample filtration/purification. The three dimensional (3D) interdigitated electrodes (IDEs) along with self-assembled gold-nickel (Au-Ni) nanostructures significantly amplify the sensitivity by increasing the sensing area almost three-fold. The developed microsystem is integrated with an agar-based growth medium and monitors the metabolism of bacterial cells, enabling bacterial detection in approximately one hour after inoculation, i.e. in the lag-phase. Incorporation of a secondary agar layer as a biocompatible passivation layer protects the IDEs from potential Faradaic reactions and enhances sensitivity to modulation of the non-Faradaic impedance due to cellular metabolism. The resultant label-free sensor is capable of selective identification of metabolizing cells (vs. dead cells) across a wide linear range (10-1000 cells/µl). These results help pave the way for rapid antibacterial susceptibility testing at the point-of-need, which is currently a major challenge in healthcare.

Mapping and application of enhancer-trap flippase expression in larval and adult Drosophila CNS.

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shi(ts), regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tub(P)>Gal80> ('flip out') and tub(P)>stop>Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

A genetic mosaic approach for neural circuit mapping in Drosophila.

Transgenic manipulation of subsets of brain cells is increasingly used for studying behaviors and their underlying neural circuits. In Drosophila, the GAL4-upstream activating sequence (UAS) binary system is powerful for gene manipulation, but GAL4 expression is often too broad for fine mapping of neural circuits. Here, we describe the development of unique molecular genetic tools to restrict GAL4 expression patterns. Building on the GAL4-UAS system, our method adds two components: a collection of enhancer-trap recombinase, Flippase (ET-FLP), transgenic lines that provide inheritable, reproducible, and tissue-specific FLP and an FRT-dependent GAL80 "flip-in" construct that converts FLP expression into tissue-specific repression of GAL4 by GAL80. By including a UAS-encoded fluorescent protein, circuit morphology can be simultaneously marked while the circuit function is assessed using another UAS transgene. In a proof-of-principle analysis, we applied this ET-FLP-induced intersectional GAL80/GAL4 repression (FINGR) method to map the neural circuitry underlying fly wing inflation. The FINGR system is versatile and powerful in combination with the vast collection of GAL4 lines for neural circuit mapping as well as for clonal analysis based on the infusion of the yeast-derived FRT/FLP system of mitotic recombination into Drosophila. The strategies and tactics underlying our FINGR system are also applicable to other genetically amenable organisms in which transgenes including the GAL4, UAS, GAL80, and FLP factors can be applied.