Mechanistic Insight into Electrode Processes by Operando Visualization of Interfacial pH Using Fluorescent Nanosensors.

Operando visualization of interfacial pH is crucial, yet challenging in electrochemical processes. Herein, we report the fabrication and utilization of ratiometric, fluorescent pH-sensitive nanosensors for operando quantification of fast-dynamic, interfacial pH changes in electrochemical processes and environments where unprotected fluorescent dyes would be degraded. Spatio-temporal pH changes were detected using an electrochemically coupled laser scanning confocal microscope (EC-LSCM) during the electrocoagulation treatment of model and field samples of oil-sands-produced water. Operando visualization of interfacial pH provided new insights into the electrode processes, including ion speciation, electrode fouling, and Faradaic efficiency. We provide compelling evidence that formed metal complexes precipitate at the edge of the pH boundary layer and that there is a strong coupling between the thickness of the interfacial pH layer and the electrode fouling. Furthermore, these findings provide a powerful pathway for optimizing the operating conditions, minimizing electrode passivation, and enhancing the efficiency of electrochemical processes, e.g., electrocoagulation, flow batteries, capacitive deionization, and electrolyzes.

In-Operando Mapping of pH Distribution in Electrochemical Processes.

In aqueous electrochemical processes, the pH evolves spatially and temporally, and often dictates the process performance. Herein, a new method for the in-operando monitoring of pH distribution in an electrochemical cell is demonstrated. A combination of pH-sensitive fluorescent dyes, encompassing a wide pH range from ≈1.5 to 8.5, and rapid electrochemically coupled laser scanning confocal microscopy is used to observe pH changes in the cell. Using electrocoagulation as an example process, we show that the method provides new insights into the reaction mechanisms. The pH close to the aluminium electrode surface is influenced by the applied current density, hydrolysis of aluminium cations, and gas evolution. Through quantification of the pH at the anode, along with gas analysis, we find that hydrogen is evolved at the anode due to a non-Faradaic chemical reaction. This leads to increased production of coagulant, which may open new routes to enhance the process performance. This method for in-operando dynamic visualization of pH paves the way for studies of electrochemical processes, including other water treatment, electrosynthesis, and batteries.

HPTLC Method for the Ultrasensitive Detection of Triamterene in Plasma.

A high-performance thin-layer chromatographic (HPTLC) method has developed for the selective detection of a diuretic drug, triamterene (TRIAM), in pure form, tablets and human plasma. The method was based on chromatographic separation of TRIAM using HPTLC plates, precoated with silica gel, and a mobile phase consisted of ethyl acetate: dimethylformamide: ammonia (7.0: 2.7: 0.3, by volume). The native fluorescence signal of TRIAM was detected at 440 nm and used to quantify TRIAM using the proposed method, improving the method sensitivity to ~250-folds in comparison to that reported in previous HPTLC studies. The developed method enabled the detection of TRIAM in pure drug and biological samples (human plasma) within linear concentrations ranged from 0.8 to 60 ng/band or 1.0 to 60 ng/band for pure drug and plasma samples, respectively. Furthermore, the method was validated according to the official guidelines to permit its applicability in quality control and clinical laboratories.

Gold-Oligonucleotide Nanoconstructs Engineered to Detect Conserved Enteroviral Nucleic Acid Sequences.

Enteroviruses are ubiquitous mammalian pathogens that can produce mild to life-threatening disease. We developed a multimodal, rapid, accurate and economical point-of-care biosensor that can detect nucleic acid sequences conserved amongst 96% of all known enteroviruses. The biosensor harnesses the physicochemical properties of gold nanoparticles and oligonucleotides to provide colourimetric, spectroscopic and lateral flow-based identification of an exclusive enteroviral nucleic acid sequence (23 bases), which was identified through in silico screening. Oligonucleotides were designed to demonstrate specific complementarity towards the target enteroviral nucleic acid to produce aggregated gold-oligonucleotide nanoconstructs. The conserved target enteroviral nucleic acid sequence (≥1 × 10-7 M, ≥1.4 × 10-14 g/mL) initiates gold-oligonucleotide nanoconstruct disaggregation and a signal transduction mechanism, producing a colourimetric and spectroscopic blueshift (544 nm (purple) > 524 nm (red)). Furthermore, lateral-flow assays that utilise gold-oligonucleotide nanoconstructs were unaffected by contaminating human genomic DNA, demonstrated rapid detection of conserved target enteroviral nucleic acid sequence (<60 s), and could be interpreted with a bespoke software and hardware electronic interface. We anticipate that our methodology will translate in silico screening of nucleic acid databases to a tangible enteroviral desktop detector, which could be readily translated to related organisms. This will pave the way forward in the clinical evaluation of disease and complement existing strategies to overcome antimicrobial resistance.

A clinical study for the evaluation of pharmacokinetic interaction between daclatasvir and fluoxetine.

A simple and sensitive chromatographic method has been developed for the quantitative analysis of an antiviral agent, daclatasvir (DCV), that commonly prescribed for the treatment of hepatitis C viral (HCV) infection. The method was applied to detect DCV in human plasma and real blood samples collected from patients diagnosed with HCV and treated with DCV. The analysis strategy was based on recording the native fluorescence of DCV in plasma, after pre-column treatment to precipitate the plasma proteins using a readily applicable protocol. Chromatographic conductions, factors influencing the fluorescence and stability studies were also investigated. Furthermore, the method was validated according to the International Conference on Harmonization (ICH) guidelines and could be used to detect DCV in plasma over a linear range of 1.0-4000 ng/mL, with an acceptable sensitivity as the limit of detection (LOD) was 0.025 ng/mL. In addition, the study was extended to evaluate the pharmacokinetic interaction between DCV and a co-prescribed antidepressant drug, fluoxetine (FLX) in real blood samples, collected from volunteering patients who were diagnosed with HCV and treated with DCV alone or combined with FLX. The results showed a significant influence of FLX on the pharmacokinetic profile of DCV. The findings observed in this study could be used by clinical pharmacists to adjust the DCV dose, when combined with FLX, during the HCV treatment.

Enhanced distance-dependent fluorescence quenching using size tuneable core shell silica nanoparticles.

Silica nanoparticles (SNPs) have been used as favoured platforms for sensor, drug delivery and biological imaging applications, due to their ease of synthesis, size-control and bespoke physico-chemical properties. In this study, we have developed a protocol for the synthesis of size-tuneable SNPs, with diameters ranging from 20 nm to 500 nm, through the optimisation of experimental components required for nanoparticle synthesis. This protocol was also used to prepare fluorescent SNPs, via covalent linkages of fluorophores, to the nanoparticle matrix using 3-aminopropyltriethoxysilane (APTES). This enabled the fabrication of ratiometric, fluorescent, pH-sensitive nanosensors (75 nm diameter) composed SNPs covalently linked to two pH-sensitive fluorescent dyes Oregon Green (OG) and 5(6)-carboxyfluorescein (FAM) and a reference fluorescent dye 5-(6)-carboxytetramethylrhodamine (TAMRA), extending the dynamic range of measurement from pH 3.5 to 7.5. In addition, size-tuneable, core-shell SNPs, covalently linked to a fluorescent TAMRA core were synthesised to investigate distance-dependant fluorescence quenching between TAMRA and black hole quencher 2 (BHQ2®) using nanometre-sized silica shells as physical spacers. The results showed a significant fluorescence quenching could be observed over greater distances than that reported for the classical distance-dependent molecular fluorescence quenching techniques, e.g. the Förster (fluorescence) resonance energy transfer (FRET). The methods and protocols we have detailed in this manuscript will provide the basis for the reproducible production of size tunable SNPs, which will find broad utility in the development of sensors for biological applications.

Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors.

Intracellular pH is a key parameter that influences many biochemical and metabolic pathways that can also be used as an indirect marker to monitor metabolic and intracellular processes. Herein, we utilise ratiometric fluorescent pH-sensitive nanosensors with an extended dynamic pH range to measure the intracellular pH of yeast (Saccharomyces cerevisiae) during glucose metabolism in real-time. Ratiometric fluorescent pH-sensitive nanosensors consisting of a polyacrylamide nanoparticle matrix covalently linked to two pH-sensitive fluorophores, Oregon green (OG) and 5(6)carboxyfluorescein (FAM), and a reference pH-insensitive fluorophore, 5(6)carboxytetramethylrhodamine (TAMRA), were synthesised. Nanosensors were functionalised with acrylamidopropyltrimethyl ammonium hydrochloride (ACTA) to confer a positive charge to the nanoparticle surfaces that facilitated nanosensor delivery to yeast cells, negating the need to use stress inducing techniques. The results showed that under glucose-starved conditions the intracellular pH of yeast population (n ≈ 200) was 4.67 ± 0.15. Upon addition of d-(+)-glucose (10 mM), this pH value decreased to pH 3.86 ± 0.13 over a period of 10 minutes followed by a gradual rise to a maximal pH of 5.21 ± 0.26, 25 minutes after glucose addition. 45 minutes after the addition of glucose, the intracellular pH of yeast cells returned to that of the glucose starved conditions. This study advances our understanding of the interplay between glucose metabolism and pH regulation in yeast cells, and indicates that the intracellular pH homestasis in yeast is highly regulated and demonstrates the utility of nanosensors for real-time intracellular pH measurements.