Potentiometric pH Nanosensor for Intracellular Measurements: Real-Time and Continuous Assessment of Local Gradients.

We present a pH nanosensor conceived for single intracellular measurements. The sensing architecture consisted of a two-electrode system evaluated in the potentiometric mode. We used solid-contact carbon nanopipette electrodes tailored to produce both the indicator (pH nanosensor) and reference electrodes. The indicator electrode was a membrane-based ion-selective electrode containing a receptor for hydrogen ions that provided a favorable selectivity for intracellular measurements. The analytical features of the pH nanosensor revealed a Nernstian response (slope of -59.5 mV/pH unit) with appropriate repeatability and reproducibility (variation coefficients of <2% for the calibration parameters), a fast response time (<5 s), adequate medium-term drift (0.7 mV h-1), and a linear range of response including physiological and abnormal cell pH levels (6.0-8.5). In addition, the position and configuration of the reference electrode were investigated in cell-based experiments to provide unbiased pH measurements, in which both the indicator and reference electrodes were located inside the same cell, each of them inside two neighboring cells, or the indicator electrode inside the cell and the reference electrode outside of (but nearby) the studied cell. Finally, the pH nanosensor was applied to two cases: (i) the tracing of the pH gradient from extra-to intracellular media over insertion into a single PC12 cell and (ii) the monitoring of variations in intracellular pH in response to exogenous administration of pharmaceuticals. It is anticipated that the developed pH nanosensor, which is a label-free analytical tool, has high potential to aid in the investigation of pathological states that manifest in cell pH misregulation, with no restriction in the type of targeted cells.

Mass Spectrometry Imaging Shows Modafinil, A Student Study Drug, Changes the Lipid Composition of the Fly Brain.

Modafinil, a widely used psychoactive drug, has been shown to exert a positive impact on cognition and is used to treat sleep disorders and hyperactivity. Using time-of-flight secondary ion mass spectrometric imaging, we studied the changes of brain lipids of Drosophila melanogaster induced by modafinil to gain insight into the functional mechanism of modafinil in the brain. We found that upon modafinil treatment, the abundance of phosphatidylcholine and sphingomyelin species in the central brain of Drosophila is significantly decreased, whereas the levels of phosphatidylethanolamine and phosphatidylinositol in the brains show significant enhancement compared to the control flies. The alteration of brain lipids caused by modafinil is consistent with previous studies about cognition-related drugs and offers a plausible mechanism regarding the action of modafinil in the brain as well as a potential target for the treatment of certain disorders.

Combined electrochemistry and mass spectrometry imaging to interrogate the mechanism of action of modafinil, a cognition-enhancing drug, at the cellular and sub-cellular level.

Modafinil is a mild psychostimulant-like drug enhancing wakefulness, improving attention and developing performance in various cognitive tasks, but its mechanism of action is not completely understood. This is the first combination of amperometry, electrochemical cytometry and mass spectrometry to interrogate the mechanism of action of a drug, here modafinil, at cellular and sub-cellular level. We employed single-cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC) to investigate the alterations in exocytotic release and vesicular catecholamine storage following modafinil treatment. The SCA results reveal that modafinil slows down the exocytosis process so that, the number of catecholamines released per exocytotic event is enhanced in the modafinil-treated cells. Also, IVIEC results offer an upregulation effect of modafinil on the vesicular catecholamine storage. Mass spectrometry imaging by time-of-flight secondary ion mass spectrometry (ToF-SIMS) illustrates that treatment with modafinil reduces the cylindrical-shaped phosphatidylcholine at the cellular membrane, while the high curvature lipids with conical structures such as phosphatidylethanolamine and phosphatidylinositol are elevated after modafinil treatment. Combining the results obtained by SCA, IVIEC and ToF-SIMS suggests that modafinil-treated cells release a larger portion of their vesicular content at least in part by changing the lipid composition of the cell membrane, suggesting regulation of cognition.

Direct Measurement of Total Vesicular Catecholamine Content with Electrochemical Microwell Arrays.

We have designed and fabricated a microwell array chip (MWAC) to trap and detect the entire content of individual vesicles after disruption of the vesicular membrane by an applied electrical potential. To understand the mechanism of vesicle impact electrochemical cytometry (VIEC) in microwells, we simulated the rupture of the vesicles and subsequent diffusion of entrapped analytes. Two possibilities were tested: (i) the vesicle opens toward the electrode, and (ii) the vesicle opens away from the electrode. These two possibilities were simulated in the different microwells with varied depth and width. Experimental VIEC measurements of the number of molecules for each vesicle in the MWAC were compared to VIEC on a gold microdisk electrode as a control, and the quantified catecholamines between these two techniques was the same. We observed a prespike foot in a significant number of events (∼20%) and argue this supports the hypothesis that the vesicles rupture toward the electrode surface with a more complex mechanism including the formation of a stable pore intermediate. This study not only confirms that in standard VIEC experiments the whole content of the vesicle is oxidized and quantified at the surface of the microdisk electrode but actively verifies that the adsorbed vesicle on the surface of the electrode forms a pore in the vicinity of the electrode rather than away from it. The fabricated MWAC promotes our ability to quantify the content of vesicles accurately, which is fundamentally important in bioanalysis of the vesicles.

Intracellular injection of phospholipids directly alters exocytosis and the fraction of chemical release in chromaffin cells as measured by nano-electrochemistry.

Using a nano-injection method, we introduced phospholipids having different intrinsic geometries into single secretory cells and used single cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC) with nanotip electrodes to monitor the effects of intracellular incubation on the exocytosis process and vesicular storage. Combining tools, this work provides new information to understand the impact of intracellular membrane lipid engineering on exocytotic release, vesicular content and fraction of chemical release. We also assessed the effect of membrane lipid alteration on catecholamine storage of isolated vesicles by implementing another amperometric technique, vesicle impact electrochemical cytometry (VIEC), outside the cell. Exocytosis analysis reveals that the intracellular nano-injection of phosphatidylcholine and lysophosphatidylcholine decreases the number of released catecholamines, whereas phosphatidylethanolamine shows the opposite effect. These observations support the emerging hypothesis that lipid curvature results in membrane remodeling through secretory pathways, and also provide new evidence for a critical role of the lipid localization in modulating the release process. Interestingly, the IVIEC data imply that total vesicular content is also affected by in situ supplementation of the cells with some lipids, while, the corresponding VIEC results show that the neurotransmitter content in isolated vesicles is not affected by altering the vesicle membrane lipids. This suggests that the intervention of phospholipids inside the cell has its effect on the cellular machinery for vesicle release rather than vesicle structure, and leads to the somewhat surprising conclusion that modulating release has a direct effect on vesicle structure, which is likely due to the vesicles opening and closing again during exocytosis. These findings could lead to a novel regulatory mechanism for the exocytotic or synaptic strength based on lipid heterogeneity across the cell membrane.

Modified fractal iron oxide magnetic nanostructure: A novel and high performance platform for redox protein immobilization, direct electrochemistry and bioelectrocatalysis application.

A novel biosensing platform based on fractal-pattern of iron oxides magnetic nanostructures (FIOMNs) and mixed hemi/ad-micelle of sodium dodecyl sulfate (SDS) was designed for the magnetic immobilization of hemoglobin (Hb) at a screen printed carbon electrode (SPCE). The FIOMNs was successfully synthesized through hydrothermal approach and characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and X-ray diffraction (XRD). In order to provide guidelines for the mixed hemi/ad-micelle formation, zeta-potential isotherms were investigated. The construction steps of the biosensor were evaluated by electrochemical impedance spectroscopy, cyclic voltammetry and Fourier transform infrared spectroscopy. Direct electron transfer of Hb incorporated into the biocomposite film was realized with a pair of quasi-reversible redox peak at the formal potential of -0.355V vs. Ag/AgCl attributing to heme Fe(III)/Fe(II) redox couple. The results suggested that synergistic functions regarding to the hyper-branched and multidirectional structure of FIOMNs and the dual interaction ability of mixed hemi/ad-micelle array of SDS molecules not only induce an effective electron transfer between the Hb and the underlying electrode (high heterogeneous electron transfer rate constant of 2.08s(-1)) but also provide powerful and special microenvironment for the adsorption of the redox proteins. Furthermore, the biosensor displayed an excellent performance to the electrocatalytic reduction of H2O2 with a detection limit of 0.48µM and Michaelis-Menten constant (Km) value of 44.2µM. The fabricated biosensor represented the features of sensitivity, disposable design, low sample volume, rapid and simple preparation step, and acceptable anti-interferences, which offer great perspectives for the screen-determination of H2O2 in real samples.

Mixed Hemi/Ad-Micelle Sodium Dodecyl Sulfate-Coated Magnetic Iron Oxide Nanoparticles for the Efficient Removal and Trace Determination of Rhodamine-B and Rhodamine-6G.

Mixed hemi/ad-micelle sodium dodecyl sulfate (SDS)-coated magnetic iron oxide nanoparticles (MHAMS-MIONPs) were used as an efficient adsorbent for both removal and preconcentration of two important carcinogenic xanthine dyes named rhodamine-B (RB) and rhodamine-6G (RG). To gain insight in the configuration of SDS molecules on the surface of MIONPs, zeta potential measurements were performed in different [SDS]/[MIONP] ratios. Zeta potential data indicated that mixed hemi/ad-micelle MHAM was formed in [SDS]/[MIONP] ratios over the range of 1.1 to 7.3. Parameters affecting the adsorption of dyes were optimized as removal efficiency by one variable at-a-time and response surface methodology; the obtained removal efficiencies were ∼100%. Adsorption kinetic and equilibrium studies, under the optimum condition (pH = 2; amount of MIONPs = 87.15 mg; [SDS]/[MIONP] ratio = 2.9), showed that adsorption of both dyes are based on the pseudo-second-order and the Langmuir isotherm models, respectively. The maximum adsorption capacities for RB and RG were 385 and 323 mg g(-1), respectively. MHAMS-MIONPs were also applied for extraction of RB and RG. Under optimum conditions (pH = 2; amount of damped MHAMS-MIONPs = 90 mg; eluent solvent volume = 2.6 mL of 3% acetic acid in acetonitrile), extraction recoveries for 0.5 mg L(-1) of RB and RG were 98% and 99%, with preconcentration factors of 327 and 330, respectively. Limit of detection obtained for rhodamine dyes were <0.7 ng mL(-1). Finally, MHAMS-MIONPs were successfully applied for both removal and trace determination of RB and RG in environmental and wastewater samples.

Optimization of magnetic stirring assisted dispersive liquid-liquid microextraction of rhodamine B and rhodamine 6G by response surface methodology: Application in water samples, soft drink, and cosmetic products.

An exact, rapid and efficient method for the extraction of rhodamine B (RB) and rhodamine 6G (RG) as well as their determination in three different matrices was developed using magnetic stirring assisted dispersive liquid-liquid microextraction (MSA-DLLME) and HPLC-Vis. 1-Octanol and acetone were selected as the extraction and dispersing solvents, respectively. The potentially variables were the volume of extraction and disperser solvents, pH of sample solution, salt effect, temperature, stirring rate and vortex time in the optimization process. A methodology based on fractional factorial design (2(7)(-2)) was carried out to choose the significant variables for the optimization. Then, the significant factors (extraction solvent volume, pH of sample solution, temperature, stirring rate) were optimized using a central composite design (CCD). A quadratic model between dependent and independent variables was built. Under the optimum conditions (extraction solvent volume=1050µL, pH=2, temperature=35°C and stirring rate=1500rpm), the calibration curves showed high levels of linearity (R(2)=0.9999) for RB and RG in the ranges of 5-1000ngmL(-1) and 7.5-1000ngmL(-1), respectively. The obtained extraction recoveries for 100ngmL(-1) of RB and RG standard solutions were 100% and 97%, and preconcentration factors were 48 and 46, respectively. While the limit of detection was 1.15ngmL(-1) for RB, it was 1.23ngmL(-1) for RG. Finally, the MSA-DLLME method was successfully applied for preconcentration and trace determination of RB and RG in different matrices of environmental waters, soft drink and cosmetic products.

Magnetic stirring-assisted dispersive liquid-liquid microextraction followed by high performance liquid chromatography for determination of phthalate esters in drinking and environmental water samples.

A simple, fast and efficient method for the preconcentration of phthalate esters (PEs) in water samples was developed using magnetic stirring-assisted dispersive liquid-liquid microextraction (MSA-DLLME) followed by high performance liquid chromatography coupled with ultraviolet detection (HPLC-UV). This novel microextraction method is based on the fast injection of extracting solvent into the aqueous solution, which is being stirred by a magnetic stirrer, to form a cloudy binary component solvent (aqueous solution:extracting solvent) system. The extraction parameters such as type and volume of extracting solvent, pH of sample, salt addition, extraction time and stirring rate were optimized. Under the optimal conditions (extracting solvent: 200 µL dodecane; pH of sample: 6.5; extraction time: 5 min; stirring rate: 1000 rpm), linearity was observed in the range of 2-1000 µg L(-1) for dimethyl phthalate (DMP) and 1-1000 µg L(-1) for diethyl phthalate (DEP), benzyl butyl phthalate (BBP) and di-n-butyl phthalate (DBP) with correlation determination values above 0.99 for them. The limits of detection and quantification were ranged from 0.13 to 0.38 µg mL(-1) and 0.43 to 1.27 µg mL(-1), respectively. The ranges of intra-day and inter-day precisions (n=5) at 100 µg L(-1) of PEs were 1.50-2.65% and 2.31-3.35%, respectively. Finally, the MSA-DLLME method was successfully applied for preconcentration of PEs in drinking and environmental water samples.

Preconcentration of trace amounts of methadone in human urine, plasma, saliva and sweat samples using dispersive liquid-liquid microextraction followed by high performance liquid chromatography.

A simple, rapid and efficient method for the preconcentration of methadone was developed using dispersive liquid-liquid microextraction (DLLME) followed by high performance liquid chromatography with ultra violet detection (HPLC-UV). The extraction method is based on the fast injection of a mixture of extracting and disperser solvents into the aqueous solution to form a cloudy ternary component solvent (aqueous solution:extracting solvent:disperser solvent) system. The extraction parameters such as nature and volume of extracting and disperser solvents, pH of sample, and extraction time were studied for optimization. Under the optimal conditions (extracting solvent: chloroform, 250 µL; disperser solvent: methanol, 2.5 mL and pH of sample: 10.0) a linear calibration curve was obtained in the range of 0.5-5000 ng mL(-1) with r(2)=0.9995. To demonstrate analytical performance, figures of merits of the proposed method in four different biological matrices (urine, plasma, saliva and sweat) spiked with methadone were investigated. The limits of detection and quantification in these matrices were ranged from 4.90 to 24.85 ng mL(-1) and 16.32 to 82.75 ng mL(-1), respectively. The extraction recoveries were above 97% and the preconcentration factors of methadone in distilled water, urine, plasma, saliva, and sweat samples were 196.52, 10.03, 9.93, 1.97 and 1.99, respectively. While the precision for inter-day was ≤6.43 (n=5), it was ≤2.26 (n=5) for intra-day assay. Finally, the method was successfully applied in the determination of methadone in the human urine, plasma, saliva and sweat samples.

Utilization of inverted dispersive liquid-liquid microextraction followed by HPLC-UV as a sensitive and efficient method for the extraction and determination of quercetin in honey and biological samples.

A sensitive, rapid and efficient method for the extraction of quercetin as well as its determination in honey and biological samples was developed using inverted dispersive liquid-liquid microextraction (IDLLME) and HPLC-UV. The extraction method is based on the application of an extracting solvent lighter than water in the ternary component solvent (aqueous solution: extracting solvent: disperser solvent) system. The extraction parameters such as type and volume of extracting and disperser solvent, pH of sample, stirring rate and extraction time were optimized. Under the optimal conditions (extracting solvent: 100 µL 1-octanol; disperser solvent: 300 µL acetonitrile; pH of sample: 4.5 and stirring rate: 1000 rpm) a linear calibration curve was obtained in the range of 0.5-1000 ng mL(-1) with R(2)=0.9993 (n=10). The limits of detection and quantification were 0.26 and 0.78 ng mL(-1), respectively. The extraction recovery was 97% and the preconcentration factor was 243. While the relative standard deviation for 25 ng mL(-1) was 3.51 (n=5), it was 2.12 (n=5) for 500 ng mL(-1) of quercetin. The method was successfully applied for the preconcentration and determination of quercetin in honey, urine and plasma samples.

Application of dispersive liquid-liquid microextraction and spectrophotometric detection to the rapid determination of rhodamine 6G in industrial effluents.

A rapid and effective preconcentration method for extraction of rhodamine 6G was developed by using a dispersive liquid-liquid microextraction (DLLME) prior to UV-vis spectrophotometry. In this extraction method, a suitable mixture of acetone (disperser solvent) and chloroform (extractant solvent) was injected rapidly into a conical test tube containing aqueous solution of rhodamine 6G. Therefore, a cloudy solution was formed. After centrifugation of the cloudy solution, sedimented phase was evaporated, reconstituted with methanol and measured by UV-vis spectrophotometry. Different operating variables such as type and volume of extractant solvent, type and volume of disperser solvent, pH of the sample solution, salt concentration and extraction time were investigated. The optimized conditions (extractant solvent: 300 microL of chloroform, disperser solvent: 3 mL of acetone, pH: 8 and without salt addition) resulted in a linear calibration graph in the range of 5-900 ng mL(-1) of rhodamine 6G in initial solution with R(2)=0.9988 (n=5). The Limits of detection and quantification were 2.39 and 7.97 ng mL(-1), respectively. The relative standard deviation for 50 and 250 ng mL(-1) of rhodamine 6G in water were 2.88% and 1.47% (n=5), respectively. Finally, the DLLME method was applied for determination of rhodamine 6G in different industrial waste waters.