Voltammetric and pharmacological characterization of dopamine release from single exocytotic events at rat pheochromocytoma (PC12) cells

Although rat pheochromocytoma (PC12) neurotransmitter storage vesicles are known to contain a variety of neurotransmitters including catecholamines, there is little evidence that the molecular species detected during amperometric monitoring of exocytosis is a catecholamine. Rather, as these are catecholamine-containing cells, one assumes catecholamines are released. Additionally, although the total amount of transmitter released can be quantified, it has been extremely difficult to evaluate the concentration at the point of release for each exocytosis event. Interpreting voltammograms obtained in the attoliter volume affected between the electrode and the cell and defined by the size of the exocytosis pore during exocytosis is an extreme analytical challenge. Here we use voltammetry of approximately 10(-19) mol released from individual exocytosis events to identify, along with pharmacological evidence, the released compound at PC12 cells as a catecholamine, most likely dopamine. The area of the electrode at which oxidation occurs following an exocytosis event is proportional to the temporal delay prior to acquisition of a voltammogram. This model allows determination of relative concentrations from individual release events and has been used to examine events at control cells and cells incubated with the dopamine precursor, L-3,4-dihydroxyphenylalanine (L-DOPA). Exposure to L-DOPA (100 microM for 1 h) results in 145 detectable events for 11 cells compared to 77 events for 29 control cells, clearly indicating that vesicles can be "loaded" with dopamine. However, the concentrations measured at the electrode surface provide similar distributions for both L-DOPA-treated and control cells. Cyclic voltammetric measurements of relative concentration for zeptomole levels of transmitter in attoliter volumes provide evidence that loading vesicles by increased transmitter synthesis does not lead to elevated concentrations at individual release sites.     

Microbioassay system for antiallergic drug screening using suspension cells retaining in a poly(dimethylsiloxane) microfluidic device

This article describes an antiallergic drug-screening system by the detection of histamine released from mast cells (suspension cells) on a multilayer microchip. In this study, the elastmeric material, poly(dimethylsiloxane) (PDMS), was employed to fabricate microchannels and microchambers. The microchip consists of two sections: a histamine-releasing one, which has a cell chamber, and a histamine-derivatizing one. Both were laminated to one microchip. Rat peritoneal mast cells were retained in the cell chamber (1.2 microL) with a filtering system using a cellulose nitrate membrane. This filtering system could easily retain suspension cells without cell damage. Mast cells were viable for a sufficient time to conduct the assay on the cell chamber. The cells were stimulated with a chemical release compound 48/80 (C48/80), and then histamine flowed into the lower layer, where it was derivatized to the fluorescent molecules with o-phthalaldehyde and its fluorescence was detected on the microchip. This flow system could detect the time course of the histamine release, and this microchip system required only 20 min for the assay. By this integrated system, 51 pmol of histamine released from 500 cells was detected, and the number of cells required for the assay was reduced to 1% compared with conventional bulk systems. By comparing the released histamine levels with and without drugs, their effect could be evaluated. The inhibition ratio of C48/80 induced-histamine release using an antiallergic drug, disodium cromoglicate (DSCG), was related to the concentration of DSCG. This flow system was applicable for antiallergy drug screening by rapid measurement of the inhibition of histamine release from a very small amount of mast cells.     

Improving data acquisition for fast-scan cyclic voltammetry.

Described is an improved data acquisition system for fast-scan cyclic voltammetry (FSCV). The system was designed to significantly diminish noise sources that were identified in previously recorded FSCV measurements for the detection of neurotransmitters. Minimized noise is necessary to observe the low concentrations of neurotransmitters that are physiologically important. The system was based on a high-speed, 16-bit AD/DA acquisition board that allowed high scan rates and better resolved the small faradaic currents which remained after background subtraction. Irregularities that occur when independent timing sources are used for generation of the voltage waveform and collection of the current can create large noise artifacts near the voltage limits during FSCV. These were eliminated by the use of a single acquisition board that generated the voltage waveform and collected the current. Noise from frequency drift of the power line was eliminated through the use of a phase-locked loop. To demonstrate the improved performance of the system, data were collected using carbon-fiber microelectrodes in a flow injection analysis system and in brain slices. This new data acquisition system performed significantly better than another system previously used in our laboratory without these features. The improved detection limits of the new system allowed clearly resolved current spikes featuring pre-release "feet" to be recorded adjacent to individual mast cells following chemical stimulation. When combined with false-color plots, the low-noise system facilitated identification of dopamine release in a freely moving animal.     

Cell chip with nano-scale peptide layer to detect dopamine secretion from neuronal cells

A cell chip with a nano-scaled thin film of cysteine modified synthetic oligopeptide C(RGD)4 was fabricated to detect dopamine secretion from neuronal cells. Thin C(RGD)4 peptide layer was fabricated on chip surface for increasing the binding affinity of cells to gold electrode surface, which is essential for the electrochemical detection of dopamine released from PC12 cells. The structural formation of the peptide thin film was confirmed by both atomic force microscopy (AFM) and scanning electron microscopy (SEM). Redox characteristics of chemical dopamine were firstly characterized by voltammetric tool to compare the dopamine released from PC12 cells. Cells grown on the chip were then subjected to cyclic voltammetric (CV) analysis after 48 hours of incubation. The intensities of reduction peaks were found to be increased with increasing the concentrations of PC12 cells. In addition, the electrochemical redox signal increased more in the cells treated with glucose and potassium compared to the control group. Hence, the developed cell chip can be used to determine the effects of drugs on living cells electrochemically.     

Release Kinetics of Acyclovir from a Suspension of Acyclovir Incorporated in a Cubic Phase Delivery System

Acyclovir is a widely used agent in the treatment of herpes virus infections of the skin, but owing to its poor physicochemical properties in terms of bioavailability and suboptimal formulations, the treatment is far from optimal. The liquid crystalline cubic phase system has been reported to act as a bioadhesive drug delivery system. In the present study, acyclovir was suspended in a cubic phase of glycerol monooleate (GMO) and water 65%:35% w/w, and the phase behavior and release kinetics were examined. X-ray diffraction and differential scanning calorimetry (DSC) measurements demonstrated that the cubic phase containing 1%-10% (w/w) acyclovir retains its phase condition in the temperature range investigated (20°C-70°C). Acyclovir can be incorporated in high amounts (∼40% w/w) without causing phase transition, as is shown in polarized light. This is probably because of its low solubility (∼0.1% w/w) in the cubic phase. The release characteristics of acyclovir incorporated as a suspension (1%-5% w/w) into a cubic phase were investigated using Franz diffusion cells. Acyclovir was quantified by high-performance liquid chromatography (HPLC). The drug was readily released from the system, and the release increased with the initial drug load concentration. About 25%-50% was released after 24 h. The release is dependent on the square root of time, and the kinetics can be described by the Higuchi theory. The rate-limiting step in the release process is most likely diffusion. The suggested theory is further supported by identical release data obtained for micronized and nonmicronized acyclovir. The fluxes for 1% and 5% w/w were 380 and 900 μg/h^1/2, respectively. Comparison of the release rates of acyclovir delivered from a cubic phase and from the commercial product, Zovir® cream, showed the rate to be six times faster from the cubic phase. The results indicate that the cubic phase is a promising drug delivery system for acyclovir.     

Investigation on magnetically controlled delivery of doxorubicin from superparamagnetic nanocarriers of gelatin crosslinked with genipin

Gelatin (Type B) nanoparticles were prepared by a single W/O emulsion technique and characterized by infrared (IR) spectra, transmission electron micrographs (TEM), surface potential measurements and magnetization studies. Whereas the IR spectra clearly confirmed the presence of gelatin, genipin and doxorubicin in the loaded nanoparticles, the transmission electron micrographs (TEM) image depicts smooth surface, spherical shape and non-uniform size of nanoparticles (up to 100 nm). The prepared nanoparticles were loaded with doxorubicin, a well known anticancer drug, and in vitro release dynamics of entrapped drug was investigated as a function of various experimental factors such as percent loading of the drug, chemical architecture of the nanocarriers, and pH, temperature, ionic strength and nature of the release medium in presence and absence of magnetic field. The nanoparticles were also studied for their water sorption capacity. The drug release process was analyzed kinetically using Ficks power law and a correlation was established between the quantity of released drug and swelling of the nanoparticles.     

Achieving high detection sensitivity (14 zmol) of biomolecular ions in bioaerosol mass spectrometry

Bioaerosol mass spectrometry (BAMS) performs single-cell analysis in real time. However, the specificity of BAMS mass signatures has been limited by low sensitivity at high masses. To increase the mass range and sensitivity of BAMS, a novel design was developed that utilizes a linear flight tube with delayed extraction and an electrostatic ion guide. This study quantifies the sensitivity limits of the novel BAMS design and evaluates the feasibility of BAMS to detect higher mass biomarkers from single cells. All experiments were carried out using MALDI aerosol particles that were nebulized from solution. Sensitivity was assessed by generating particles with decreasing amounts of analyte via serial dilutions. The amount of analyte contained within each particle was calculated based on particle size, density, and molarity of the analyte within solution. A variety of biomolecular ions were studied and signals obtained from particles containing 300 zmol of maltopentaose, 132 zmol of alpha-cyclodextrin, and 14 zmol (approximately 8400 molecules) of gramicidin S are reported. The detection of 14 zmol of gramicidin S is to the best of our knowledge a record in sensitivity for MALDI TOF-MS.     

Hydrogels based on chitosan and dextran as potential drug delivery systems

The release of human growth hormone (GH) from bioartificial polymeric materials in the form of hydrogels, was measured in vitro for up to 3 weeks. Poly(vinyl-alcohol) (PVA) was blended, in different ratios, with two biological polymers, dextran and chitosan respectively. These blends were used to prepare hydrogels, using a freeze–thawing method. The hydrogels were loaded with GH, and their potential use as delivery systems was investigated. The release with time of PVA, in aqueous medium, was also monitored and evaluated. Scanning electron microscopy was used to investigate the structure of the hydrogels. The results obtained indicated that GH can be released from both dextran/PVA and chitosan/PVA hydrogels. The initial GH concentration used for sample loading affected the total quantity of GH released but not the pattern of release. The amount of GH released was affected by the content of the biological component. The percentage of PVA released was low but it was, however, related to the content of chitosan and dextran in the blends. ©1999 Kluwer Academic Publishers     

Application of ganglioside-sensitized liposomes in a flow injection immunoanalytical system for the determination of cholera toxin

Cholera, an acute infectious disease associated with water and seafood contamination, is caused by the bacterium Vibrio cholerae, which lives and colonizes in the small intestine and secretes cholera toxin (CT), a causative agent for diarrhea in humans. Based on earlier lateral flow assays, a flow injection liposome immunoanalysis (FILIA) system with excellent sensitivity was developed in this study for the determination of CT at zeptomole levels. Ganglioside (GM1), found to have specific affinity toward CT, was inserted into the phospholipid bilayer during the liposome synthesis. These GM1-sensitized, sulforhodamine B (SRB) dye-entrapping liposomes were used as probes in the FILIA system. Anti-CT antibodies were immobilized in its microcapillary. CT was detected by the formation of a sandwich complex between the immobilized antibody and GM1 liposomes. During the assay, the sample was introduced first into the column, and then liposomes were injected to bind to all CT captured by the antibody in the microcapillary. Subsequently, the SRB dye molecules were released from the bound liposomes via the addition of the detergent octyl glucopyranoside. The released dye molecules were transported to a flow-through fluorescence detector for quantification. The FILIA system was optimized with respect to flow rate, antibody concentration, liposome concentration, and injected sample volume. The calibration curve for CT had a linear range of 10-16 to 10-14 g mL-1. The detection limit of this immunosensor was 6.6 x 10(-17) g mL-1 in 200-microL samples (equivalent to 13 ag or 1.1 zmol).     

Compact microelectrode array system: tool for in situ monitoring of drug effects on neurotransmitter release from neural cells

This paper presents a compact microelectrode array (MEA) system, to study potassium ion-induced dopamine release from PC12 neural cells, without relying on a micromanipulator and a microscope. The MEA chip was integrated with a custom-made "test jig", which provides a robust electrical interfacing tool between the microchip and the macroenvironment, together with a potentiostat and a microfluidic syringe pump. This integrated system significantly simplifies the operation procedures, enhances sensing performance, and reduces fabrication costs. The achieved detection limit for dopamine is 3.8 x 10-2 muM (signal/noise, S/N = 3) and the dopamine linear calibration range is up to 7.39 +/- 0.06 muM (mean +/- SE). The effects of the extracelluar matrix collagen coating of the microelectrodes on dopamine sensing behaviors, as well as the influences of K+ and l-3,4-digydroxyphenylalanine concentrations and incubation times on dopamine release, were extensively studied. The results show that our system is well suited for biologists to study chemical release from living cells as well as drug effects on secreting cells. The current system also shows a potential for further improvements toward a multichip array system for drug screening applications.     

Kinetics of Cephalexin Release from Eudragit-Hydroxypropylcellulose Membranes

The release of cephalexin dispersed in films composed of different ratios of Eudragit-RS and hydroxyropylcellulose (HPC) was investigated. The results indicate that drug release from matrix follows a diffusion-controlled model, where the quantity released per unit area is proportional to the square roof of time. The release rate was found to be proportional to diethylphthalate (DEP) content, drug concentration, and HPC fraction in the film. Addition of plasticizer is indispensable for the improvement of film characteristics. The aggregation of drug particles was increased with DEP contents in film. The release rate was enhanced by adding the HPC to the rate-controlling membrane.     

Toward the Next Generation of Retinal Neuroprosthesis: Visual Computation with Spikes

A neuroprosthesis is a type of precision medical device that is intended to manipulate the neuronal signals of the brain in a closed-loop fashion, while simultaneously receiving stimuli from the environment and controlling some part of a human brain or body. Incoming visual information can be processed by the brain in millisecond intervals. The retina computes visual scenes and sends its output to the cortex in the form of neuronal spikes for further computation. Thus, the neuronal signal of interest for a retinal neuroprosthesis is the neuronal spike. Closed-loop computation in a neuroprosthesis includes two stages: encoding a stimulus as a neuronal signal, and decoding it back into a stimulus. In this paper, we review some of the recent progress that has been achieved in visual computation models that use spikes to analyze natural scenes that include static images and dynamic videos. We hypothesize that in order to obtain a better understanding of the computational principles in the retina, a hypercircuit view of the retina is necessary, in which the different functional network motifs that have been revealed in the cortex neuronal network are taken into consideration when interacting with the retina. The different building blocks of the retina, which include a diversity of cell types and synaptic connections—both chemical synapses and electrical synapses (gap junctions)—make the retina an ideal neuronal network for adapting the computational techniques that have been developed in artificial intelligence to model the encoding and decoding of visual scenes. An overall systems approach to visual computation with neuronal spikes is necessary in order to advance the next generation of retinal neuroprosthesis as an artificial visual system.     

Flame etching enhances the sensitivity of carbon-fiber microelectrodes

Small sensors are useful for in vivo measurements and probing small spaces. In this paper, we compare two methods of fabrication of small, cylindrical carbon-fiber microelectrodes: flame-etching and electrochemical etching. With both methods, microelectrodes can be fabricated with tip diameters of 1 to 3 microm. Electrodes were tested with fast-scan cyclic voltammetry. Flame etching resulted in electrodes that have larger S/N ratios and higher currents per unit area for 1 microM dopamine than normal carbon-fiber microelectrodes or electrochemically etched electrodes. Therefore, the increased sensitivity is not just a property of size. The flame-etched surfaces had nanometer-scale surface features that were not observed on the other electrodes and exhibited increased sensitivity for other electroactive compounds found in the brain, including ascorbic acid, DOPAC, and serotonin. Faster kinetics and a faster response to a step change in dopamine were also observed, when the applied waveform was -0.4 to 1.0 V and back at 400 V/s. The sensitivity of the flame-etched electrodes was enhanced by overoxidizing the surface. The flame-etched electrodes were used to detect dopamine release in anesthetized rats after a single stimulation pulse. The small flame-etched electrodes will facilitate measurements of low concentrations in discrete brain regions or small organisms.     

Amperometric detection of quantal catecholamine secretion from individual cells on micromachined silicon chips

We have fabricated electrochemical electrodes in picolitersized wells for measuring catecholamine release from individual cells with millisecond resolution. Each well-electrode roughly conforms to the shape of the cell in order to capture a large fraction of released catecholamine with high time resolution. Using this device, we can resolve spikes in amperometric current corresponding to quantal catecholamine release via exocytosis. In addition, we have combined amperometric recording on the chip with patch-clamp recordings of membrane capacitance as an assay of exocytosis. A quantitative comparison of the two methods suggests that a large fraction of catecholamine release is oxidized on the surface of the well-electrode. This technology has applications in cell-based biosensor development, high-throughput screening of drugs, and basic science investigations.     

Kinetics of Cephalexin Release from Eudragit-Hydroxypropylcellulose Membranes

Abstract

The release of cephalexin dispersed in films composed of different ratios of Eudragit-RS and hydroxyropylcellulose (HPC) was investigated. The results indicate that drug release from matrix follows a diffusion-controlled model, where the quantity released per unit area is proportional to the square roof of time. The release rate was found to be proportional to diethylphthalate (DEP) content, drug concentration, and HPC fraction in the film. Addition of plasticizer is indispensable for the improvement of film characteristics. The aggregation of drug particles was increased with DEP contents in film. The release rate was enhanced by adding the HPC to the rate-controlling membrane.     

Calcium alginate nanocarriers as possible vehicles for oral delivery of insulin

In the present investigation, alginate nanoparticles have been prepared and characterised by various techniques such as FTIR, SEM, particle size analysis and surface charge measurements. It was found from both the SEM and particle size analysis that average size of the particle was about 40?nm. The particles were loaded with insulin and the release kinetics of insulin was studied in PBS medium. The results indicated that when percent loading increases from 11.7 to 38.9, the released amount of insulin increased from 18% to 60% of the loaded drug. The effect of composition of nanoparticles, pH and temperature of the release medium was examined on the amount of released insulin. It was observed when that amount of alginate in the feed mixture was varied from 1.0 to 2.0?g, the prepared nanoparticles showed a decreasing tendency to release insulin. Similarly, upon increasing the concentration of crosslinker in the range 0.5–1.1?mM, the release of insulin constantly decreased. The chemical stability of the loaded drug was assessed especially under highly acidic conditions of artificial gastric juice and it was noticed that even in harsh acidic environment (pH 1.2) the insulin remains chemically stable. The invitro blood compatibility of nanoparticles was also investigated and it was found that for a definite composition of nanoparticles, protein adsorption and percent haemolysis were minimum which suggested for an optimum blood compatibility of alginate nanoparticles of definite composition. Thus, it can be conclusively stated that the calcium alginate nanoparticles prepared by emulsion crosslinking method show potential to be developed as oral formulation for insulin delivery.     

On the relation of setting and early-age strength development to porosity and hydration in cement-based materials

Previous research has demonstrated a linear relationship between compressive strength (mortar cubes and concrete cylinders) and cumulative heat release normalized per unit volume of (mixing) water for a wide variety of cement-based mixtures at ages of 1 d and beyond. This paper utilizes concurrent ultrasonic reflection and calorimetry measurements to further explore this relationship from the time of specimen casting to 3 d. The ultrasonic measurements permit a continuous evaluation of thickening, setting, and strength development during this time period for comparison with the ongoing chemical reactions, as characterized by isothermal calorimetry measurements. Initially, the ultrasonic strength-heat release relation depends strongly on water-to-cement ratio, as well as admixture additions, with no universal behavior. Still, each individual strength-heat release curve is consistent with a percolation-based view of the cement setting process. However, beyond about 8 h for the systems investigated in the present study, the various strength-heat release curves merge towards a single relationship that broadly characterizes the development of strength as a function of heat released (fractional space filled), demonstrating that mortar and/or concrete strength at early ages can be effectively monitored using either ultrasonic or calorimetry measurements on small paste or mortar specimens.     

Dopamine transport into a single cell in a picoliter vial

The analysis of chemical events in small volumes requires careful manipulation of samples and sensitive detection methods. Here, we describe the measurement of the neurotransmitter dopamine in a picoliter vial with electrochemical techniques. The vials were fabricated from fused-silica capillaries that provided a transparent container suitable for the observation and manipulation of a biological cell, sample solutions, and electrodes. Evaporation of the sample was prevented with a mineral oil layer, allowing for experiments lasting for several minutes. The small volume of these vials (100-200 pL) allows rapid mixing of all of the solution reagents. Similarly, the small volume allows exhaustive electrolysis of the vial contents with a 3-microm radius, disk-shaped carbon fiber microelectrode within 60 s. Fast-scan cyclic voltammetry at carbon fiber microelectrodes was used to monitor the concentration of analyte in the vial without depleting its contents. The concentration of dopamine introduced by pneumatic injection remained stable when sampled by cyclic voltammetry, and no evidence for adsorption to the walls was observed. However, when the vial contained a single HEK-293 cell transfected to express the dopamine transporter, the dopamine concentration decreased with time at a rate consistent with the uptake kinetics mediated by the transporter located on the cell membrane.     

Genetically encoded optical probe for detecting release of proteins from mitochondria toward cytosol in living cells and mammals

We developed a genetically encoded bioluminescence indicator for monitoring the release of proteins from the mitochondria in living cells. The principle of this method is based on reconstitution of split Renilla reniformis luciferase (Rluc) fragments by protein splicing with an Ssp DnaE intein. A target mitochondrial protein connected with an N-terminal fragment of Rluc and an N-terminal fragment of DnaE is expressed in mammalian cells. If the target protein is released from the mitochondria toward the cytosol upon stimulation with a specific chemical, the N-terminal Rluc meets the C-terminal Rluc connected with C-terminal DnaE in the cytosol, and thereby, the full-length Rluc is reconstituted by protein splicing. The extent of release of the target fusion protein is evaluated by measuring activities of the reconstituted Rluc. To test the feasibility of this method, here we monitored the release of Smac/DIABLO protein from mitochondria during apoptosis in living cells and mice. The present method allowed high-throughput screening of an apoptosis-inducing reagent, staurosporine, and imaging of the Smac/DIABLO release in cells and in living mice. This rapid analysis can be used for screening and assaying chemicals that would increase or inhibit the release of mitochondrial proteins in living cells and animals.     

Response times of carbon fiber microelectrodes to dynamic changes in catecholamine concentration

The electrode response time and the measured concentrations during dynamic catecholamine changes were compared using constant potential amperometry and fast-scan cyclic voltammetry. The amperometric response to a rectangular pulse of catecholamine is more rectangular than the cyclic voltammetric response; however, the response times are very similar when, during cyclic voltammetry, the temporal lag due to adsorption and desorption of catecholamine to the electrode is removed by deconvolution. Deconvolution of cyclic voltammetry data was applied to stimulated dopamine release in vivo, allowing for modeling of release and uptake kinetics and to measure catecholamine release from single cells, resulting in better resolution of peaks from single vesicles. In vitro postcalibrations were performed to calculate concentrations of catecholamine measured with cyclic voltammetry and amperometry. The addition of 600 microM ascorbic acid to the postcalibration buffer, allowing a catalytic reaction to regenerate dopamine, resulted in similar calculated concentrations for stimulated release of dopamine using amperometry and cyclic voltammetry. Using deconvoluted cyclic voltammetry to remove the response time lag and adding ascorbic acid to the calibration buffer, the shape and concentration of dynamic catecholamine changes are very similar when measured with constant potential amperometry and cyclic voltammetry.