RESUMEN
A near-infrared (NIR) fluorescent probe NS667 was developed using a novel synthetic strategy by integrating an electron-rich 1,2,3,4-tetrahydroquinoxaline (THQ) into the scaffold from NS510, which binds to catecholamines with high affinity. The fluorophore core was constructed with a tandem nucleophilic aromatic substitution. Upon binding to catecholamines, the fluorescence of this probe shifted, with the emission in the NIR region. Live cell imaging results demonstrate that NS667 can effectively image norepinephrine in chromaffin cells with shifted fluorescence, which highlights the potential of the probe for neuroimaging in tissues.
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Catecolaminas , Colorantes Fluorescentes , Norepinefrina , FluorescenciaRESUMEN
Nucleic acids can undergo conformational changes upon binding small molecules. These conformational changes can be exploited to develop new therapeutic strategies through control of gene expression or triggering of cellular responses and can also be used to develop sensors for small molecules such as neurotransmitters. Many analytical approaches can detect dynamic conformational change of nucleic acids, but they need labeling, are expensive, and have limited time resolution. The nanopore approach can provide a conformational snapshot for each nucleic acid molecule detected, but has not been reported to detect dynamic nucleic acid conformational change in response to small -molecule binding. Here we demonstrate a modular, label-free, nucleic acid-docked nanopore capable of revealing time-resolved, small molecule-induced, single nucleic acid molecule conformational transitions with millisecond resolution. By using the dopamine-, serotonin-, and theophylline-binding aptamers as testbeds, we found that these nucleic acids scaffolds can be noncovalently docked inside the MspA protein pore by a cluster of site-specific charged residues. This docking mechanism enables the ion current through the pore to characteristically vary as the aptamer undergoes conformational changes, resulting in a sequence of current fluctuations that report binding and release of single ligand molecules from the aptamer. This nanopore tool can quantify specific ligands such as neurotransmitters, elucidate nucleic acid-ligand interactions, and pinpoint the nucleic acid motifs for ligand binding, showing the potential for small molecule biosensing, drug discovery assayed via RNA and DNA conformational changes, and the design of artificial riboswitch effectors in synthetic biology.
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Aptámeros de Nucleótidos , Nanoporos , Riboswitch , Ligandos , Conformación de Ácido Nucleico , ARN , Aptámeros de Nucleótidos/químicaRESUMEN
Maintaining homeostasis of metabolites such as amino acids is critical for cell survival. Dysfunction of nutrient balance can result in human diseases such as diabetes. Much remains to be discovered about how cells transport, store, and utilize amino acids due to limited research tools. Here we developed a novel, pan-amino acid fluorescent turn-on sensor, NS560. It detects 18 of the 20 proteogenic amino acids and can be visualized in mammalian cells. Using NS560, we identified amino acids pools in lysosomes, late endosomes, and surrounding the rough endoplasmic reticulum. Interestingly, we observed amino acid accumulation in large cellular foci after treatment with chloroquine, but not with other autophagy inhibitors. Using a biotinylated photo-cross-linking chloroquine analog and chemical proteomics, we identified Cathepsin L (CTSL) as the chloroquine target leading to the amino acid accumulation phenotype. This study establishes NS560 as a useful tool to study amino acid regulation, identifies new mechanisms of action of chloroquine, and demonstrates the importance of CTSL regulation of lysosomes.
RESUMEN
A fluorescent sensor for catecholamines, NS510, is presented. The sensor is based on a quinolone fluorophore incorporating a boronic acid recognition element that gives it high affinity for catecholamines and a turn-on response to norepinephrine. The sensor results in punctate staining of norepinephrine-enriched chromaffin cells visualized using confocal microscopy indicating that it stains the norepinephrine in secretory vesicles. Amperometry in conjunction with total internal reflection fluorescence (TIRF) microscopy demonstrates that the sensor can be used to observe destaining of individual chromaffin granules upon exocytosis. NS510 is the highest affinity fluorescent norepinephrine sensor currently available and can be used for measuring catecholamines in live-cell assays.
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Técnicas Biosensibles/métodos , Células Cromafines/metabolismo , Exocitosis/fisiología , Colorantes Fluorescentes/química , Norepinefrina/análisis , Animales , Calcio/metabolismo , Bovinos , Células Cultivadas , Norepinefrina/metabolismoRESUMEN
BACKGROUND: Quantal exocytosis of oxidizable neurotransmitters can be detected as spikes of amperometric current using electrochemical microelectrodes. Measurements of spike parameters indicate the maximal transmitter flux, flux duration, and amount of transmitter released from individual vesicles. Automated analysis algorithms need to reject spikes that overlap in time. In addition, many spikes are preceded by small amplitude "foot" signals, attributed to slow release of transmitter through a fusion pore. Accurate pre-spike baseline determination is essential for estimating fusion-pore duration and the amount of transmitter released through the fusion pore. NEW METHOD: We developed an estimation approach that is based on fitting a multi-exponential function to the data. Our previously described matched-filter algorithm is used to identify the sections of data to fit and provides seed values to facilitate convergence of the iterative fit. The new estimation algorithm includes overlap rejection, a two-step fitting procedure and a novel baseline estimation procedure. RESULTS: Histograms of spike parameters demonstrate excellent agreement of the new approach with manually computed parameters. COMPARISON WITH EXISTING METHODS: Parameter estimates generated using the new approach are closer to blind manual estimates than commonly used existing methods. The improved performance is due to better detection of valid spikes and rejection of overlapping spikes. Moreover, since the complete time course of the spike is fit to a function, more complete information about the spike time course is captured. CONCLUSIONS: The matched-filter seeded algorithm reliably rejects overlaps and estimates spike and foot signal parameters in a fully automated manner.
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Electrofisiología/métodos , Exocitosis/fisiología , Procesamiento de Señales Asistido por Computador , Algoritmos , Animales , Células Cromafines/fisiología , Electrofisiología/instrumentación , Humanos , Análisis de los Mínimos Cuadrados , Microelectrodos , Reconocimiento de Normas Patrones Automatizadas/métodosRESUMEN
BACKGROUND: Electrochemical microelectrodes located immediately adjacent to the cell surface can detect spikes of amperometric current during exocytosis as the transmitter released from a single vesicle is oxidized on the electrode surface. Automated techniques to detect spikes are needed in order to quantify the spike rate as a measure of the rate of exocytosis. NEW METHOD: We have developed a Matched Filter (MF) detection algorithm that scans the data set with a library of prototype spike templates while performing a least-squares fit to determine the amplitude and standard error. The ratio of the fit amplitude to the standard error constitutes a criterion score that is assigned for each time point and for each template. A spike is detected when the criterion score exceeds a threshold and the highest-scoring template and the time of peak score is identified. The search for the next spike commences only after the score falls below a second, lower threshold to reduce false positives. The approach was extended to detect spikes with double-exponential decays with the sum of two templates. RESULTS: Receiver Operating Characteristic plots (ROCs) demonstrate that the algorithm detects >95% of manually identified spikes with a false-positive rate of â¼2%. COMPARISON WITH EXISTING METHODS: ROCs demonstrate that the MF algorithm performs better than algorithms that detect spikes based on a derivative-threshold approach. CONCLUSIONS: The MF approach performs well and leads into approaches to identify spike parameters.
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Potenciales de Acción , Algoritmos , Microelectrodos , Neuronas/fisiología , Reconocimiento de Normas Patrones Automatizadas/métodos , Glándulas Suprarrenales/fisiología , Animales , Carbono , Fibra de Carbono , Bovinos , Células Cultivadas , Células Cromafines/fisiología , Exocitosis , Reacciones Falso Positivas , Análisis de los Mínimos Cuadrados , Ratones , Curva ROC , Procesamiento de Señales Asistido por Computador , Programas InformáticosRESUMEN
Carbon-fiber electrodes (CFEs) are the gold standard for quantifying the release of oxidizable neurotransmitters from single vesicles and single cells. Over the last 15 years, microfabricated devices have emerged as alternatives to CFEs that offer the possibility of higher throughput, subcellular spatial resolution of exocytosis, and integration with other techniques for probing exocytosis including microfluidic cell handling and solution exchange, optical imaging and stimulation, and electrophysiological recording and stimulation. Here we review progress in developing electrochemical electrode devices capable of resolving quantal exocytosis that are fabricated using photolithography.
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Células Cromafines/metabolismo , Técnicas Electroquímicas/métodos , Exocitosis , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , Animales , Carbono , Fibra de Carbono , Técnicas Electroquímicas/instrumentación , Humanos , Microelectrodos , Técnicas Analíticas Microfluídicas/instrumentación , Imagen Óptica/instrumentación , Imagen Óptica/métodosRESUMEN
An electrochemical microelectrode located immediately adjacent to a single neuroendocrine cell can record spikes of amperometric current that result from exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. Here, we report the development of an efficient method where the same electrochemical microelectrode is used to electropermeabilize an adjacent chromaffin cell and then measure the consequent quantal catecholamine release using amperometry. Trains of voltage pulses, 5-7 V in amplitude and 0.1-0.2 ms in duration, were used to reliably trigger release from cells using gold electrodes. Amperometric spikes induced by electropermeabilization had similar areas, peak heights and durations as amperometric spikes elicited by depolarizing high K(+) solutions, therefore release occurs from individual secretory granules. Uptake of trypan blue stain into cells demonstrated that the plasma membrane is permeabilized by the voltage stimulus. Voltage pulses did not degrade the electrochemical sensitivity of the electrodes assayed using a test analyte. Surprisingly, robust quantal release was elicited upon electroporation in the absence of Ca(2+) in the bath solution (0 Ca(2+)/5 mM EGTA). In contrast, electropermeabilization-induced transmitter release required Cl(-) in the bath solution in that bracketed experiments demonstrated a steep dependence of the rate of electropermeabilization-induced transmitter release on [Cl(-)] between 2 and 32 mM. Using the same electrochemical electrode to electroporate and record quantal release of catecholamines from an individual chromaffin cell allows precise timing of the stimulus, stimulation of a single cell at a time, and can be used to load membrane-impermeant substances into a cell.
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Técnicas Electroquímicas/métodos , Electroporación , Células Neuroendocrinas/citología , Células Neuroendocrinas/metabolismo , Neurotransmisores/análisis , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/metabolismo , Animales , Bovinos , Células Cultivadas , Técnicas Electroquímicas/instrumentación , Microelectrodos , Neurotransmisores/metabolismo , Análisis de la Célula IndividualRESUMEN
A method for the selective labeling and imaging of catecholamines in live and fixed secretory cells is reported. The method integrates a tailored approach using a novel fluorescence-based turn-on molecular sensor (NeuroSensor 521) that can exploit the high concentration of neurotransmitters and acidic environment within secretory vesicles for the selective recognition of norepinephrine and dopamine. The utility of the method was demonstrated by selectively labeling and imaging norepinephrine in secretory vesicles such that discrimination between norepinephrine- and epinephrine-enriched populations of chromaffin cells was observed. This method was validated in fixed cells by co-staining with an anti-PNMT antibody.
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Células Cromafines/química , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Norepinefrina/análisis , Animales , Sitios de Unión/fisiología , Catecolaminas/análisis , Bovinos , Células CultivadasRESUMEN
The design, fabrication and test of a microfluidic cell trapping device to measure single cell exocytosis were reported. Procedures on the patterning of double layer template based on repetitive standard photolithography of AZ photoresist were investigated. The replicated poly(dimethyl siloxane) devices with 2.5 µm deep channels were proved to be efficient for stopping cells. Quantal exocytosis measurement can be achieved by targeting single or small clumps of chromaffin cells on top of the 10 µm × 10 µm indium tin oxide microelectrodes arrays with the developed microdevice. And about 72 % of the trapping sites can be occupied by cells with hydrodynamic trapping method and the recorded amperometric signals are comparable to the results with traditional carbon fiber microelectrodes. The method of manufacturing the microdevices is simple, low-cost and easy to perform. The manufactured device offers a platform for the high throughput detection of quantal catecholamine exocytosis from chromaffin cells with sufficient sensitivity and broad application.
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Separación Celular/instrumentación , Dimetilpolisiloxanos/química , Exocitosis , Hidrodinámica , Técnicas Analíticas Microfluídicas/instrumentación , Compuestos de Estaño/química , Animales , Bovinos , Electroquímica , MicroelectrodosRESUMEN
We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrodes were fabricated from three different conducting materials: Indium-tin-oxide (ITO), nitrogen-doped diamond-like carbon (DLC) deposited on top of ITO, or very thin (12-17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release. The lower sensitivity of ITO electrodes was confirmed by chronoamperometry measurements comparing the currents in the presence of different analytes with the different electrode materials.
RESUMEN
Electrochemical microelectrodes are commonly used to record amperometric spikes of current that result from oxidation of transmitter released from individual vesicles during exocytosis. Whereas the exquisite sensitivity of these measurements is well appreciated, a better understanding of the noise sources that limit the resolution of the technique is needed to guide the design of next-generation devices. We measured the current power spectral density (S(I)) of electrochemical microelectrodes to understand the physical basis of dominant noise sources and to determine how noise varies with the electrode material and geometry. We find that the current noise is thermal in origin in that S(I) is proportional to the real part of the admittance of the electrode. The admittance of microelectrodes is well described by a constant phase element model such that both the real and imaginary admittance increase with frequency raised to a power of 0.84-0.96. Our results demonstrate that the current standard deviation is proportional to the square root of the area of the working electrode, increases â¼linearly with the bandwidth of the recording, and varies with the choice of the electrode material with Au ≈ carbon fiber > nitrogen-doped diamond-like carbon > indium-tin-oxide. Contact between a cell and a microelectrode does not appreciably increase noise. Surface-patterned microchip electrodes can have a noise performance that is superior to that of carbon-fiber microelectrodes of the same area.
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Técnicas Electroquímicas , Exocitosis , Carbono/química , Fibra de Carbono , Diamante/química , Microelectrodos , Compuestos de Estaño/químicaRESUMEN
Biphasic insulin secretion in response to glucose consists of a transient first phase followed by a progressive second phase. It is a well described feature of whole perfused pancreases as well as isolated pancreatic islets of Langerhans. Applying to single cell assays of exocytosis (capacitance monitoring and amperometry) to single canine Beta-cells we have examined the time courses of granule exocytosis in response to voltage-clamp depolarizations that mimic two modes of glucose-induced electrical activity, and then compared these to biphasic insulin secretion. Action potentials evoked in short trains at frequencies similar those recorded during first phase insulin secretion trigger phasic exocytosis from a small pool of insulin granules that are likely docked near voltage-activated Ca²âº channels. In contrast, prolonged voltage-clamp pulses mimicking plateau depolarizations occur during second phase insulin secretion and trigger tonic or continuous exocytosis. Comparing the latter results with ones obtained using photorelease of caged Ca²âº in other insulin-secreting cells, we suggest that tonic exocytosis likely results from granule release from a highly Ca²âº-sensitive pool of insulin granules, likely located further from Ca²âº channels. Both phasic and tonic modes of exocytosis are enhanced by glucose, via its metabolism. Hence, in canine Beta-cells we propose that two distinct modes of exocytosis, tuned to two types of electrical activity, may underlay biphasic insulin secretion.
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Exocitosis , Insulina/metabolismo , Animales , Perros , Secreción de Insulina , Islotes Pancreáticos/metabolismoRESUMEN
Electrochemical microelectrodes are commonly used to detect spikes of amperometric current that correspond to exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. We are developing transparent multielectrochemical electrode arrays on microchips in order to automate measurement of quantal exocytosis. Here, we report development of an improved device to target individual cells to each microelectrode in an array. Efficient targeting (~75%) is achieved using cell-sized microwell traps fabricated in SU-8 photoresist together with patterning of poly(l-lysine) in register with electrodes to promote cell adhesion. The surface between electrodes is made resistant to cell adhesion using poly(ethylene glycol) in order to facilitate movement of cells to electrode "docking sites". We demonstrate the activity of the electrodes using the test analyte ferricyanide and perform recordings of quantal exocytosis from bovine adrenal chromaffin cells on the device. Multiple cell recordings on a single device demonstrate the consistency of spike measurements, and multiple recordings from the same electrodes demonstrate that the device can be cleaned and reused without degradation of performance. The new device will enable high-throughput studies of quantal exocytosis and may also find application in rapidly screening drugs or toxins for effects on exocytosis.
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Electroquímica/instrumentación , Exocitosis , Análisis de la Célula Individual/instrumentación , Animales , Catecolaminas/metabolismo , Bovinos , Células Cromafines/metabolismo , Diseño de Equipo , Equipo Reutilizado , Microelectrodos , Propiedades de SuperficieRESUMEN
Here we describe a method to fabricate a multi-channel high-throughput microchip device for measurement of quantal transmitter release from individual cells. Instead of bringing carbon-fiber electrodes to cells, the device uses a surface chemistry approach to bring cells to an array of electrochemical microelectrodes. The microelectrodes are small and "cytophilic" in order to promote adhesion of a single cell whereas all other areas of the chip are covered with a thin "cytophobic" film to block cell attachement and facilitate movement of cells to electrodes. This cytophobic film also insulates unused areas of the conductive film, thus the alignment of cell docking sites to working electrodes is automatic. Amperometric spikes resulting from single-granule fusion events were recorded on the device and had amplitudes and kinetics similar to those measured using carbon-fiber microelectrodes. Use of this device will increase the pace of basic neuroscience research and may also find applications in drug discovery or validation.
RESUMEN
The release of neurotransmitters and hormones from secretory vesicles plays a fundamental role in the function of the nervous system including neuronal communication. High-throughput testing of drugs modulating transmitter release is becoming an increasingly important area in the fields of cell biology, neurobiology, and neurology. Carbon-fiber amperometry, provides high-resolution measurements of amount and time course of transmitter release from single vesicles, and their modulation by drugs and molecular manipulations. However, such methods do not allow the rapid collection of data from a large number of cells. To allow such testing, we have developed a CMOS potentiostat circuit that can be scaled to a large array. In this paper, we present two post-CMOS fabrication methods to incorporate the electrochemical electrode material. We demonstrate by proof of principle the feasibility of on-chip electrochemical measurements of dopamine, and catecholamine release from adrenal chromaffin cells. The measurement noise is consistent with the typical electrode noise in recordings with external amplifiers. The electronic noise of the potentiostat in recordings with 400 mus integration time is ~0.11 pA and is negligible compared to the inherent electrode noise.
RESUMEN
Neurons and endocrine cells secrete neurotransmitter and hormones in discrete packets in a process called quantal exocytosis. Electrochemical microelectrodes can detect spikes in current resulting from the oxidation of individual quanta of transmitter only if the electrodes are small and directly adjacent to release sites on the cell. Here we report development of a microchip device that uses microfluidic traps to automatically target individual or small groups of cells to small electrochemical electrodes. Microfluidic channels and traps were fabricated by multi-step wet etch of a silicon wafer whereas Pt electrodes were patterned in register with the trap sites. We demonstrate high-resolution amperometric measurement of quantal exocytosis of catecholamines from chromaffin cells on the device. This reusable device is a step towards developing high-throughput lab-on-a-chip instruments for recording quantal exocytosis to increase the pace of basic neuroscience research and to enable screening of drugs that target exocytosis.
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Catecolaminas/análisis , Células Cromafines/citología , Electroquímica/instrumentación , Exocitosis , Técnicas Analíticas Microfluídicas/instrumentación , Animales , Bovinos , Diseño de EquipoRESUMEN
Electrochemical measurement of transmitter or hormone release from individual cells on microchips has applications both in basic science and drug screening. High-resolution measurement of quantal exocytosis requires the working electrode to be small (cell-sized) and located in immediate proximity to the cell. We examined the ability of candidate electrode materials to promote the attachment of two hormone-secreting cell types as a mechanism for targeting cells for to recording electrodes with high precision. We found that nitrogen-doped diamond-like carbon (DLC:N) promoted cell attachment relative to other materials tested in the rank order of DLC:N>In(2)O(3)/SnO(2) (ITO), Pt>Au. In addition, we found that treating candidate electrode materials with polylysine did not increase attachment of chromaffin cells to DLC:N, but promoted cell attachment to the other tested materials. We found that hormone-secreting cells did not attach readily to Teflon AF as a potential insulating material, and demonstrated that patterning of Teflon AF leads to selective cell targeting to DLC:N "docking sites". These results will guide the design of the next generation of biochips for automated and high-throughput measurement of quantal exocytosis.
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Carbono/metabolismo , Diamante/química , Exocitosis , Nitrógeno/química , Animales , Bovinos , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cromafines/citología , Células Cromafines/efectos de los fármacos , Electrodos , Exocitosis/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Metales , Polilisina/farmacología , Politetrafluoroetileno/química , RatasRESUMEN
Carbon-based electrode materials have been widely used for many years for electrochemical charge storage, energy generation, and catalysis. We have developed an electrode material with high specific capacitance by entrapping graphite nanoparticles into a sol-gel network. Films from the resulting colloidal suspensions were highly porous due to the removal of the entrapped organic solvents from sol-gel matrix giving rise to high Brunauer-Emmett-Teller (BET) specific surface areas (654 m(2)/g) and a high capacitance density ( approximately 37 F/g). An exponential increase of capacitance was observed with decreasing scan rates in cyclic voltammetry studies on these films suggesting the presence of pores ranging from micro (< 2 nm) to mesopores. BET surface analysis and scanning electron microscope images of these films also confirmed the presence of the micropores as well as mesopores. A steep drop in the double layer capacitance with polar electrolytes was observed when the films were rendered hydrophilic upon exposure to a mild oxygen plasma. We propose a model whereby the microporous hydrophobic sol-gel matrix perturbs the hydration of ions which moves ions closer to the graphite nanoparticles and consequently increase the capacitance of the film.
RESUMEN
Carbon electrodes are widely used in electrochemistry due to their low cost, wide potential window, and low and stable background noise. Carbon-fiber electrodes (CFE) are commonly used to electrochemically measure "quantal" catecholamine release via exocytosis from individual cells, but it is difficult to integrate CFEs into lab-on-a-chip devices. Here we report the development of nitrogen doped diamond-like carbon (DLC:N) microelectrodes on a chip to monitor quantal release of catecholamines from cells. Advantages of DLC:N microelectrodes are that they are batch producible at low cost, and are harder and more durable than graphite films. The DLC:N microelectrodes were prepared by a magnetron sputtering process with nitrogen doping. The 30 microm by 40 microm DLC:N microelectrodes were patterned onto microscope glass slides by photolithography and lift-off technology. The properties of the DLC:N microelectrodes were characterized by AFM, Raman spectroscopy and cyclic voltammetry. Quantal catecholamine release was recorded amperometrically from bovine adrenal chromaffin cells on the DLC:N microelectrodes. Amperometric spikes due to quantal release of catecholamines were similar in amplitude and area as those recorded using CFEs and the background current and noise levels of microchip DLC:N electrodes were also comparable to CFEs. Therefore, DLC:N microelectrodes are suitable for microchip-based high-throughput measurement of quantal exocytosis with applications in basic research, drug discovery and cell-based biosensors.