Detection of nitric oxide release from single cells by using constant-distance-mode scanning electrochemical microscopy.
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Cell Line↗
Biomedical subjects
Wolfgang Schuhmann
Publications and source records attributed to Wolfgang Schuhmann.
Feedback-independent Pt nanoelectrodes for shear force-based constant-distance mode scanning electrochemical microscopy.
A new generation of platinum nanoelectrodes for constant-distance mode scanning electrochemical microscopy (CD-SECM) has been prepared, characterized, and used for high spatial resolution electrochemical measurements and visualization of electrochemically induced concentration gradients in microcavities. The probes have long (1-2 cm), narrow quartz tips that were conically polished and have a Pt nanoelectrode that is slightly offset from center. Because of the size and location of the electrode on the probe, it does not exhibit SECM feedback while approaching the analyzed sample surfaces even to distances within a few hundred nanometers. The probe was positioned near the surface while scanning and performing electrochemical measurements through use of nonoptical shear force control of the tip-to-sample distance. Test structures consisted of cylindrically shaped microcavities that are 50 microm in diameter with three individually addressable electrodes: a gold disk at 8-microm depth, a crescent-shaped gold ring at 4-microm depth along the wall, and a top gold electrode at the rim. Different electrodes within the microcavity were used to reduce and oxidize redox species in 250 microL of a solution of 5 mM hexaamineruthenium(III) chloride and 0.1 M potassium chloride, protected from evaporation by mineral oil, while the SECM tip followed the topography of the structures and monitored the current from the oxidation of [Ru(NH3)6]2+. Electrochemically generated concentration profiles were obtained from these complex test structures that are not possible with any other SECM technology at this time.
Electrochemistry↗
Parallel synthesis of libraries of anodic and cathodic functionalized electrodeposition paints as immobilization matrix for amperometric biosensors.
The integration of flexible anchoring groups bearing imidazolyl or pyridyl substituents into the structure of electrodeposition paints (EDP) is the basis for the parallel synthesis of a library containing 107 members of different cathodic and anodic EDPs with a high variation in polymer properties. The obtained EDPs were used as immobilization matrix for biosensor fabrication using glucose oxidase as a model enzyme. Amperometric glucose sensors based on the different EDPs showed a wide variation in their sensor characteristics with respect to the apparent Michaelis-Menten constant (KM(app)) representing the linear measuring range and the maximum current (Imax(app)). Based on these results first assumptions concerning the impact of different side chains in the EDP on the expected biosensor properties could be obtained allowing for an improved rational optimization of EDPs used as immobilization matrix in amperometric biosensors.
Biosensing Techniques↗
SECM visualization of spatial variability of enzyme-polymer spots. 1. Discretisation and interference elimination using artificial neural networks.
Enzyme-polymer layers immobilized on an electrode surface often serve as basis for amperometric biosensors. Caused by the formation process they show spatial variability in the polymer thickness which corresponds to a variability of immobilized enzyme activity. The relationship between topography and localized enzymatic activity of enzyme-polymer spots was studied using scanning electrochemical microscopy (SECM) in the feedback mode and generator-collector mode. Discretisation with a grid size corresponding to the scanning parameters defined substructures which can be treated as individual microsensors with specific response characteristics. The local responses are mainly governed by the polymer thickness but also influenced by neighbouring sites. Thus, discretisation allowed us to treat an enzyme-polymer spot with dimensions of about 300 microm diameter like an array of more than 400 individual microsensors. Using suitable selection criteria and multivariate calibration it was possible to identify sensing sites which are optimal for the determination of glucose. It was demonstrated that an artificial neural network which was trained with the data provided by SECM images well predicted glucose concentration in the presence of ascorbic acid.
Biosensing Techniques↗
Simultaneous detection of L-glutamate and nitric oxide from adherently growing cells at known distance using disk shaped dual electrodes.
An ex vivo system for simultaneous detection of nitric oxide (NO) and L-glutamate using integrated dual 250 microm platinum disk electrodes modified individually with suitable sensing chemistries has been developed. One of the sensors was coated with an electrocatalytic layer of Ni tetrasulfonate phthalocyanine tetrasodium salt (Ni-TSPc) covered by second layer of Nafion, which stabilises on the one hand the primary oxidation product NO(+) and prevents interferences from negatively charged compounds such as NO(2)(-). For glutamate determination, the second electrode was modified with a crosslinked redox hydrogel consisting of Os complex modified poly(vinylimidazol), glutamate oxidase and peroxidase. A manual x-y-z micromanipulator on top of an inverted optical microscope was used to position the dual electrode sensor at a defined distance of 5 microm from a cell population under visual control. C6 glioma cells were stimulated simultaneously with bradykinin or VEGF to release NO while KCl was used to invoke glutamate release. For evaluation of the glutamate sensors, in some experiments HN10 cells were used. To investigate the sensitivity and reliability of the system, several drugs were applied to the cells, e.g. Ca(2+)-channel inhibitors for testing Ca(2+)-dependence of the release of NO and glutamate, rotenone for inducing oxidative stress and glutamate antagonists for analysing glutamate release. With these drugs the NO and glutamate release was modulated in a similar way then expected from previously described systems or even in-vivo measurements. We therefore conclude that our system is suitable to analyse stress-induced mechanisms in cell lines.
Animals↗
AFM study of conducting polymer polypyrrole nanoparticles formed by redox enzyme - glucose oxidase - initiated polymerisation.
Redox enzyme - glucose oxidase E.C. 1.1.3.4 from Penecillum vitale (GOx) - initiated polypyrrole (Ppy) synthesis was applied for the formation of polypyrrole based nanoparticles. The increase in optical absorbance at lambda=460 nm was exploited for the monitoring of polypyrrole polymerisation process. The shape and size of the formed Ppy nanoparticles was also monitored by means of contact mode AFM. The highest increase in the diameter of the formed Ppy nanoparticles was detected during 15-day period. AFM imaging was performed in contact mode to investigate the shape and flexibility of particles deposited on the SiO2 and Pt surfaces. Contact mode AFM investigations allowed us to conclude that after drying at 50 degrees C the formed Ppy particles are more flexibly deposited on the Pt electrode if compared to those deposited on the SiO2 substrate. The application of well-shaped Ppy nanoparticles in biomedicine, chromatography and bioanalysis may be predicted.
Electric Conductivity↗
Electrochemical high-content screening of nitric oxide release from endothelial cells.
Release of nitric oxide (NO) is of high importance for regulating endothelial cell functions during vasodilatation, vascular remodeling, and angiogenesis. Thus, a direct and reliable real-time method for NO detection that takes into account time-dependent variations of the NO concentration in the complex reaction within the diffusion zone above the cells is vital for obtaining information about the role of NO in intracellular endothelial signal transduction and its impact on the surrounding cells. In this study, the time course of vascular endothelial growth factor E (VEGF-E) stimulated NO release from transformed human umbilical vein endothelial cells (T-HUVEC) was investigated by means of metalloporphyrin-based NO sensors employed in an electrochemical robotic system. The NO sensor was obtained by electrochemically induced deposition of Ni(II) tetrakis(p-nitrophenylporphyrin) on a 50-microm diameter platinum disk electrode which was integrated, together with a 25-microm diameter platinum disk, in a double-barrel electrode arrangement. The second electrode was used as a guidance sensor for the automatic and highly reproducible positioning of the NO sensor at a known distance from a layer of adherently growing cells by using z-approach curves in the negative feedback mode of scanning electrochemical microscopy (SECM). The electrochemical robotic system allows the fully automated detection of NO with high sensitivity and selectivity to be performed in real time within 96-well microtiter plates. A functional cell assay was established to allow the standardized detection of NO released upon stimulation from T-HUVEC with a sensor positioned at a known distance above the endothelial cells. The overall system was evaluated by automatic detection of NO release from T-HUVEC upon stimulation with VEGF-E after incubation with a variety of drugs that are known to act on different sites in the complex signal-transduction pathway that finally invokes NO release.
Biological Assay↗
Fabrication of a potentiometric/amperometric bifunctional enzyme microbiosensor.
We report the fabrication and functional characterization of a needle-type bifunctional enzyme microbiosensor that has, as technical novelty, simultaneously integrated a potentiometric and amperometric detection of an enzyme-catalyzed reaction at the tip of a pulled glass micropipet. The construction involved immobilizing an enzyme onto the platinized outer tip surface using the precipitation of electrodeposition paint with direct entrapment of the biocomponent in the slowly growing polymer film. Products of enzyme-substrate reaction could then be targeted in a dual-detection mode on one hand with the covered Pt layer at the tip region as amperometric detector and on the other hand with a proton-selective liquid membrane-based potentiometric sensor inside the open pipet tip. Completing and testing bifunctional glucose microsensors demonstrated the functionality of the proposed strategy. Synchronized amperometric and potentiometric detection of the addition of a glucose standard to a buffer solution became evident by observing stepwise increases in the amperometric H2O2 oxidation current and corresponding increases in the potential of the pH-selective sensor, which translates to a local pH decrease around the tip due to hydrolysis of enzymatically formed gluconic acid.
Aspergillus niger↗
Robotic sequential analysis of a library of metalloporphyrins as electrocatalysts for voltammetric nitric oxide sensors.
A library of 83 metalloporphyrins with varying substitution pattern at the meso-position of the porphyrins and different central metal ions in the core region has been synthesized in small quantities using a parallel synthesis strategy. By means of a specially designed electrochemical robotic device integrating a 96-well microtitre plate and an easily movable assembly of working, counter and reference electrodes, the different porphyrins were automatically applied in sequence to an in-well electrochemical preparation and testing of NO sensors. Screening the entire compound collection suggested initial considerations concerning the influence of varied functionalities of the metalloporphyrins on their electrocatalytic properties for the oxidation of NO and helped to identify the quality of the investigated catalyst candidates. As compared to manually performed quality tests, the proposed strategy of automation has advantages in convenience, rapidity and especially reproducibility avoiding any inaccuracies introduced by manually performing all steps of the complex sensor formation and testing sequence.
Animals↗
Glutamate detection from nerve cells using a planar electrodes array integrated in a microtiter plate.
There is an increasing interest in new strategies for replacing animal tests in research. The use of cell cultures and integrated electrodes is seen as a promising alternative that could potentially solve this problem. In this work, we present a L-glutamate sensor based on a bienzyme redox hydrogel, capable of detecting the release of this excitatory neurotransmitter from adherently growing cells upon stimulation. The low working potential required for the operation of the sensor decreases the possibility of interference by easily oxidizable compounds always present in complex biological samples. A low detection limit of 0.5 microM L-glutamate, a response time of about 35 s, and a linear range of up to 60 microM are the main characteristics of the sensor. The system has been successfully employed to monitor the release of l-glutamate from HN10 and C6 cells upon stimulation with K(+)-ions. The developed integrated electrochemical platform will be used in future for drug screening and potentially for replacing animal models in neurological experiments.
Biological Assay↗
Simultaneous detection of the release of glutamate and nitric oxide from adherently growing cells using an array of glutamate and nitric oxide selective electrodes.
The simultaneous detection of nitric oxide and glutamate using an array of individually addressable electrodes, in which the individual electrodes in the array were suitably modified with a highly sensitive nitric oxide sensing chemistry or a glutamate oxidase/redox hydrogel-based glutamate biosensor is presented. In a sequence of modification steps one of the electrodes was covered first with a positively charged Ni porphyrin entrapped into a negatively charged electrodeposition paint followed by the manual modification of the second working electrode by a bienzyme sensor architecture based on crosslinked redox hydrogels with entrapped peroxidase and glutamate oxidase. Adherently growing C6-glioma cells were grown on membrane inserts and placed in close distance to the modified sensor surfaces. The current responses recorded at each electrode after stimulation of glutamate and NO release by means of K+ and bradykinin clearly demonstrate the ability of the individual electrode in the array to detect the analyte towards which its sensitivity and selectivity was targeted without interference from the neighbouring electrode or other analytes present in the test mixture.
Animals↗
Cell-compatible array of three-dimensional tip electrodes for the detection of nitric oxide release.
An array of electrodes on which cells could be grown directly was fabricated using silicon anisotropic etching and a thick-photoresist process and employed for the detection of nitric oxide (NO) released from a population of adherently growing human umbilical vein endothelial cells (HUVEC). The electrodes are tip-shaped and are 40 microm high of which only the top 15 microm are exposed Pt-tips. After electrochemical induced modification of the exposed Pt tips using Ni phthalocyanine the individual addressable electrode tips were sensitive and selective for the detection of NO at an applied constant potential of 750 mV. The silicon nitride insulation of the lower part of the tip electrodes prevented the death of the cells upon the application of the working potential at which NO was detected. It also helped to avoid the perturbation of the integrity of the sensing chemistry imparted on the electrode surface that could have resulted from the contact of the adherently growing cells with the active electrode surface. The release of nitric oxide from HUVEC was successfully monitored with different numbers of tip electrodes simultaneously connected as combined working electrode.
Cell Culture Techniques↗
A novel L-lactate-selective biosensor based on flavocytochrome b2 from methylotrophic yeast Hansenula polymorpha.
A novel amperometric biosensor highly selective to L-lactate has been developed using L-lactate-cytochrome c oxidoreductase (flavocytochrome b2) isolated for the first time from thermotolerant methylotrophic yeast Hansenula polymorpha as biorecognition element. Different immobilization methods and low-molecular free-diffusing redox mediators have been tested for optimising the electrochemical communication between the immobilized enzyme and the electrode surface. Moreover, the possibility of direct electron transfer from the reduced form of FCb2 to carbon electrodes has been evaluated. The bioanalytical properties of FCb2-based biosensors, such as signal rise time, dynamic range, dependence of the sensor output on the pH value, the temperature and the storage stability were investigated, and the proposed biosensor demonstrated a very fast response and a high sensitivity and selectivity for L-lactate determination.
Biosensing Techniques↗
Electrochemical nitric oxide sensor preparation: a comparison of two electrochemical methods of electrode surface modification.
Platinum electrodes modified with Mn(II) 5-(N-(8-pyrrole-yl-3,6-dioxa-1-aminooctane)phenylamide-10,15,20-trimethoxyphenylporphyrin (Mn(II)triOMeTCPPyP) using multi-sweep cyclic voltammetry and differential pulse amperometry were evaluated as electrocatalytic surfaces for the oxidation of nitric oxide. The electrodes modified using the pulse amperometric approach were more sensitive towards the detection of nitric oxide. The increased sensitivity led to the attainment of a wider linear dynamic range for the quantification of nitric oxide.
Electrodes↗
Imaging immobilised ssDNA and detecting DNA hybridisation by means of the repelling mode of scanning electrochemical microscopy (SECM).
The supposed repelling mode of scanning electrochemical microscopy (SECM) allows truly label-free electrochemical recognition of the presence and hybridisation of nucleic acids that are immobilised on conducting DNA chips. Basically, the SECM-based detection of single- and double-stranded DNA profits from the electrostatic repulsion between deprotonated phosphate groups at the backbone of the oligonucleotides and a free-diffusing negatively charged redox mediator (e.g. [Fe(CN)(6)](3-/4-)). In electrolytes of proper pH and ionic strength, this coulomb interaction is heavily influencing the diffusion properties of the mediator in the vicinity of the surface-anchored DNA strands. This charge interaction modulates the diffusional mass transport for the charged redox species in the DNA modified regions, and thus locally decreases the positive feedback currents measured with a SECM tip placed within the electrochemical nearfield of the chip surface. This approach was used to study arrays of synthetic 20-base oligonucleotide probes that were immobilised on monolayer-modified gold surfaces. Evidence is provided that the density of probes, the ionic strength of solution and the tip-to-sample distance have a strong impact on the capability of the repelling mode of SECM to visualise probe spots and hybridisation while the concentration of the chosen mediator did not significantly affect detection.
Coated Materials, Biocompatible↗
Dual microelectrodes for distance control and detection of nitric oxide from endothelial cells by means of scanning electrochemical microscope.
Dual Pt disk microelectrodes consisting of a 10-microm distance sensor and a 50-microm nitric oxide sensor were prepared. The 50-microm electrode was modified with Ni(4-N-tetramethyl)pyridyl porphyrin enclosed in the polymer network of a negatively charged electrodeposition paint. This paint prevented the dissolution of the otherwise soluble porphyrin in the aqueous test medium due to charge interactions. It also denied negatively charged ions in the analyte solution access to the electrode surface by electrostatic repulsion, thereby preventing interference from anions such as nitrite, nitrate, and ascorbate. With the aid of a scanning electrochemical microscope, it was possible to use the distance sensor by recording the negative feedback effect on the reduction of molecular oxygen to "guide" the nitric oxide sensor to various known distances from a layer of adherently growing human umbilical vein endothelial cells for the detection of nitric oxide released from the cells upon stimulation with bradykinin. The use of the distance sensor made it possible to preserve the integrity of the adherently growing cells concomitantly with the modified electrode by preventing the deterioration of the modifying layer during the distance adjustment step.
Cells, Cultured↗
Constant-distance mode scanning potentiometry. 1. Visualization of calcium carbonate dissolution in aqueous solution.
Constant-distance mode scanning potentiometry was established by integrating potentiometric microsensors as ion-selective scanning probes into a SECM setup that was equipped with a piezoelectric shear force-based tip-to-sample distance control. The combination of specially designed micrometer-sized potentiometric tips with an advanced system for tip positioning allowed simultaneous acquisition of both topographic and potentiometric information at solid/liquid interfaces with high spatial resolution. The performance of the approach was evaluated by applying Ca(2+)-selective constant-distance mode potentiometry to monitor the dissolution of calcium carbonate occurring either at the (104) surface of calcite crystals or in proximity to the more complex surface of cross sections of a calcium carbonate shell of Mya arenaria exposed to slightly acidic aqueous solutions. Micrometer-scale heterogeneities in the apparent calcium activity profiles have successfully been resolved for both samples.
Calcium Carbonate↗
Label-free electrochemical recognition of DNA hybridization by means of modulation of the feedback current in SECM.
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Base Sequence↗