Electrochemical push-pull probe: from scanning electrochemical microscopy (SECM) to multimodal altering of cell microenvironment

To understand biological processes at the cellular level, a general approach is to alter the cells’ environment and to study their chemical responses. Herein, we present the implementation of an electrochemical push-pull probe, which combines a microfluidic system with a microelectrode, as a tool for locally altering the microenvironment of few adherent living cells by working in two different perturbation modes, namely electrochemical (i.e. electrochemical generation of a chemical effector compound) and microfluidic (i.e. infusion of a chemical effector compound from the pushing microchannel, while aspirating it through the pulling channel thereby focusing the flow between the channels). The effect of several parameters such as flow rate, working distance and probe inclination angle on the affected area of adherently growing cells was investigated both theoretically and experimentally. As a proof of concept, localized fluorescent labeling and pH changes were purposely introduced to validate the probe as a tool for studying adherent cancer cells through the control over the chemical composition of the extracellular space with high spatiotemporal resolution. A very good agreement between experimental and simulated results showed for instance, that the electrochemical perturbation mode enables to affect precisely only few living cells localized in a high-density cell culture

Amperometric biosensor-based microsystems for detecting analytes of biomedical importance

Monitoring in the biomedical field is rapidly emerging as the most important application area of miniaturized analytical devices. Diagnostics, disease monitoring, drug development, and medical research are important areas taking full advantage of the short response time, reduced sample consumption, low costs, and reduced power requirements of miniaturized analytical tools. Such tools can either integrate several standard analytical steps (micro-Total Analysis System, µTAS, or Lab-on-a-Chip) or consist only of detection units, which do not need sample preparation steps (e.g. microsystems based on biosensors). Electrochemical detectors are of special interest in developing miniaturized analytical devices, because they are simple, easy to miniaturize with already established technologies, do not require labeling procedures, and yield reliable results often without previous separation steps. Amperometric biosensor-based microsystems are analytical devices combining at micrometer scale the advantages of electrochemical detection with the selectivity of biological or biologically-derived sensing elements (e.g. the selectivity of an enzyme for its substrate). Several such microsystems are meeting the requirements of in vivo or ex vivo monitoring in the biomedical field. Needle type amperometric microsensors are currently used to monitor neurotransmitters in vivo in animal brain, microdialysis probes combined with flow-through biosensor-based microdetectors are used to monitor glucose and lactate from head trauma patients in neurointensive care, and ex vivo monitoring of glucose with portable amperometric biosensors is part of daily life for those suffering of diabetes. In addition, some of the new applications of amperometric microsensors (e.g. Scanning Electrochemical Microscopy, SECM) allow investigation of biochemical processes even at single cell level. This thesis is focusing on several aspects of amperometric biosensor-based microsystems: (i) biosensor improvement by use of both novel enzymes, and enzyme immobilization methods suitable for microelectrodes, (ii) new detection procedures/ schemes, based on surface patterned enzyme microstructures and Scanning Electrochemical Microscope, and (iii) device design, fabrication and evaluation

Design, viualisation and utilisation of enzyme microstructures built on solid surfaces

Abstract in Undetermined Immobilized bioelements offer additional sensitivity and/or selectivity in analytical techniques, in biotechnological devices, and in different medical applications. Great research interest has recently been focused in all these fields on miniaturization techniques, special attention being accorded to the patterning of biomolecules (e.g. enzymes) on solid surfaces with micrometer resolution. Novel microanalysis systems consider the use of enzyme microstructures as e.g. part of mu- Total Analysis System platforms as biochemical microreactors or detection units. This mini-review highlights recent advances in the creation of enzyme microstructures by indirect methods (photolithography, microcontact printing. etc.) and active placement methods (electrospraying, microdispensing, etc.). Some key visualization techniques of enzyme microstructures (fluorescence microscopy, scanning electrochemical microscopy, etc.) are also mentioned together with examples of their applications or application possibilities

Miniaturized on-line digestion system for the sequential identification and characterization of protein analytes

A miniaturized on-column digestion system constructed for the sequential analysis of semi-purified protein analytes is presented. By utilizing fused silica capillary (diameter 150 mu m) packed with a zone of trypsin-modified Eupergit C beads and a second zone of reversed-phase C18 material, a linear column set-up was constructed. The protein analytes (pmol amounts) were first digested in the 600 nl trypsin reactor portion of the system. Next, the generated peptides were trapped in the C18 column shaped as an electrospray emitter. Finally, after washing the matrix free from salts and other hydrophilic impurities present in the sample, peptides were eluted. A stepwise increased concentration profile of organic solvent, created by a dual syringe pump system, promoted the release of bound peptides, which were identified by electrospray ionization MS/MS. This approach proved to be very efficient, achieving almost complete digestion of the proteins studied, with suitable operational stability maintained for more than 1 week. Further, a small nebulizer was designed and fitted to the electrospray emitter. A significant improvement of the spray stability was observed and droplet build-up on the capillary was avoided, even at flow rates well above 1500 nl/min. The proteins chloroperoxidase, staphylococcal enterotoxin B and protein A (injection volume 0.3 mu l, salt concentration 0.2-1 M) were sequentially digested, desalted, eluted, detected and conclusively identified by bioinformatics web tools with an analytical cycle time of 10 min

Amperometric biosensor-based flow-through microdetector for microdialysis applications

A new flow-through electrochemical microcell was fabricated, in order to be used as an on-line detector in microdialysis-based investigations. The microcell is obtained by sandwich-like assembling the electrode system patterned on a glass chip and a silicon rubber gasket bearing the fluidic elements (inlet, outlet, flow channel, and containers for reference and counter electrode). Four Pt microelectrodes (100 mum x 100 mum squares), a Pt counter electrode, and a Ag reference electrode were patterned on a 9 mm x 14 mm glass chip by thin film technology. The silicon rubber cover was fabricated using a polydimethylsiloxane based precursor and a plastic mold, and it avoids significant dilution of the sample before getting in contact with the sensing electrodes. The microdetector can be directly connected to the outlet of the microdialysis probe reducing significantly the delay between sampling and detection. The microelectrodes were modified with enzyme-based chemistries in order to detect glucose, glutamate, and choline by electrooxidation of the hydrogen peroxide produced in the reaction of the analytes with their corresponding oxidases. Problems concerning appropriate linear range, interference elimination, and cross-talk elimination were addressed. (C) 2004 Elsevier B.V. All rights reserved

Bienzyme biosensors for glucose, ethanol and putrescine built on oxidase and sweet potato peroxidase

Amperometric biosensors for glucose, ethanol, and biogenic amines (putrescine) were constructed using oxidase/peroxidase bienzyme systems. The H2O2 produced by the oxidase in reaction with its substrate is converted into a measurable signal via a novel peroxidase purified from sweet potato peels. All developed biosensors are based on redox hydrogels formed of oxidases (glucose oxidase, alcohol oxidase, or amine oxidase) and the newly purified sweet potato peroxidase (SPP) cross-linked to a redox polymer. The developed electrodes were characterized (sensitivity, stability, and performances in organic medium) and compared with similarly built ones using the 'classical' horseradish peroxidase (HRP). The SPP-based electrodes displayed higher sensitivity and better detection limit for putrescine than those using HRP and were also shown to retain their activity in organic phase much better than the HPR based ones. The importance of attractive or repulsive electrostatic interactions between the peroxidases and oxidases (determined by their isoelectric points) were found to play an important role in the sensitivity of the obtained sensor

High temporal resolution monitoring of fermentations using an on-line amperometric flow-through microdetector

An on-line microdetector containing amperometric biosensors was used for high temporal resolution (t(90%) approximate to 120 s) monitoring of glucose and ethanol concentrations during small scale fermentations. ne ability of the microdetector to report on the effect of different experimental conditions was tested in fermentation processes carried out at 30 and at 37 degrees C. An increased ethanol production rate accompanied by an increased glucose consumption rate in the fermentation carried out at 37 degrees C was promptly revealed. Therefore, the microdetector proved to be an especially useful tool to monitor fermentations where the investigated processes are too fast to be followed by classical analytical approaches

Comparison of two glutathione S-transferases used in capacitive biosensors for detection of heavy metals

This work describes the development of a heavy-metal biosensor based on either recombinant 6His-Tag glutathione S-transferase (GST-(His)(6)) or glutathione S-transferase Theta 2-2 (GST-theta 2-2), and a capacitive transducer. The dynamic range of the pure bovine liver GST-Theta 2-2 biosensor was 1 fM to 1mM for Zn2+, and 10pM to 1mM for Cd2+. The GST-(His) 6 biosensor was able to detect Zn2+ and Cd2+ in the range of 1 fM to 10 mu M, and Hg2+ in the range of 1 fM to 10mM. The bovine liver GST Theta 2-2 biosensor displays an increased selectivity and a wider dynamic range for Zn2+ compared with the GST-(His) 6 biosensor. Therefore, by using different GST isozymes, it is possible to modulate important characteristics of capacitive biosensors for the detection of heavy metals

Visualization of micropatterned complex biosensor sensing chemistries by means of scanning electrochemical microscopy

Redox hydrogel-based micropatterned complex biosensor architectures, used as sensing chemistries in amperometric ethanol or glucose biosensors, were deposited on gold, graphite or glass. Well-localized immobilization of active hydrogels with variable compositions was achieved by dispensing 100 pl droplets of cocktails containing alcohol or glucose dehydrogenase, redox polymer (PVI(13)dmeOs) and crosslinker (PEGDGE) while moving the target surface relative to the position of the nozzle of a piezo-actuated microdispenser. The resulting structures were microscopic patterns of enzyme-containing lines of a redox hydrogel with a line width of about 100 mum. Scanning electrochemical microscopy (SECM) in the amperometric feedback mode was used to visualize the immobilized enzyme microstructures and their localized biochemical activity was observed with high lateral resolution by detecting the enzymatically consumed substrate using K-4[Fe(CN)(6)] as a free-diffusing electron-transfer mediator. (C) 2003 Elsevier B.V. All rights reserved

Ultramicrobiosensor for the selective detection of glutamate

Carbon fiber microelectrodes, able to detect catecholamine release from single cells, have significantly contributed to our present understanding of the mechanism of secretory neurotransmission. In spite of their obvious advantages, there are only a few amperometric sensors (characterized by appropriate size, sensitivity, and selectivity) able to measure the release of other (not easily oxidizable) neurotransmitters at cellular level. The present work describes the fabrication and characterization of an ultramicrobiosensor for the selective detection of glutamate. ne developed sensor has a size of 2.5 - 15 mu m in diameter, a sensitivity of 0.62 mA mM(-1) cm(-2), and a detection limit of 5 mu M. The excellent selectivity of the sensor (achieved using electrodeposition of Ru, Rh, and poly(m-phenylenediamine)) makes it a promising candidate for monitoring glutamate release at single cell level