Enzyme-modified Microelectrodes Research Articles

Enzyme-Modified Microelectrode for Simultaneous Local Measurements of O2 and pH.

The use of miniaturized probes opens a new dimension in the analysis of (bio)chemical processes, enabling the possibility to perform measurements with local resolution. In addition, multiparametric measurements are highly valuable for a holistic understanding of the investigated process. Therefore, different strategies have been suggested for simultaneous local measurements of various parameters. Electroanalytical methods are a powerful strategy in this direction. However, they have been mainly restricted to coupling concurrent independent measurements with different miniaturized probes. Here, we present an enzymatic microbiosensor for the simultaneous detection of O2 and pH. The sensing strategy is based on the pH-dependent bioelectrocatalytic process associated with O2 reduction at a gold microelectrode modified with a multicopper oxidase. After initial investigations of the bioelectrocatalytic reaction over gold macroelectrodes, the fabrication and characterization of micrometer-sized probes are presented. The microbioelectrode exhibits a linear current increase with O2 concentration extending to 17.2 mg L-1, with a sensitivity of (5.56 ± 0.13) nA L mg-1 and a limit of detection of (0.5 ± 0.3) mg L-1. Moreover, a linear response allowing pH detection is obtained between pH 5.2 and 7.5 with a slope of -(47 ± 8) mV per pH unit. In addition, two proof-of-concept analytical examples are shown, demonstrating the capability of the developed sensing system for simultaneous local measurements of O2 and pH. Compared with other miniaturized probes reported before for simultaneous detection, our strategy stands out as the two investigated parameters are acquired from the very same measurement. This strategy greatly simplifies the analytical setup and for the first time provides truly simultaneous local detection in the micrometer scale.

Biocatalytic metal nanopatterning through enzyme-modified microelectrodes

The formation of narrow-size distribution nanomaterials on surfaces in defined patterns is a research area of great interest due to its relevance in many applications such as catalysis, optoelectronics, and sensing devices. Patterning surface with nanostructures has been achieved by numerous techniques including electron-beam lithography, microcontact printing, constructive lithography, and different scanning probe microscopy techniques. Here, we present a different approach by which gold patterns are formed by an enzyme-catalyzed reaction followed by a surface-catalyzed process. Our study takes the advantage of scanning electrochemical microscopy (SECM) where the tip is modified with an enzyme and generates a reductant. The latter participates in an electroless deposition reaction, where AuCl4− is reduced catalyzed by a Pd surface. The result is local deposition of gold patterns made of nanoparticles as soon as the reductant generated by the tip, i.e., hydroquinone, approaches the Pd surface. Two enzymes were used: glucose oxidase (GOx) and alkaline phosphatase (ALP). The entire process was carefully studied and optimized, which enabled a good control of the patterns formed.

Enzyme-modified microelectrodes for electrochemical detection of sphingomyelin in the plasma membranes of single cells

In this communication, sphingomyelin (SM) in the plasma membrane of single cells is detected electrochemically using an enzyme-modified Pt microelectrode for the first time. The enzymes immobilized at the electrode surface, which include sphingomyelinase (SMase), alkaline phosphatase (ALP), and choline oxidase, react with SM in a series of reactions to generate hydrogen peroxide. The electrochemical oxidation of hydrogen peroxide induces an elevation in the current, which offers a quantitative determination of SM in a range from 140 to 560 µM. Further contact of the microelectrode with one oocyte cell results in the reaction of SM in the plasma membrane with the enzymes at the electrode. The resulting current increase is correlated with the content of SM in the plasma membrane, confirming the electrochemical detection of membrane SM in single cells. This success in single cell electrochemical analysis of membrane SM expands the application of microelectrodes in single cell studies, which will help in the elucidation of cellular heterogeneity in lipid trafficking.

Self-Organized Nanostructure Modified Microelectrode for Sensitive Electrochemical Glutamate Detection in Stem Cells-Derived Brain Organoids.

Neurons release neurotransmitters such as glutamate to communicate with each other and to coordinate brain functioning. As increased glutamate release is indicative of neuronal maturation and activity, a system that can measure glutamate levels over time within the same tissue and/or culture system is highly advantageous for neurodevelopmental investigation. To address such challenges, we develop for the first time a convenient method to realize functionalized borosilicate glass capillaries with nanostructured texture as an electrochemical biosensor to detect glutamate release from cerebral organoids generated from human embryonic stem cells (hESC) that mimic various brain regions. The biosensor shows a clear catalytic activity toward the oxidation of glutamate with a sensitivity of 93 ± 9.5 nA·µM−1·cm−2. It was found that the enzyme-modified microelectrodes can detect glutamate in a wide linear range from 5 µM to 0.5 mM with a limit of detection (LOD) down to 5.6 ± 0.2 µM. Measurements were performed within the organoids at different time points and consistent results were obtained. This data demonstrates the reliability of the biosensor as well as its usefulness in measuring glutamate levels across time within the same culture system.

Carbon-fiber microelectrodes for in vivo applications

Carbon-fiber microelectrodes (CFMEs) have been a useful tool for measuring rapid changes in neurotransmitters because of their small size, sensitivity, and good electrochemical properties. In this article, we highlight recent advances using CFMEs for measuring neurotransmitters in vivo. Dopamine has been a primary neurotransmitter of interest but direct electrochemical detection of other neurochemicals including nitric oxide and adenosine has also been investigated. Surface treatments have been studied to enhance electrode sensitivity, such as covalent modification or the addition of a layer of carbon nanotubes. Enzyme-modified microelectrodes that detect non-electroactive compounds further extend the usefulness of CFMEs beyond the traditional monoamines. CFMEs continue to be used in vivo to understand basic neurobiological mechanisms and the actions of pharmacological agents, including drugs of abuse. Advances in sensitivity and instrumentation now allow CFMEs to be used for measurements of natural dopamine release that occur during behavioral experiments. A new technique combining electrochemistry with electrophysiology at a single microelectrode facilitates a better understanding of neurotransmitter concentrations and their effects on cell firing. Future research in this field will likely concentrate on fabricating smaller electrodes and electrode arrays, as well as expanding the use of CFMEs in neuroscience beyond dopamine.

Electrochemical detection of acetylcholine and choline: application to the quantitative nonradiochemical evaluation of choline transport

The development of analytical methods for determining the cholinergic biomarkers acetylcholine (ACh) and choline (Ch) is important for assessing their role in neurological and cognitive functions. In this review, electrochemical (EC) strategies to detect ACh and Ch are summarized and compared to other analysis methods. Recent research focusing on the development of a versatile nonradiochemical in vitro assay to evaluate Ch transport is also described. The assay coupled to analysis by capillary electrophoresis (CE) with indirect EC detection at an enzyme-modified microelectrode affords exceptional selectivity and sensitivity. Femtomole or lower mass detection limits for ACh (1 fmol) and Ch (100 amol) have been readily achieved, opening up a new range of possible experiments for investigating transport or turnover of Ch and ACh in neurobiological systems. The value of this method is illustrated through the evaluation of the pharmacological efficacy and mode of inhibition of a new class of quaternary ammonium alkyl-substituted catechol-based inhibitors of high-affinity choline transport (CHT). This microanalytical approach is particularly useful when knowledge of endogenous concentrations of Ch or ACh is desired or when the amount of available compounds or the sample size is limited. A brief description of the principles of CE is also provided.

Inhibition of choline transport by redox-active cholinomimetic bis-catechol reagents

Both N, N′-(2,3-dihydroxybenzyl)- N, N, N′, N′-tetramethyl-1,6-hexanediamine dibromide (DTH, 6) and N, N′-(2,3-dihydroxybenzyl)- N, N, N′, N′-tetramethyl-1,10-decanediamine dibromide (DTD, 7), which are symmetrical bis-catechol substituted hexamethonium and decamethonium analogues, respectively, were found to inhibit high-affinity choline transport in mouse brain synaptosomes. Inhibitory properties were evaluated using an extraordinarily sensitive capillary electrophoresis method employing electrochemical detection at an enzyme-modified microelectrode. Dose–response curves were generated for each inhibitor and IC 50 values were determined to be 76 μM for 6 and 21 μM for 7. Lineweaver–Burk analysis revealed that both molecules inhibit high-affinity choline uptake by a mixed inhibition mechanism. The K I values for 6 and 7 were determined to be 73 ± 1 and 31 ± 2 μM, respectively. The inhibition properties were further compared to a series of mono-catechol analogues, 3-[(trimethylammonio)methyl]catechol ( 1), N, N-dimethylepinephrine ( 4) and 6-hydroxy- N, N-dimethylepinephrine ( 5), as well as the well-characterized hemicholinium inhibitors, hemicholinium-15 (HC-15, 8) and hemicholinum-3 (HC-3, 9).

Voltammetric analysis of glucose using poly 4-vinylpyridine modified carbon fiber electrode

An enzyme-modified microelectrode has been constructed and evaluated. The 4-vinlypyridine was polymerized anodically at its optimum onto a carbon fiber (8 µm diameter) when its concentration was 0.003 M, pH 3.0, scan rate 50 mV s−1 and at a constant potential of +0.40 V vs. SCE. Initial investigation indicated that the type of supporting electrolyte and solvent used affected this polymerization. Glucose oxidase was then dip-coated onto the polymer modified electrode for glucose determination. The dip-coating enzyme immobilization technique has produced the best current response. The glucose analysis was only done in vitro for a whole blood sample. The effects of common interferents in glucose determination are also reported. The operational life of the electrode spanned over two weeks of constant use under normal laboratory condition. The in-vivo glucose analysis has yet to be done but preliminary work on this looked very promising. This topic will be discussed in future articles.

Classification of the mode of inhibition of high-affinity choline uptake using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode

A nonradiochemical in vitro assay using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode has been developed to evaluate the inhibition of high-affinity choline transport in synaptosomes. Quantitative analysis of high-affinity choline transporter rates as a function of inhibitor and substrate concentrations allowed determination of the mode of inhibition for the quaternary ammonium-catechol-based inhibitors 3-[(trimethylammonio)methyl]catechol, N,N-dimethylepinephrine, and 6-hydroxy- N,N-dimethylepinephrine. The results are compared to the well-characterized inhibitor of choline transport, hemicholinium-3.

Microfluidic device integrated with pre-reactor and dual enzyme-modified microelectrodes for monitoring in vivo glucose and lactate

A microfluidic device integrated with a micro-pre-reactor and glucose- and lactate-sensing microelectrodes in a dual channel was fabricated for in vivo monitoring using a micromachining technique. The device consists of two glass plates bonded together with UV-curable resin. The microelectrodes were made of carbon film, and a thin layer flow channel and a flow separator between the glucose and lactate electrodes were fabricated from 23-μm-thick positive photoresist. One carbon electrode was modified with a bilayer of glucose oxidase (GOD)/osmium-poly(vinylpyridine) wired horseradish peroxidase (Os-gel-HRP) and the other was modified with a bilayer of lactate oxidase (LOX)/Os-gel-HRP. The other glass plate has two rectangular flow channels created by chopper cutting with a dicing saw to enable a microdialysis (MD) probe and an outlet tube to be fitted. The cathodic currents of both the glucose and lactate electrodes started to increase rapidly after a microdialysis probe was dipped in glucose or lactate solution, and quickly reached a steady state value. We observed no chemical crosstalk between the glucose and lactate electrodes under our measurement conditions despite the short distance between them. By combining the device with a microdialysis probe, we obtained a linear relationship between the cathodic currents and glucose and lactate concentrations of 5 μM to 5 mM, which covers a concentration range sufficient for measuring brain glucose and lactate. The effect of l-ascorbic acid ( l-AA), which is known to cause major interference during in vivo measurement, was eliminated when ascorbate oxidase was incorporated in the device upstream of the thin-layer flow channel. We successfully observed a glucose concentration decrease and lactate concentration increase in a rat brain when we perfused the striatum with veratridine solution.

Enhanced measurement stability and selectivity for choline and acetylcholine by capillary electrophoresis with electrochemical detection at a covalently linked enzyme-modified electrode.

Enzyme-modified microelectrodes were developed for the indirect amperometric detection of acetylcholine and choline following separation by capillary electrophoresis. Electrodes were prepared by first sequentially electrodepositing polypyrrole and polytyramine from the monomers on a 200-microm platinum electrode. The polymer bilayer provides enhanced selectivity and pendant amino groups for covalent coupling of acetylcholinesterase (AChE) and choline oxidase (ChO) by their reaction with glutaraldehyde. The AChE/ChO-modified electrode was then employed as an end-column detector to determine acetylcholine and choline with and without the internal standard butyrylcholine. Excellent operational stability during 2 days of continuous use was observed, with detection limits of 2 microM or 50 fmol for both acetylcholine and choline. The response from potential interferences, such as dopamine, catechol, and norepinephrine, were significantly reduced in comparison to a bare platinum electrode. The utility of this approach was demonstrated by monitoring the uptake of choline into synaptosomes.

Rapid fabrication of electrochemical enzyme sensor chip using polydimethylsiloxane microfluidic channel

Applicability of polydimethylsiloxane (PDMS) for easy and rapid fabrication of enzyme sensor chips, based on electrochemical detection, is examined. The sensor chip consists of PDMS substrate with a microfluidic channel fabricated in it, and a glass substrate with enzyme-modified microelectrodes. The two substrates are clamped together between plastic plates. The sensor chip has shown no leakage around the microelectrodes under continuous solution flow (34 μl/min). Amperometric response of the sensor chips developed in this work suggest that various types of enzyme sensors can be designed by using PDMS microfluidic channels.

Real-time electrochemical imaging using an individually addressable multi-channel electrode

We developed a real-time electrochemical imaging method that uses a multiple enzyme-modified microelectrode. The method will enable the investigation of the functions of biological materials and cells. To test its effectiveness, we imaged the two-dimensional concentration distribution for hydrogen peroxide and l-glutamate in a standard solution. The multiple electrode consists of an 8×8 array of 30×30 μm 2 carbon micro electrode. Each electrode was connected to a 64-channel potentiostat that could apply a potential to all electrodes at the same time. The multiple electrode was coated with an Os-polyvinylpyridine based polymer (Os-gel) containing horse radish peroxidase (HRP) to detect hydrogen peroxide, which is a very common product of oxidase enzyme. When measuring glutamate, which is a well-known neurotransmitter in the mammalian central nerve system, we modified the electrode with a bilayer of Os-gel-HRP and GluOx. The detection limit of our method was 1 μM and images of the glutamate concentration-distribution changes induced by local injection of glutamate through microcapillary were obtained in real time.

Localized Visualization of Chemical Cross-Talk in Microsensor Arrays by Using Scanning Electrochemical Microscopy

An investigation of an array of four Pt microband electrodes 25 μm wide and spaced 25 μm apart was performed with the scanning electrochemical microscope (SECM). Where possible the SECM measurements were confirmed with conventional electrochemical measurements. It is shown how the sensiti- vity of the SECM recycling current to the activity of the underlying surface can be used to probe the homogeneity of enzyme-modified microelectrodes. The diffusion of H2O2 between these micro enzyme- electrodes and unmodified electrodes was investigated and it was demonstrated how the SECM can be a powerful tool in the elucidation of the properties of these electrodes.

An enzyme-modified microelectrode that detects choline injected locally into brain tissue

An enzyme-modified microelectrode that detects choline injected locally into brain tissue