Acetycholine and choline ion-selective microelectrodes

Detection of acetylcholine (ACh þ ) with high sensitivity and selectivity in the presence of interfering ions including choline (Ch þ ) poses a significant analytical challenge. A cucurbit[6]uril derivative, (allyloxy) 12 CB[6], forms a stable 1:1 inclusion complex with ACh þ but does not interact significantly with Ch þ in solution. Based on this result, we prepared ion-selective electrodes (ISEs) for ACh þ using (allyloxy) 12 CB[6] as an ionophore. The (allyloxy) 12 CB[6]-based ISEs detect ACh þ with unprecedentedly high selectivity over Ch þ as well as other interfering ions such as dopamine, NH 4 þ , NðCH 3 Þ þ 4 and NðCH 2 CH 3 Þ þ 4 .

ACS Sensors, 2018

The neurotransmitter acetylcholine (ACh) plays a key role in the pathophysiology of brain disorders such as Alzheimer's disease. Understanding the dynamics of ACh concentration changes and kinetics of ACh degradation in the living brain is crucial to unravel the pathophysiology of such diseases and the rational design of therapeutics. In this work, an electrochemical sensor capable of dynamic, label-free, selective, and in-situ detection of ACh in a range of 1 nM to 1 mM (with temporal resolution of less than one second) was developed. The sensor was employed for the direct detection of ACh in artificial cerebrospinal fluid and rat brain homogenate, without any prior separation steps. A potentiometric receptor-doped ion-selective electrode (ISE) with selectivity for ACh was designed by taking advantage of the positive charge of ACh. The dynamic range, limit of detection (LOD), and the selectivity of the sensor were optimized stepwise by (i) screening of hydrophobic biomimetic calixarenes to identify receptors that strongly bind to ACh based on shape-selective multitopic recognition, (ii) doping of the ISE sensing membrane with an ACh-binding hydrophobic calixarene to enable selective detection of ACh in complex matrices, (iii) utilizing a hydrophilic calixarene in the inner filling solution of the ISE to buffer the concentration of ACh and, thereby, lower the LOD of the sensor, and (iv) introducing a surface treatment step prior to the measurement by placing the sensor for ∼1 min in a solution of a hydrophilic calixarene to lower the LOD of the sensor even further.

Applied biochemistry and biotechnology, 2013

Technology always has been an indispensible part in the development of biosensors. The performance of biosensors is being tremendously improved using new materials as transducer as well as binding material in their construction. The use of new materials allowed innovation on transduction technology in biosensor preparations. Because of the submicron dimensions of these sensors, simple and rapid analyses in vitro as well as in vivo are now possible. Portable instruments capable of analysing multiple components are becoming available, too. Sensors that provide excellent temporal and spatial resolution for in vivo monitoring such as for measurement of neurotransmitters have become prominent. The interest to improve the stability, sensitivity and selectivity of the sensors is paramount. This study tries to give an overview of the present status of the material-based biosensor design and new generation of choline/acetylcholine neurotransmitter biosensors.

European Journal of Neuroscience, 2004

Conventional microdialysis methods for measuring acetylcholine (ACh) efflux do not provide sufficient temporal resolution to relate cholinergic transmission to individual stimuli or behavioral responses, or sufficient spatial resolution to investigate heterogeneities in such regulation within a brain region. In an effort to overcome these constraints, we investigated a ceramic-based microelectrode array designed to measure amperometrically rapid changes in extracellular choline as a marker for cholinergic transmission in the frontoparietal cortex of anesthetized rats. These microelectrodes exhibited detection limits of 300 nm for choline and selectivity (> 100 : 1) of choline over interferents such as ascorbic acid. Intracortical pressure ejections of choline (20 mm, 66-400 nL) and ACh (10 and 100 mm, 200 nL) dose-dependently increased choline-related signals that were cleared to background levels within 10 s. ACh, but not choline-induced signals, were significantly attenuated by co-ejection of the acetylcholinesterase inhibitor neostigmine (Neo; 100 mm). Pressure ejections of drugs known to increase cortical ACh efflux, potassium (KCl; 70 mm, 66, 200 nL) and scopolamine (Scop; 10 mm, 200 nL), also markedly increased extracellular choline signals, which again were inhibited by Neo. Scopinduced choline signals were also found to be tetrodotoxin-sensitive. Collectively, these findings suggest that drug-induced increases in current measured with these microelectrode arrays reflect the oxidation of choline that is neuronally derived from the release and subsequent hydrolysis of ACh. Choline signals assessed using enzyme-selective microelectrode arrays may represent a rapid, sensitive and spatially discrete measure of cholinergic transmission.

Neuroscience Letters, 1985

A new technique of simultaneously recording from ion-selective and voltammetric microelectrodes in vivo allows one to measure concomitant ionic and monoamine neurotransmitter fluxes in the extracellular fluid space of the brain. Examples are presented of the results obtained with various drug and stimulus applications. The time correlations of the ionic and neurotransmitter fluxes are especially useful in attempting to better understand the possible interactions of these chemical dynamics with neuronal functioning. There are marked fluxes of ions, neurotransmitters and their metabolites, peptides and probably a host of as yet unspecified chemical species, accompanying neuronal activity. These chemical events are reflected as concentration changes, A[X]o,. in the extracellular fluid (ECF) of the brain. The time and space distributions of some of these species may alter certain spheres of synaptic activity. Several authors have conceptualized the functional significance of such influences on target neurons [2, 9, 11, 15], and increasing attention is being given to understanding this dynamic chemistry of the ECF space. Such studies depend upon reliable probes to provide in vivo monitoring of the chemical dynamics. The measurement of extracellular contributions of potassium, calcium and other ions by ion-selective microelectrodes (ISMs) is well established [6, 10, 16]. Voltammetric electrodes which record signals related to the release of monoamine neurotransmitters are finding increasing acceptance [1, 4, 7]. We have combined these two methodologies in order to simultaneously record ion and neurotransmitter fluxes together with the focal electrical activity. The present studies were designed to illustrate that new and potentially important information can be obtained by this simultaneous recording technique. Several pharmacological agents whose overall actions in the CNS are fairly well established were chosen for study. The ISM and voltammetric responses were in overall accord with the known

Analytical Chemistry, 1996

Many of the molecules involved in biological signaling processes are easily oxidized and have been monitored by electrochemical methods. Temporal response, spatial considerations, and sensitivity of the electrodes must be optimized for the specific biological application. To monitor exocytosis from single cells in culture, constant potential amperometry offers the best temporal resolution, and a low-noise picoammeter improves the detection limits. Smaller electrodes, with 1-µm diameters, provided spatial resolution sufficient to identify the locations of release sites on the surface of single cells. For the study of neurotransmitter release in vivo, larger cylindrical microelectrodes are advantageous because the secreted molecules come from multiple terminals near the electrode, and the greater amounts lead to a larger signal that emerges from the Johnson noise of the current amplifier. With this approach, dopamine release elicited by two electrical stimulus pulses at 10 Hz was detected with fastscan cyclic voltammetry in vivo. Nafion-coated elliptical electrodes have previously been shown to be incapable of detecting such concentration changes without extensive signal averaging. In addition, we demonstrate that highpass filtering (200 Hz) of cyclic voltammograms recorded at 300 V/s decreases the background current and digitization noise at these microelectrodes, leading to an improved signal. Also, high-pass filtering discriminated against ascorbic acid, DOPAC, and acidic pH changes, three common interferences in vivo. Many chemical messengers that relay information between biological cells can be electrochemically oxidized. These molecules include catecholamines, 1-3 histamine, 4 5-hydroxytryptamine, 5-7 and insulin. 8 For this reason, microsensors designed from carbon fibers have been shown to be quite useful to monitor secretion of these species in a variety of biological systems. 1-8 As progress is made in these measurements, it is clear that the optimum type of electrode and electroanalytical technique depend upon the particular biological preparation investigated. Four major issues in electrochemical measurements are electrode response time and electrode size, as well as sensitivity and selectivity of the method. These issues are often intertwined. For example, early studies showed that differential pulse voltammetry at electrochemically modified electrodes had high selectivity but could not resolve the rapid concentration changes that neurotransmitters undergo. 9-12 This was due to the slow response time of the electrodes as well as the slow scan rate of the technique. The two techniques most widely used today are amperometry 3,14-18 and fast-scan cyclic voltammetry. 5,19-22 Amperometry allows measurement of the most rapid concentration changes and is theoretically limited by the double-layer capacitance of the electrode and the resistance of the surrounding solution, but it provides no chemical identification of the species detected. 3,14-16 Nevertheless, amperometry has provided unique information on the kinetics of secretion and subsequent biochemical fates of the easily oxidized chemical messengers in situations where the biological environment is well controlled. Such situations arise in cell cultures containing a single cell type, 1,18,23,24 or in the brain when specific nerve terminals are activated. 3,14 Fastscan cyclic voltammetry is particularly useful because the voltammogram provides information to identify the detected substance. 25,26 However, cyclic voltammetry's response time, selectivity, and sensitivity critically depend on the electrode's surface state. 6,27

The Analyst, 1996

Lipophilic, alkylated cyclodextrins (CDs) are aromatic hosts which are excellent ionophores for reversibly binding size-matched, charge-diffuse cations. The application of lipophilic p-CDs as ionophores for sensing acetylcholine chloride using a potentiometric ISE and an amperometric biosensor is described. Potentiometric ISEs, using 2,6-didodecyl-P-CD as ionophore, showed a Nernstian response with a limit of detection -log[C] = 5.0 and selectivity coefficient -1ogKOf = 4.2 (in serum levels of Na+, K+ and Ca2+). A Nernstian response is maintained in the presence of bovine serum albumin for a PVC-and a polyurethane-based electroactive membrane on initial contact. However, on prolonged contact, the polyurethane-based membranes showed a lower shift in contact with analyte solution) than PVC. Amperometric biosensors were assembled by modifying screen-printed electrodes with a ferrocenyl charge shuttle, enzymes (horseradish peroxidase, choline oxidase and/or acetylcholine esterase) and a thin film comprising a polyurethane matrix, 2,3,6-triethyl-P-CD, a plasticizer and a large anionic salt. The resulting sensor, which was capable of detecting subpicomolar levels of acetylcholine, was highly specific and was stable to storage in air and in solution. Interference from endogenous electroactive compounds was minimal.

Electrochemical sensors are of increasing interest, especially those developed for direct application in biological areas such as pharmacology and neuroscience. This divulgation article evidences the results obtained by nanotechnology researchers using modified nanotube-microelectrodes, which can be used for neurotransmitter measurements in the brain. Resumen Los sensores electroquímicos son de interés creciente, en especial los elaborados para su aplicación directa en las áreas biológicas, como la neurociencia y la farmacología. Este artículo de divulgación estudiantil evidencia los resultados obtenidos por un grupo de investigadores en nanotecnología usando microelectrodos modificados con nanotubos, que pueden ser utilizados para mediciones de neurotransmisores en el cerebro.

Sensors, 2008

ACS Applied Materials & Interfaces, 2020

The malfunctioning in the release of acetylcholine (ACh +), leading to consequential damages in the neural system, has become an impulsion for the development of numerous progressive transport and detection gadgets. However, several challenges, such as laterality and complexity of transport devices, low precision of amperometric detection systems, and sumptuous, multistaged enzymatic quantification methods, have not yet been overcome. Herein, ionomers, because of their selective ion transporting nature, are chosen as suitable candidates for being implemented into both targeted ACh + delivery and sensing systems. Based on these two approaches, for the very first time in the literature, the disulfonated poly(arylene ether sulfone) membrane is concurrently (i) used in the mimicry of transduction of the electrical-to-ionic signal in a neural network as "Acetylcholine Pen" (ACh + Pen) and (ii) operated as a highly sensitive, conductivity-based ACh + quantifier. Our dual device, being able to operate under an actual action potential of 55 mV bias , shows a strong potential of future applicability in real-time ionic delivery-andsensing systems.