Acetylcholine detection

Numerous postcolumn enzymatic reactors have been designed for LCEC. Enzymes can be used to produce an electroactive compound from the analyte of interest or, alternatively, to generate an electroactive species that is proportional to analyte concentration. An example of the latter is the detection of acetylcholine [46]. In this case, acetylcholinesterase is used to convert acetylcholine to choline. The resulting choline is reacted with choline oxidase to produce hydrogen peroxide. The amount of hydrogen peroxide produced is directly proportional to the initial concentration of acetylcholine. Detection limits are in the 100 femto-mole range. 

The binding of C-3 by a simple caUx[4]arene host in nonpolar media can be improved by a factor of 25 by the formation of host clusters on the surfaces of gold nanoparticles. Applications of host-guest complexes are mainly directed towards the development of sensors. For example, acetylcholine detection at micromolar concentration is based on inclusion of a fluorophore containing the pyridinium unit (tranj-4[4-(dimethylamino)... 

N. Korbakov, P. Timmerman, N. Lidich, B. Urbach, A. Sa ar, S. Yitzchaik, Acetylcholine detection at micromolar concentrations with the use of an artificial receptor-based fluorescence switch, Langmuir, 2008, 24, 2580-2587. 

Fig. 1. Schematic for chemoreceptor-modified ISFET biosensor for detection of acetylcholine where the source and drain are both n-ty e siHcon.

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

Lazareno, S., and Birdsall, N. J. M. (1995). Detection, quantitation, and verification of allosteric interactions of agents with labeled and unlabeled ligands at G protein-coupled receptors Interactions of strychnine and acetylcholine at muscarinic receptors. Mol. Pharmacol. 48 362-378. 

Turek, J.W., Kang, C.H., Campbell, J.E., Americ, S.P., Sullivan, J.P. A sensitive technique for the detection of the alpha 7 neuronal nicotinic acetylcholine receptor antagonist, methyllycaconitine, in rat plasma and brain. J. Neurosci. Methods. 61 113, 1995. 

Trettnak W., Reininger F., Zinterl E., Wolfbeis O.S., Fiber Optic Remote Detection of Pesticides and Related Inhibitors of the Enzyme Acetylcholine Esterase, Sensor Actuat B-Chem 1993 11 87. 

Absorbance- and reflectance-based measurements are widespread, as there are many enzymatic reaction products or intermediates that are colored or if not, can react with the appropriate indicator. Sensors using acetylcholinesterase for carbamate pesticides detection are an example of indirect optical fiber biosensors. This enzyme catalyses the hydrolysis of acetylcholine with concomitant decrease in pH41 ... 

Acetylcholine + H20 AChE> Choline + Acetic acid Choline + Oz + HzO COx >- Betaine + H202 FIGURE 2.3 Bienzymatic reaction for pesticide detection. 

M. Snejdakova, L. Svobodova, D.P. Nikolelis, J. Wang, and D. Hianik, Acetylcholine biosensor based on dendrimer layers for pesticides detection. Electroanalysis 15, 1185—1191 (2003). 

A particular interest for clinical applications was a possibility for detection of dopamine by its oxidation on nickel [19], cobalt [65], and osmium [66] hexacyanofer-ates. Except for oxidation of dopamine, cobalt and osmium hexacyanoferrates were active in oxidation of epinephrine and norepinephrine. For clinical analysis it is also important to carry out the detection of morphine on cobalt [67] and ferric [68] hexacyanoferrates, as well as the detection of oxidizable amino acids (cystein, methionine) by manganous [69] and ruthenium [70] hexacyanoferrate-modified electrodes. In general, oxidation of thiols was first shown for Prussian blue [71] and nickel hexacyanoferrate [72], This approach has been used for the detection of thiols in rat striatum microdialysate [73], Alternatively, the detection of thiocholine with Prussian blue was employed for pesticide determination in acetylcholine-esterase test [74],... 

The same group reported in 1986 a sensitive and selective HPLC method employing CL detection utilizing immobilized enzymes for simultaneous determination of acetylcholine and choline [187], Both compounds were separated on a reversed-phase column, passed through an immobilized enzyme column (acetylcholine esterase and choline oxidase), and converted to hydrogen peroxide, which was subsequently detected by the PO-CL reaction. In this period, other advances in this area were carried out such as the combination of solid-state PO CL detection and postcolumn chemical reaction systems in LC [188] or the development of a new low-dispersion system for narrow-bore LC [189],... 

On the other hand, several oxidases are known to generate hydrogen peroxide, acting as an oxidant in the CL system, from corresponding substrates. IMERs in which the oxidases are immobilized on adequate supporting materials such as glass beads have been developed. IMERs are often used for flow injection with CL detection of uric acid and glucose, and are also applicable to the CL determination of acetylcholine, choline, polyamines, enzyme substrates, etc., after online HPLC separation. 

Measuring muscle-evoked responses to repetitive motor nerve electrical stimulation permits detection of presyn-aptic neuromuscular junction dysfunction. In botulism and the Lambert-Eaton syndrome, repetitive stimulation elicits a smaller than normal skeletal muscle response at the beginning of the stimulus train, due to impaired initial release of acetylcholine-containing vesicles from presyn-aptic terminals of motor neurons followed by a normal or accentuated incremental muscle response during repeated stimulation. This incremental response to repetitive stimulation in presynaptic neuromuscular disorders can be distinguished from the decremental response that characterizes autoimmune myasthenia gravis, which affects the postsynaptic component of neuromuscular junctions. 

Urine catecholamines may also serve as biomarkers of disulfoton exposure. No human data are available to support this, but limited animal data provide some evidence of this. Disulfoton exposure caused a 173% and 313% increase in urinary noradrenaline and adrenaline levels in female rats, respectively, within 72 hours of exposure (Brzezinski 1969). The major metabolite of catecholamine metabolism, HMMA, was also detected in the urine from rats given acute doses of disulfoton (Wysocka-Paruszewska 1971). Because organophosphates other than disulfoton can cause an accumulation of acetylcholine at nerve synapses, these chemical compounds may also cause a release of catecholamines from the adrenals and the nervous system. In addition, increased blood and urine catecholamines can be associated with overstimulation of the adrenal medulla and/or the sympathetic neurons by excitement/stress or sympathomimetic drugs, and other chemical compounds such as reserpine, carbon tetrachloride, carbon disulfide, DDT, and monoamine oxidase inhibitors (MAO) inhibitors (Brzezinski 1969). For these reasons, a change in catecholamine levels is not a specific indicator of disulfoton exposure. 

Increased levels of urinary catecholamines may also be associated with accumulation of acetylcholine that resulted from acetylcholinesterase inhibition by disulfoton. No human data were located to support this, but limited animal data provide some evidence. Disulfoton exposure caused a 173% and 313% increase in urinary noradrenaline and adrenaline levels in rats, respectively, within 72 hours (Brzezinski 1969). The major metabolite of catecholamine metabolism, HMMA, was also detected in the urine from rats given acute doses of disulfoton (Wysocka-Paruszewska 1971). 

Wang, X.-F., et ah, Signat-on electrochemiluminescence biosensors based on CdS-carbon nanotube nanocomposite for the sensitive detection of choline and acetylcholine. Advanced Functional Materials, 2009.19(9) p. 1444-1450. 

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