Electrocatalytic Microelectrode Detectors for Choline and Acetylcholine Following Separation by Capillary Electrophoresis

Abstract

Two electrocatalytic enzyme modified microelectrode systems were employed as end-column amperometric detectors of choline (Ch) and acetylcholine (ACh) following separation by capillary electrophoresis (CE). Horseradish peroxidase crosslinked in an Os based redox polymer hydrogel (HRP-Os) was physically adsorbed on Au microelectrodes followed by chemical crosslinking of the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO). An alternative approach utilized the deposition of the transition metal catalyst, Prussian Blue (PB), on Pt microelectrodes as the electrocatalyst. Utilizing butyrylcholine (BuCh) as an internal standard, the HRP-Os/AChE-ChO and PB/AChE-ChO electrodes exhibited excellent linear responses from 2-2000 μM and 10-2000 μM, respectively, for both Ch and ACh. Detection limits of 0.1 μM or 38 amol were determined for the HRP-Os/AChE-ChO electrode. The limit of detection for ACh and Ch at the PB/AChE-ChO electrode was 5 μM or 9.5 fmol. The electrodes were operated at potentials of +0.10 and −0.10 V vs. Ag/AgCl (3M NaCl), respectively, and thus minimized the potential response from oxidizable interferences. In addition, both electrocatalytic electrodes showed good operational stability for more than 70 hours. The enhanced detection capability of the HRP-Os/AChE-ChO and PB/AChE-ChO electrodes in combination with efficient CE separation of Ch and ACh provides a new sensitive and selective strategy for monitoring and quantifying these cholinergic biomarkers in biological fluids.

Bioguided Isolation and Structure Identification of Acetylcholinesterase Enzyme Inhibitors from Drynariae Rhizome

“…Compared with the reported data [25][26][27][28], the eight compounds were flavonoids and identified as naringenin (28 mg), eriodictyol (19 mg), kaempferol (24 mg), luteolin (9 mg), astragalin (11 mg), luteolin-7-O-β-D-glucoside (7 mg), naringin (51 mg), and neoeriocitrin (68 mg) by UV, IR, MS, 1 H-NMR, 13 C-NMR, and 2D-NMR. e 13 C-NMR data of these compounds are given in Table 2.…”

“…However, the matrix effect and background color of complex herb extracts is uncertain, often interfering with the detection of the secondary reaction, and easily induces results bias. To improve this fault, the contents of Ch of the reaction system could be determined by a variety of ways, such as thin-layer chromatography [10], fluorimetric assay [11], high-performance liquid chromatography [12], capillary electrophoresis [13], and mass spectrometry [14]. Among these methods, mass spectrometry coupled with ultra-performance liquid chromatography showed advantages in higher sensitivities, high efficiency, better separation, less amount of enzyme, and isotope-label free, which is more suitable for the screening of potential AChE inhibitors from complex systems, such as natural herb extracts [15,16].…”

Application of Micro-Fluidic Devices for Biomarker Analysis in Human Biological Fluids

“…Biomarkers of the cholinergic system are choline and acetylcholine, which Mukherjee and Kirchhoff detected and quantified using CE coupled with electrochemical detection. (Mukherjee & Kirchhoff, 2009) This sensitive system was able to detect biomarkers in the range of fmol to atmol, which far exceeds previous detection limits and makes the system particularly applicable to the detection of these neuronal biomarkers in human samples. The measurement of free cortisol in urine was carried out by Olędzka et al (Olędzka et al, 2010) Using a solid phase extraction (SPE)-coupled MEKC with UV detection, free cortisol was detected and quantified with a limit of quantification in the 5 ng/mL range.…”

“…Electrochemical detection offers excellent selectivity and sensitivity and the ability to modify microelectrodes to gain further selectivity for targeted analysis. (Mukherjee & Kirchhoff, 2009) Capacitively coupled contactless conductivity detection (C 4 D) offers further acceleration and simplification of CE analyses. It detects analytes in their native state and does not require time-consuming sample derivatization.…”

Biosensor for organophosphorus pesticides based on the acetylcholine esterase inhibition mediated by choline oxidase bioelectrocatalysis

“…Recently, electroactive mediators such as 2,6-dichlorophenoldophenol 8-10 , tetracyanoquinodimethane [11][12][13] , and related compounds 14,15 have been adjusted for OP detection based on the AChE/ChOx bienzyme system.…”