A liquid chromatography (LC) detector based on a fast response and sensitive bienzyme amperometric biosensor for acetylcholine (ACh) and choline (Ch) is described. The detector fabrication consisted of glutaraldehyde co-crosslinking of acetylcholinesterase and choline oxidase with bovine serum albumin on the Pt working electrode of a conventional thin-layer electrochemical flow cell. The influence of some experimental parameters (e.g. enzyme loading, thickness of the bienzyme layer, flow rate) on the detector characteristics has been studied in order to optimize the analyte response while minimizing band broadening and distortion. A mobile phase consisting of a phosphate buffer (I 0.1 M, pH 6.5) containing 5 mM sodium hexane sulphonate and 10 mM tetramethylammonium phosphate was found to give very satisfactory resolution and peak shape in ion-pair, reversed-phase LC. Linear responses were observed over at least four decades and absolute detection limits (at a signal to noise ratio of 3) were 12 and 27 fmole injected for Ch and ACh, respectively. After one month of intensive use in the LC system, the detector retained about 70% of its initial sensitivity. The potential of the described approach is demonstrated by the simultaneous determination of Ch and ACh in rat brain tissue homogenates.

An aceylcholinesterase/choline oxidase-based amperometric biosensor as a liquid chromatography detector for acetylcholine and choline determination in brain tissue

GUERRIERI, Antonio;
2001

Abstract

A liquid chromatography (LC) detector based on a fast response and sensitive bienzyme amperometric biosensor for acetylcholine (ACh) and choline (Ch) is described. The detector fabrication consisted of glutaraldehyde co-crosslinking of acetylcholinesterase and choline oxidase with bovine serum albumin on the Pt working electrode of a conventional thin-layer electrochemical flow cell. The influence of some experimental parameters (e.g. enzyme loading, thickness of the bienzyme layer, flow rate) on the detector characteristics has been studied in order to optimize the analyte response while minimizing band broadening and distortion. A mobile phase consisting of a phosphate buffer (I 0.1 M, pH 6.5) containing 5 mM sodium hexane sulphonate and 10 mM tetramethylammonium phosphate was found to give very satisfactory resolution and peak shape in ion-pair, reversed-phase LC. Linear responses were observed over at least four decades and absolute detection limits (at a signal to noise ratio of 3) were 12 and 27 fmole injected for Ch and ACh, respectively. After one month of intensive use in the LC system, the detector retained about 70% of its initial sensitivity. The potential of the described approach is demonstrated by the simultaneous determination of Ch and ACh in rat brain tissue homogenates.
File in questo prodotto:
File Dimensione Formato  
An Acetylcholinesterase-Choline Oxidase-Based Amperometric Biosensor as a Liquid Chromatography.pdf

accesso aperto

Tipologia: Documento in Post-print
Licenza: DRM non definito
Dimensione 100.21 kB
Formato Adobe PDF
100.21 kB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11563/2627
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 69
  • ???jsp.display-item.citation.isi??? 65
social impact