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Title: Biosensor Based on Self-Assembling Acetylcholinesterase on Carbon Nanotubes for Flow injection/Amperometric Detection of Organophosphate Pesticides and Nerve Agents

Abstract

A highly sensitive flow-injection amperometric biosensor for organophosphate pesticides and nerve agents based on self-assembly of acetylcholinesterase (AChE) on carbon nanotube (CNT)-modified glassy carbon (GC) electrode is described. AChE is immobilized on the negatively-charged CNT surface by alternatively assembling a cationic polydiallyldimethylammonium chloride (PDDA) layer and an AChE layer. Transmission electron microscopy images confirm the formation of layer-by-layer nanostructures on carboxyl functionalized CNTs. The unique sandwich-like structure (PDDA/AChE/PDDA) on the CNT surface formed by self-assembly provides a favorable microenvironment to keep the bioactivity of AChE and to prevent enzyme molecule leakage. The electrocatalytic activity of CNT leads to a greatly improved electrochemical detection of the enzymatically generated thiocholine product, including a low oxidation overvoltage (+150 mV), higher sensitivity, and stability. The developed PDDA/AChE/PDDA/CNT/GC biosensor integrated into a flow injection system was used to monitor organophosphate pesticides and nerve agents, such as paraoxon. The sensor performance, including inhibition time and regeneration conditions, was optimized with respect to operating conditions. Under the optimal conditions, the biosensor was used to measure as low as 0.4 pM paraoxon with a 6-min inhibition time. The biosensor had excellent operational lifetime stability with no decrease in the activity of enzymes for more than 20 repeated measurementsmore » over a 1-week period. The developed biosensor system is an ideal tool for online monitoring of organophosphate pesticides and nerve agents.« less

Authors:
;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
877552
Report Number(s):
PNNL-SA-46970
12490; 6899; TRN: US200608%%415
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Analytical Chemistry; Journal Volume: 78; Journal Issue: 3
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; 54 ENVIRONMENTAL SCIENCES; 45 MILITARY TECHNOLOGY, WEAPONRY, AND NATIONAL DEFENSE; PESTICIDES; BIOLOGICAL WARFARE AGENTS; DETECTION; AMPEROMETRY; PROBES; CARBOXYLESTERASES; IMMOBILIZED ENZYMES; CARBON; NANOSTRUCTURES; Carbon nanotubes; biosensor; pesticides; nerve agents; Environmental Molecular Sciences Laboratory

Citation Formats

Liu, Guodong, and Lin, Yuehe. Biosensor Based on Self-Assembling Acetylcholinesterase on Carbon Nanotubes for Flow injection/Amperometric Detection of Organophosphate Pesticides and Nerve Agents. United States: N. p., 2006. Web. doi:10.1021/ac051559q.
Liu, Guodong, & Lin, Yuehe. Biosensor Based on Self-Assembling Acetylcholinesterase on Carbon Nanotubes for Flow injection/Amperometric Detection of Organophosphate Pesticides and Nerve Agents. United States. doi:10.1021/ac051559q.
Liu, Guodong, and Lin, Yuehe. Wed . "Biosensor Based on Self-Assembling Acetylcholinesterase on Carbon Nanotubes for Flow injection/Amperometric Detection of Organophosphate Pesticides and Nerve Agents". United States. doi:10.1021/ac051559q.
@article{osti_877552,
title = {Biosensor Based on Self-Assembling Acetylcholinesterase on Carbon Nanotubes for Flow injection/Amperometric Detection of Organophosphate Pesticides and Nerve Agents},
author = {Liu, Guodong and Lin, Yuehe},
abstractNote = {A highly sensitive flow-injection amperometric biosensor for organophosphate pesticides and nerve agents based on self-assembly of acetylcholinesterase (AChE) on carbon nanotube (CNT)-modified glassy carbon (GC) electrode is described. AChE is immobilized on the negatively-charged CNT surface by alternatively assembling a cationic polydiallyldimethylammonium chloride (PDDA) layer and an AChE layer. Transmission electron microscopy images confirm the formation of layer-by-layer nanostructures on carboxyl functionalized CNTs. The unique sandwich-like structure (PDDA/AChE/PDDA) on the CNT surface formed by self-assembly provides a favorable microenvironment to keep the bioactivity of AChE and to prevent enzyme molecule leakage. The electrocatalytic activity of CNT leads to a greatly improved electrochemical detection of the enzymatically generated thiocholine product, including a low oxidation overvoltage (+150 mV), higher sensitivity, and stability. The developed PDDA/AChE/PDDA/CNT/GC biosensor integrated into a flow injection system was used to monitor organophosphate pesticides and nerve agents, such as paraoxon. The sensor performance, including inhibition time and regeneration conditions, was optimized with respect to operating conditions. Under the optimal conditions, the biosensor was used to measure as low as 0.4 pM paraoxon with a 6-min inhibition time. The biosensor had excellent operational lifetime stability with no decrease in the activity of enzymes for more than 20 repeated measurements over a 1-week period. The developed biosensor system is an ideal tool for online monitoring of organophosphate pesticides and nerve agents.},
doi = {10.1021/ac051559q},
journal = {Analytical Chemistry},
number = 3,
volume = 78,
place = {United States},
year = {Wed Feb 01 00:00:00 EST 2006},
month = {Wed Feb 01 00:00:00 EST 2006}
}
  • A nanoparticle-based electrochemical immunosensor has been developed for the detection of phosphorylated acetylcholinesterase (AChE) adducts, which is a potential exposure biomarker for organophosphate pesticides (OP) and chemical warfare nerve agent exposures. Zirconia nanoparticles (ZrO2 NPs) were used as selective sorbents to capture the phosphorylated AChE adduct, and quantum dots (ZnS@CdS, QDs) were used as tags to label monoclonal anti-AChE antibody to track the immunorecognition events. The sandwich-like immunoreactions were performed among the ZrO2 NPs, which were pre-coated on a screen printed electrode (SPE) by electrodeposition, phosphorylated AChE and QD-anti-AChE. The captured QD tags were determined on the SPE by electrochemicalmore » stripping analysis of its metallic component (cadmium) after an acid-dissolution step. Paraoxon was used as a model OP insecticide to prepare the phosphorylated AChE adduct to demonstrate the proof of principle for this sensor technology. The paraoxon-AChE adduct was characterized by Fourier Transform Infrared Spectrum, and the binding affinity of anti-AChE to the paraoxon-AChE was validated with an enzyme-linked immunosorbent assay. The parameters (e.g., amount of ZrO2 NP, QD-anti-AChE concentration,) that govern the electrochemical response of immunosensors were optimized. The voltammetric response of the immunosensor is highly linear over the range of 10 pM to 4 nM paraoxon-AChE, and the limit of detection is estimated to be 8 pM. This new nanoparticle-based electrochemical immunosensor thus provides a sensitive and quantitative tool for biomonitoring exposure to OP pesticides and nerve agents.« less
  • A disposable biosensor based on carbon nanotube was developed for detection of pesticides and nerve agents.
  • A flow injection amperometric glucose biosensor based on electrostatic self-assembling glucose oxidase (GOx) on a carbon nanotube (CNT)-modified glassy carbon transducer is described. GOx is immobilized on the negatively charged CNT surface by alternatively assembling a cationic polydiallyldimethylammonium chloride (PDDA) layer and a GOx layer. The unique sandwich-like layer structure (PDDA/GOx/PDDA/CNT) formed by self-assembling provides a favorable microenvironment to keep the bioactivity of GOx and to prevent enzyme molecule leakage. The direct electrochemistry behavior of GOx and electrocatalysis of H2O2 on the fabricated PDDA/GOx/PDDA/CNT electrode demonstrated that such a biosensor fabrication method preserves the activity of enzyme molecules and themore » mechanical and electrocatalytic properties of carbon nanotubes, enabling sensitive determination of glucose. Flow injection amperometric detection of glucose is carried out at -100 mV (vs Ag/AgCl) in 0.05 M phosphate buffer solution (pH 7.4) with wide linear response range of 15 uM- 6 mM and a detection limit of 7 uM. The PDDA/GOx/PDDA/CNT/GC biosensor showed excellent properties for the sensitive determination of glucose with good reproducibility, remarkable stability, and free of interference from other co-existing electroactive species. The present methods can be applied to assemble other enzyme molecules and biological molecules, such as antibody, antigen, and DNA, to the CNT surface for wide biosensor and bioassay applications.« less