Elsevier

Talanta

Volume 158, 1 September 2016, Pages 142-151
Talanta

Electrochemiluminescence biosensor for determination of organophosphorous pesticides based on bimetallic Pt-Au/multi-walled carbon nanotubes modified electrode

https://doi.org/10.1016/j.talanta.2016.05.030Get rights and content

Highlights

  • A novel ECL biosensing system for detection of OPs was developed.

  • The ECL signal of luminol was significantly amplified by nanoparticles and H2O2.

  • Sensitive and selective detection of OPs was achieved in the system.

Abstract

A novel and highly sensitive electrochemiluminescence (ECL) biosensing system was designed and developed for individual detection of different organophosphorous pesticides (OPs) in food samples. Bimetallic Pt-Au nanoparticles were electrodeposited on multi-walled carbon nanotubes (MWNTs)-modified glass carbon electrode (GCE) to increase the surface area of electrode and ECL signals of luminol. Biocomposites of enzymes from acetylcholinesterase and choline oxidase (AChE and ChOx) were immobilized onto the electrode surface to produce massive hydrogen peroxides (H2O2), thus amplifying ECL signals. Based on the dual-amplification effects of nanoparticles and H2O2 produced by enzymatic reactions, the proposed biosensor exhibits highly sensitivity. The proposed biosensing approach was then used for detecting OPs by inhibition of OPs on AChE. Under optimized experimental conditions, the ECL intensity decreased accordingly with the increase in concentration of OPs, and the inhibition rates of OPs were proportional to their concentrations in the range of 0.1–50 nmol L−1 for malathion, methyl parathion and chlorpyrifos, with detection limit of 0.16 nmol L−1, 0.09 nmol L−1 and 0.08 nmol L−1, respectively. The linearity range of the biosensor for pesticide dufulin varied from 50 to 500 nmol L−1, with the detection limit of 29.7 nmol L−1. The resulting biosensor was further validated by assessment of OPs residues in cabbage, which showed a fine applicability for the detection of OPs in the realistic sample.

Introduction

Organophosphorus pesticides (OPs) are a large group of pesticides and widely used in agronomic practice for killing insects and protecting crop production [1], [2]. As toxic chemicals once OPs contaminate humans and animals, the visual and nervous system, and sensory or cognitive function can be seriously impaired [3]. It was reported that OPs or their active metabolites can exert their detrimental effects by blocking the enzyme activity of acetylcholinesterase (AChE), which in turn leads to accumulation of the neurotransmitter acetylcholine (Ach) in synapses as well as over-stimulation of the post-synaptic cholinergic receptors with a consequence of neurotoxicity [4], [5]. Considering the environmental security and risk of OPs, it is of great importance and urgency to develop an accurate, sensitive, and rapid method to monitor the realistic environmental and food contamination. Up to now, various methodologies have been developed for assessment of OPs, such as high performance liquid chromatography (HPLC) [6], gas chromatography (GC) [7], chemiluminescence [8], and thin-layer chromatography (TLC) [9]. However, most of the methods require expensive equipments and complicated sample pretreatment. Besides, vast organic solvents used for OPs extraction from samples may not be cost-effective. Therefore, establishing a low-cost, high efficient and easy-to-use method for OPs detection is essentially important.

Recently, electrochemiluminescence (ECL) has been emerging as an alternative to the conventional methods and is able to meet demands of high sensitivity, low background signal and simple instrumentation [10]. It takes an advantage of technically merged chemiluminescence and electrochemistry [11], [12] with generation of species at electrode surfaces where electron-transfer reactions occur to form excited states of light emitting [13], [14]. A variety of ECL reagents such as luminol [15], [16], semiconductor nanocrystals (NCs)-based ECL system [17] and tris (2,2′-bipyridine) ruthenium (Ⅱ) Ru(bpy)32+ [18], [19] have been used to develop sensitive biosensor. Recently, luminol has become one of the most popular ECL reagents on account of low oxidation potential, high emission yields and inexpensive reagent consumption. The reactive oxygen species (ROSs) have been demonstrated to improve the ECL performance of luminol in neutral medium [20]. As one of the ROSs, hydrogen peroxide (H2O2) is a highly reactive species owning to its presence of unpaired valent shell electron. H2O2 in the luminol-H2O2 system was found to enhance the ECL of luminol efficiently under various experimental conditions [21]. Such intensification could reduce the limitation of ECL of luminol, such as extension of practicable pH window, need for strong alkaline solution and requirements for high exciting potential to avoid possible interference [22]. Recent studies have shown that AChE-based inhibitory biosensors served as an alternative for OPs analysis [23]. The activity of AChE has been generally used as a quantitative indicator of OPs measurement [24]. Choline is the product of the enzymatic reaction. It can be oxidized on the surface of electrode and generate electrochemical signals. Upon addition of OPs, the enzyme activity of AChE would be inhibited, which can induce the decrease of electrochemical signals. However, the product would be different when a two-enzyme approach (AChE and ChOx) was employed in assembling the biosensor. Since the hydrolysis product of AChE, choline was further oxidized by ChOx in the presence of oxygen, H2O2 is produced [25], [26]. Similarly, addition of OPs would inhibit the AChE activity, which in turn reduces H2O2, and as a consequence would weaken the ECL signal of luminol. In this regard, biosensor could be employed based on the ECL of luminol intensified by H2O2. To the best of our knowledge, there has been no report on using luminol-H2O2 approach for OPs assessment.

In order to achieve sensitive determination, nanometer-scale materials have been used in fabrication of biosensors for their large surface area, high loading capacity and uniform pore structure [3]. A previous report indicated that multi-walled carbon nanotubes (MWCNTs) displayed a fast heterogeneous charge transfer and possessed electrocatalytic properties due to the edge plane sites in MWCNTs occurring at the ends and along the tube axis [27]. Metal nanoparticle (NP) is another kind of popular material owning to its high surface-to-volume ratio and high surface energy. For example, Pt NPs and Au NPs showed unusual physical and chemical properties, depending on their size and shape [28]. Potential application of Pt and Au in electrochemistry, electron-transfer and ECL reactions has been investigated [22], [29], [30], and the promoted effect on ECL signals of luminol-H2O2 system from Au NPs was reported as well [31]. Pt NPs can not only catalyze ECL of luminol molecules in solution but also enrich luminol molecules on the surface of nanoparticles [32]. Electrodes modified with metal NPs such as Au or Pt were showed to enhance the ECL emission of luminol by 2–3 orders as compared to the original bare electrodes [21]. Inspired by the superiorities of above nanomaterials, here we present a novel biosensor modification strategy. We utilized the unique synergy properties of Au-Pt bimetallic NPs and MWCNTs to improve biosensor performance and avoid the deficiency and limitation of single nanomaterial. The novel biosensor had been developed by electrodeposition of the Pt-Au bimetallic NPs on the MWCNTs modified glass carbon electrode (GCE). The biocomposites AChE & ChOx were coimmobilized on the Pt-Au/MWCNT-modified GCE by cross-linking the enzymes and modified GCE through cysteine. Because H2O2 was generated in the bioenzyme system, the ECL signal of luminol was significantly amplified on AChE&ChOx/Pt-Au/MWCNT/GCE. Herein, the OPs malathion, methyl parathion, chlorpyrifos and dufulin were chosen as the inhibitors of AChE. On the base of the effects of OPs on the ECL signal of luminol, a new rapid and sensitive luminol-based ECL approach was developed for OPs detection.

Section snippets

Materials and chemicals

The organophosphorous pesticide dufulin (99%) was obtained from Center for Research and Development of Fine Chemicals of Guizhou University. Malathion (95%), chlorpyrifos (99%) and methyl parathion (99%) were obtained from Syngenta Nantong Crop Protection Co., Ltd. MWCNTs (10–20 nm diameter, length 10–30 µm, and >95% purity) were obtained from JC NANO, Inc. (China). Chloroauric acid (HAuCl4·4H2O, 47.8%) and chloroplatinic acid (H2PtCl6·6H2O, 37.5%) were purchased from Aladdin Co., Ltd. l-Cysteine

Principle of the ECL biosensor

Fig. 1 depicts the principle of dual-amplification strategy for OPs detection. Briefly, carboxyl-modified MWCNT was modified on the GCE surface. The bimetallic Pt-Au NPs were deposited, followed by conjugating with AChE&ChOx. In this case, ATCI in ECL detection solution was hydrolyzed into choline and acetate by AChE (Eq. (1)). Choline was further oxidized into betaine and H2O2 (Eq. (2)). As a result, the ECL signal of luminal was stimulated by bimetallic NPs and enzyme. When OPs were added,

Conclusions

A novel ECL biosensor for determination of organophosphorous pesticides has been developed. The proposed biosensor consisted of AChE&ChOx enzyme composites which were immobilized on the surface of Pt-Au/MWCNT modified glass carbon electrode through the cross-linking by cysteine. Based on the integration of these electrochemically nanometer materials and the generation of coreactant H2O2 in enzymatic reaction, the ECL signal of luminol was significantly amplified. The resulting biosensor showed

Acknowledgements

The authors acknowledge the financial support of the National Natural Science Foundation of China (Nos. 21377058, 21577064) and the financial support of the Special Fund for Agro-scientific Research in the Public Interest (No. 201203022) from the Ministry of Agriculture of China.

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