Elsevier

Analytica Chimica Acta

Volume 967, 15 May 2017, Pages 59-63
Analytica Chimica Acta

Colorimetric biosensor for the assay of paraoxon in environmental water samples based on the iodine-starch color reaction

https://doi.org/10.1016/j.aca.2017.02.028Get rights and content

Highlights

  • The iodine-starch color reaction was applied for colorimetric assay of paraoxon.

  • The assay of paraoxon can be performed in homogenous solution without reagent synthesis and enzyme immobilization.

  • A colorimetric assay for paraoxon was achieved with a limit of detection 4.7 ppb.

  • This biosensor has the potential of on-site assay of OPs residues in environmental samples.

Abstract

In this work, a new colorimetric biosensor for the assay of paraoxon was developed via the conventional iodine-starch color reaction and multi-enzyme cascade catalytic reactions. In the presence of acetylcholine chloride, acetylcholinesterase (AChE) and choline oxidase (ChO) catalyzed the formation of H2O2, which then activated horseradish peroxidase (HRP) to catalyze the oxidation of KI to produce an iodine-starch color reaction. Upon exposure to paraoxon, the catalytic activity of AChE was inhibited and less H2O2 generated, resulting in a decrease in the production of I2 and a drop in the intensity of solution color. This colorimetric biosensor showed high sensitivity for the assay of paraoxon with a limit of detection 4.7 ppb and was applied for the assay of paraoxon in spiked real samples. By employing the conventional iodine-starch color reaction, this biosensor has the potential of on-site assay of OPs residues in environmental samples.

Graphical abstract

In the presence of acetylcholine chloride, the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) catalyzed the formation of H2O2, which then activated horseradish peroxidase (HRP) to catalyze the oxidation of KI to produce an iodine-starch color reaction. Upon exposure to paraoxon, the catalytic activity of AChE was inhibited and less H2O2 generated, resulting in a decrease in the production of I2 and a drop in the intensity of the color of the solution. By employing the conventional iodine-starch color reaction, this biosensor has the potential of on-site assay of OPs residues in environmental samples.

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Introduction

Organophosphorus pesticides (OPs) are the most extensively used pesticides in modern agriculture because of their relatively low persistence and high effectiveness against insects. However, OPs residues in food products and the environment pose great threat to public health due to their high neurotoxicity [1]. Acetylcholinesterase (AChE) is a key enzyme in the central and peripheral nervous system. OPs can irreversibly inhibit the activity of AChE by phosphorylating the serine residue in the active site of AChE [2], resulting in the accumulation of the neurotransmitter acetylcholine (ACh), thus interfering with muscular responses and causing respiratory and myocardial impairment and even death [3]. Therefore, it is of great significance to develop a fast, reliable and cost-effective method to detect OPs residues for the purpose of human safety and environmental protection.

Over the past few decades, several analytical techniques such as high-performance liquid chromatography [4], gas chromatography [5], mass spectrometer [6], enzyme-linked immunosorbent assay [7], capillary electrophoresis [8], surface enhanced Raman scattering spectroscopy [9], have been developed for the detection of OPs residues in water and food samples. Although these techniques are selective and sensitive, the disadvantages such as expensiveness, requirement for well-trained personnel and time-consuming pre-treatment, limit their application in on-site analysis, especially for emergency cases such as accidental release of pesticides, acute poisoning, and so on. Therefore, simple, sensitive, portable, and low-cost methods for on-site detection of OPs residues are on great demand.

In the past few decades, biosensors based on AChE, a commercially available enzyme, have been extensively investigated due to their advantages such as simplicity, rapidity, specificity, cost effectiveness, and user-friendly operation, which are helpful for on-site analysis of OPs residues [10]. By taking advantage of the inhibition of AChE activity by OPs, they can be quantitatively measured indirectly through the detection of thiocholine or H2O2 with colorimetric [11], [12], [13], fluorescent [14], [15], [16], chemiluminescent [17], electrochemiluminescent [18], photoelectrochemical [19], and electrochemical [20], [21], [22] signals.

Among these biosensors, the colorimetric assay showed great advantages such as low cost, simplicity and practicality in on-site analysis, for the color related to the concentration of target can be observed directly by naked eyes, without the need of expensive or sophisticated instruments [23]. The most widely used colorimetric assay for OPs based on AChE was the Ellman method [11], [12]. However, this method showed low sensitivity and might result in false-positive effect [13]. Noble metal (e.g., Au and Ag) nanoparticles (NPs) are attractive as colorimetric reporters because of their particular local surface plasmon resonance property [24], [25]. Therefore, the integration of enzymatic reaction with Au or Ag NPs has recently emerged for the colorimetric assay of OPs [23], [26], [27]. However, uncontrolled aggregation of Au or Ag NPs in real samples might result in a poor specificity for the targets.

Starch is a biodegradable natural polymer of α-d-glucose contained in plants such as grain, rice, and potatoes [28]. Amylose, a main constituent of starch, can form inclusion complexes with hydrophobic guest molecules by encapsulating them within the helical cavity. An intensely blue-colored iodine-starch complex appears upon encapsulation of iodine by amylose when starch is mixed with iodine in an aqueous solution [29], [30], [31]. This phenomenon is known as the iodine-starch reaction, which was first discovered by Colin and de Claubry in 1814 [32]. Besides, as a sensitive test for iodine, the iodine-starch reaction has been used for the colorimetric detection of adenosine, glucose, α-amylase, hydrogen sulphide, and single nucleotide polymorphisms [33], [34], [35], [36], [37]. However, to the best of our knowledge, the conventional iodine-starch color reaction has not been applied for the environmental assay.

Herein, we proposed a colorimetric biosensor for on-site assay of paraoxon (a model of OPs) in environmental water samples by conventional iodine-starch color reaction. AChE and choline oxidase (ChO) catalyzed the formation of H2O2 in the presence of acetylcholine. Then the resulting H2O2 oxidized KI to I2, and subsequently formed blue-colored starch-iodine complexes via the iodine-starch reaction. When paraoxon was introduced, the inhibition of AChE activity resulted in a decrease of iodine-starch reaction. Therefore, the color of iodine-starch complexes is inversely proportional to the concentration of paraoxon residues. The assay of paraoxon can be performed in homogenous solution without the need of reagent synthesis and enzyme immobilization. Finally, this colorimetric biosensor was applied for the assay of paraoxon in environmental water samples.

Section snippets

Chemicals and materials

ACh was obtained from Tokyo Chemical Industry. AChE from Electrophorus electricus (137 U mg−1) and ChO from Alcaligenes sp. (15 U mg−1) were obtained from Sigma-Aldrich. A certified reference material (CRM) of paraoxon methanol solution (101.6 μg mL−1) with a CRM uncertainty of ±5% was purchased from AccuStandard. H2O2 (AR, 30%), Na2HPO4, and NaH2PO4 were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). Horseradish peroxidase (HRP, Rz > 1.5) was obtained from Shanghai Sangon

Principle of colorimetric assay of OPs

The colorimetric biosensor for the assay of OPs was based on the conventional iodine-starch reaction and multi-enzyme cascade catalytic reactions (Scheme 1). In a normal condition, AChE catalyzes the hydrolysis of the substrate ACh to produce choline, and ChO catalyzes the oxidation of choline to generate H2O2 in the presence of oxygen. After HRP catalyzes the oxidation of KI to I2 by H2O2, blue iodine-starch complexes are produced in the coexistence of starch. Although the structure of

Conclusion

In summary, a new colorimetric biosensor for the assay of paraoxon has been developed via the conventional iodine-starch color reaction coupled with multi-enzyme (AChE, ChO and HRP) cascade catalytic reactions. Paraoxon could inhibit AChE activity, resulting in the production of less H2O2. Therefore, the formation of iodine-starch complexes with blue color could be suppressed; the absorbance and the color intensity were inversely proportional to the concentration of paraoxon. This colorimetric

Acknowledgment

This work was financially supported by NSFC (21577017), Program for New Century Excellent Talents in Fujian Province University and Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (IRT15R11).

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