RSC Advances View Article Online PAPER View Journal | View Issue Colorimetric sensing of chlorpyrifos through negative feedback inhibition of the catalytic activity Cite this: RSC Adv., 2020, 10,13050 of silver phosphate oxygenase nanozymes† Amisha Kushwaha, Gajendar Singh and Manu Sharma * Intensive use of organophosphate chlorpyrifos pesticides in farming has become a serious issue due to their harmful effects on living beings. Most fruits, vegetables and soil contain chlorpyrifos, and it cannot be rinsed out completely by water washing. Therefore, a selective and sensitive detection of chlorpyrifos is significant. In the present study, the intriguing oxidase-mimicking activity of Ag3PO4 nanoparticles (NPs) is explored for the fast and selective detection of chlorpyrifos pesticides. Ag3PO4 NPs exhibit several advantages, such as great catalytic efficiency, high stability, monodispersity and reusability, over other expensive nanozymes via a facile one-step sensing. The size, shape, crystal planes and diffraction patterns of the Ag3PO4 NPs were observed via FESEM and HR-TEM. The surface properties and oxidation states were analyzed via XPS 0 0 Creative Commons Attribution 3.0 Unported Licence. technique. Ag3PO4 NPs possess intrinsic excellent oxidase-mimicking properties against 3,3 ,5,5 - tetramethylbezidyne (TMB). When chlorpyrifos and Ag3PO4 NP nanozymes come in proper orientation proximity, chlorpyrifos is oxidized. The oxidized chlorpyrifos produces sulfide ions and chlorpyrifos oxon. The produced sulfide ions in the reaction system interact with Ag3PO4 NPs and inhibit their catalytic activity by feedback inhibition. Indeed, neither any catalytic site is left to oxidize TMB nor any blue colour Received 19th December 2019 appears. Thus, this feedback inhibition phenomenon senses chlorpyrifos pesticides. The calculated limit Accepted 2nd March 2020 of detection (LOD) for the standard chlorpyrifos is 9.97 ppm, and the efficacy of the Ag3PO4 NPs DOI: 10.1039/c9ra10719c calculated in terms of the Km value was found to be 0.15 mM. A real sample analysis was carried out by rsc.li/rsc-advances the standard addition method with two soil samples collected from Pethapur and Chiloda villages. This article is licensed under a 1. Introduction minutes or an hour. An initial exposure of chlorpyrifos in its complete molecular state form is non-toxic, and as such it Open Access Article. Published on 01 April 2020. Downloaded 10/1/2021 1:56:17 AM. Over the last few decades, the monitoring of hazardous chlor- moves to all parts of the body. Aer the oxidation of chlorpyrifos pyrifos in the natural ecosystem has become a serious issue. in the body, the toxic chlorpyrifos oxon and sulde ions are Pesticides show harmful and adverse effects on human health produced.3 Then, chlorpyrifos permanently binds acetylcholin- and on the healthy environment of ora and fauna. Pesticides esterase and blocks its activity.4 Chlorpyrifos is also very toxic to are used to control undesirable herbs, plants, insects, fungi, bird species, such as pigeons and grackles, and is moderately nematodes, rodents, sh, bugs, microbes, etc. The organo- toxic to mallard ducks.4 When mallard ducks feed on chlor- phosphate chlorpyrifos is harmful to directly touch, inhale or pyrifos, they lay fewer eggs and fewer ducklings are born.5 The eat. Its insecticidal action causes the inhibition of the nerve eggshells also become thinner than normal, and many of the enzyme acetylcholinesterase; consequently, the neurotrans- young ducklings die.6 Among all birds, robins show the highest mitter acetylcholine accumulates at nerve endings.1 Nerve mortality from chlorpyrifos exposure. Chlorpyrifos is also very enzyme acetylcholinesterase metabolises the neurotransmitter toxic to aquatic invertebrates and sh due to bioaccumulation acetylcholine into choline and acetate in a hydrolytic manner. in their tissues.7,8 It is also toxic to honey bees,9,10 non-targeted Chlorpyrifos has a broad spectrum activity that affects the insects and earthworms up to two weeks aer it is supplied to nervous systems of humans, animals and targeted pests by soil.11 Thus, the selective and sensitive detection of chlorpyrifos progressively inhibiting acetylcholinesterase, which leads to the is very necessary. Soluble suldes generally coexist in the state À À loss of sensation.2 Exposure to even a small amount can cause of sulde ion (S ) or hydrogen sulde (HS ), which are corro- tears, runny nose, and increased saliva or drooling symptoms in sive in nature.12 Various methods have been developed for the determination of chlorpyrifos, such as electrochemical methods for the determination of chlorpyrifos on a nano-TiO2/ Central University of Gujarat, Gandhinagar, Gujarat-382030, India. E-mail: manu. cellulose acetate composite-modied glassy carbon electrode.13 [email protected] † Electronic supplementary information (ESI) available. See DOI: Gold nanoparticles have been applied for the detection of 14 10.1039/c9ra10719c. chlorpyrifos in various water samples. Chlorpyrifos attached 13050 | RSC Adv.,2020,10, 13050–13065 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper RSC Advances to quantum dots shows changes in uorescence intensity as proliferated as colorimetric tools with high catalytic properties; 15 sensed through ow cytometry. Surface-enhanced Raman however, here, the Ag3PO4 NPs oxidize TMB in one step with spectroscopy was used for the detection of chlorpyrifos in high sensitivity. Therefore, the wide range of practical applica- 16 spinach with silver colloids. A silver nanohexagon solution is tions of Ag3PO4 NPs and their high oxidative properties may used for the sensing of chlorpyrifos via UV-Vis absorption offer more importance in the eld of sensing. spectroscopy.17 These methods are sensitive and specic; however, they involve complex sample preparation, are time- consuming and expensive, and require sophisticated instru- 2. Materials and methods ments, which limit their further applications. Colorimetric 2.1 Chemicals and reagents methods are more applicable due to their direct, rapid and All chemicals and reagents were used in the entire experiment simple process of detection. Changes in colour can be observed without further purication. Silver nitrate, di-sodium hydrogen with naked eye; it does not require expensive or sophisticated phosphate, 3,30,5,50-tetramethylbenzene (TMB), DMSO, sodium instrumentation and can be applied directly in the eld. acetate, glacial acetic acid, chlorpyrifos, Endosulfan, Fenson, Recently, several studies have been reported on the colorimetric Carbofuran, Aldrin, Dieldrin and Benfuracarb were bought detection of other biomolecules and toxic ions by exploiting the from Sigma-Aldrich India and were used without further puri- oxidase-mimicking property. For instance, researchers have used cation. Milli-Q water was prepared by an integral water puri- the oxidase-like property of gold nanoclusters for the quanti ca- cation system (EMD Millipore). tion of melamine in raw milk and milk powder.18 Li et al. synthesized a cobalt and nitrogen co-doped hierarchically porous carbon hybrid with oxidase mimicking properties for the colori- 2.2 Synthesis of Ag3PO4 NPs 19 – metric detection of glutathione. Ag CoFe2O4/reduced graphene A simple co-precipitation method was used for the synthesis of 2+ 20 30 oxide nanocomposites were engineered for the detection of Hg . Ag3PO4 NPs. 60 mM (50 mL) silver nitrate added to Milli-Q Creative Commons Attribution 3.0 Unported Licence. Researchers found oxidase-like activities in cobalt oxyhydroxide water was magnetically stirred at 500 rpm for 15 min in the (CoOOH) nanoakes, which oxidise colourless o-phenylenedi- dark to obtain a transparent solution containing silver and 20 amine (OPD) to yellow oxidized OPD to sense ascorbic acid. nitrate ions. 20 mM (50 mL) Na2HPO4 in Milli-Q water was ffi Verneker et al. developed an e cient synthesis of MnFe2O4 via separately stirred for 15 min to obtain a transparent solution a co-precipitation method with controlled morphology; it showed containing sodium and phosphate ions. This solution was remarkable oxidase mimetic properties.21 Graphene quantum dot- added dropwise in a continuous manner to the prepared silver Ag nanoparticles show high oxidase and antibacterial properties nitrate solution, which was then stirred for another 6 h. The for Gram-negative, Gram-positive and drug-resistant bacteria.22 obtained lemon yellow precipitate was centrifuged, washed Cerium oxide was also studied for pH-tuneable oxidase activity for multiple times with Milli-Q water, ethanol and dried at 60 C (12 This article is licensed under a 23 cancer folate biomarker sensors. Singh et al. also studied cerium h) in the dark. The powdered Ag3PO4 NPs were stored in the molybdate-incorporated graphene oxide nanocomposites for dark for further analysis. A schematic of the mechanism of the 24 glucose sensing. Researchers also found both peroxidase and Ag3PO4 NPs synthesis is shown in Scheme 1. Open Access Article. Published on 01 April 2020. Downloaded 10/1/2021 1:56:17 AM. oxidase-like activities for AuPt alloy NPs using TMB as a chromo- genic material.25 Rope-like Co–Fe layered hydroxide nanosheets 2.3 Steady state kinetic assay for the oxidase-like activity of based on hierarchical structures were studied for intrinsic oxidase- Ag PO NPs catalytic activity.26 Citrate-capped silver nanoparticles exhibit 3 4 27 oxidase activity to detect the concentration of mercury ions (II). The oxidase-like activity of the Ag3PO4