Colorimetric sensing of chlorpyrifos through negative feedback inhibition of the catalytic activity of silver phosphate oxygenase nanozymes

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 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 appears. Thus, this feedback inhibition phenomenon senses chlorpyrifos pesticides. The calculated limit of detection (LOD) for the standard chlorpyrifos is ∼9.97 ppm, and the efficacy of the Ag3PO4 NPs calculated in terms of the Km value was found to be 0.15 mM. A real sample analysis was carried out by the standard addition method with two soil samples collected from Pethapur and Chiloda villages.


Introduction
Over the last few decades, the monitoring of hazardous chlorpyrifos in the natural ecosystem has become a serious issue. Pesticides show harmful and adverse effects on human health and on the healthy environment of ora and fauna. Pesticides are used to control undesirable herbs, plants, insects, fungi, nematodes, rodents, sh, bugs, microbes, etc. The organophosphate chlorpyrifos is harmful to directly touch, inhale or eat. Its insecticidal action causes the inhibition of the nerve enzyme acetylcholinesterase; consequently, the neurotransmitter acetylcholine accumulates at nerve endings. 1 Nerve enzyme acetylcholinesterase metabolises the neurotransmitter acetylcholine into choline and acetate in a hydrolytic manner. Chlorpyrifos has a broad spectrum activity that affects the nervous systems of humans, animals and targeted pests by progressively inhibiting acetylcholinesterase, which leads to the loss of sensation. 2 Exposure to even a small amount can cause tears, runny nose, and increased saliva or drooling symptoms in minutes or an hour. An initial exposure of chlorpyrifos in its complete molecular state form is non-toxic, and as such it moves to all parts of the body. Aer the oxidation of chlorpyrifos in the body, the toxic chlorpyrifos oxon and sulde ions are produced. 3 Then, chlorpyrifos permanently binds acetylcholinesterase and blocks its activity. 4 Chlorpyrifos is also very toxic to bird species, such as pigeons and grackles, and is moderately toxic to mallard ducks. 4 When mallard ducks feed on chlorpyrifos, they lay fewer eggs and fewer ducklings are born. 5 The eggshells also become thinner than normal, and many of the young ducklings die. 6 Among all birds, robins show the highest mortality from chlorpyrifos exposure. Chlorpyrifos is also very toxic to aquatic invertebrates and sh due to bioaccumulation in their tissues. 7,8 It is also toxic to honey bees, 9,10 non-targeted insects and earthworms up to two weeks aer it is supplied to soil. 11 Thus, the selective and sensitive detection of chlorpyrifos is very necessary. Soluble suldes generally coexist in the state of sulde ion (S À ) or hydrogen sulde (HS À ), which are corrosive 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-TiO 2 / cellulose acetate composite-modied glassy carbon electrode. 13 Gold nanoparticles have been applied for the detection of chlorpyrifos in various water samples. 14 Chlorpyrifos attached to quantum dots shows changes in uorescence intensity as sensed through ow cytometry. 15 Surface-enhanced Raman spectroscopy was used for the detection of chlorpyrifos in spinach with silver colloids. 16 A silver nanohexagon solution is used for the sensing of chlorpyrifos via UV-Vis absorption spectroscopy. 17 These methods are sensitive and specic; however, they involve complex sample preparation, are timeconsuming and expensive, and require sophisticated instruments, which limit their further applications. Colorimetric methods are more applicable due to their direct, rapid and simple process of detection. Changes in colour can be observed with naked eye; it does not require expensive or sophisticated instrumentation and can be applied directly in the eld.
Recently, several studies have been reported on the colorimetric detection of other biomolecules and toxic ions by exploiting the oxidase-mimicking property. For instance, researchers have used the oxidase-like property of gold nanoclusters for the quantication 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 colorimetric detection of glutathione. 19 Ag-CoFe 2 O 4 /reduced graphene oxide nanocomposites were engineered for the detection of Hg 2+ . 20 Researchers found oxidase-like activities in cobalt oxyhydroxide (CoOOH) nanoakes, which oxidise colourless o-phenylenediamine (OPD) to yellow oxidized OPD to sense ascorbic acid. 20 Verneker et al. developed an efficient synthesis of MnFe 2 O 4 via a co-precipitation method with controlled morphology; it showed remarkable oxidase mimetic properties. 21 Graphene quantum dot-Ag nanoparticles show high oxidase and antibacterial properties for Gram-negative, Gram-positive and drug-resistant bacteria. 22 Cerium oxide was also studied for pH-tuneable oxidase activity for cancer folate biomarker sensors. 23 Singh et al. also studied cerium molybdate-incorporated graphene oxide nanocomposites for glucose sensing. 24 Researchers also found both peroxidase and oxidase-like activities for AuPt alloy NPs using TMB as a chromogenic material. 25 Rope-like Co-Fe layered hydroxide nanosheets based on hierarchical structures were studied for intrinsic oxidasecatalytic activity. 26 Citrate-capped silver nanoparticles exhibit oxidase activity to detect the concentration of mercury ions (II). 27 Other researchers synthesized Ag@Ag 3 PO 4 microcubes as oxidase mimics for the ultrasensitive detection of Hg 2+ . 28 Previous reports show the use of expensive noble metalbased colorimetric sensors based on platinum, rubidium, and gold in their colorimetric sensing systems. All these catalysts are expensive, less stable and not easily available. To overcome these issues, we have designed an efficient Ag 3 PO 4 NP-based catalyst due to its high oxidizing properties and comparatively low cost. For a long time, Ag 3 PO 4 NPs have been considered to have antibacterial properties against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. 29 Also, it is popularly preferred as a light-sensitive material as it shows excellent photo-oxidative properties for oxygen evolution and photocatalytic dye degradation under visible light irradiation because of its narrow band gap. 30 Due to these advanced properties of Ag 3 PO 4 NPs, we were interested to use them for the rst time in colorimetric sensors for detection of chlorpyrifos. Many oxidase-mimetic nanomaterial sensors have already proliferated as colorimetric tools with high catalytic properties; however, here, the Ag 3 PO 4 NPs oxidize TMB in one step with high sensitivity. Therefore, the wide range of practical applications of Ag 3 PO 4 NPs and their high oxidative properties may offer more importance in the eld of sensing.

Synthesis of Ag 3 PO 4 NPs
A simple co-precipitation method was used for the synthesis of Ag 3 PO 4 NPs. 30 60 mM (50 mL) silver nitrate added to Milli-Q water was magnetically stirred at 500 rpm for 15 min in the dark to obtain a transparent solution containing silver and nitrate ions. 20 mM (50 mL) Na 2 HPO 4 in Milli-Q water was separately stirred for 15 min to obtain a transparent solution containing sodium and phosphate ions. This solution was added dropwise in a continuous manner to the prepared silver nitrate solution, which was then stirred for another 6 h. The obtained lemon yellow precipitate was centrifuged, washed multiple times with Milli-Q water, ethanol and dried at 60 C (12 h) in the dark. The powdered Ag 3 PO 4 NPs were stored in the dark for further analysis. A schematic of the mechanism of the Ag 3 PO 4 NPs synthesis is shown in Scheme 1.

Steady state kinetic assay for the oxidase-like activity of Ag 3 PO 4 NPs
The oxidase-like activity of the Ag 3 PO 4 NPs was conrmed by using TMB substrate as a chromogenic material. TMB is converted to the oxidised TMB (oxTMB) blue colour complex in the presence of oxidizing agents such as free radicals of H 2 O 2 , direct sunlight exposure, horseradish peroxidase (HRP) and oxidizing enzymes or nanozymes. The Ag 3 PO 4 NPs directly oxidize TMB to oxTMB (blue colour) in a one-step process. These nanoparticles act as oxidase nanozymes in the replacement of conventional HRP. Different concentrations of TMB (0.05-0.5 mM) were prepared in DMSO at room temperature. To optimize the reaction system, 60 mL (0.8 mg mL À1 ) Ag 3 PO 4 NPs were incubated with TMB in 2.0 mL sodium acetate buffer (pH 4.8). The blue oxTMB was analyzed using a UV/Visible spectrophotometer with the maximum absorbance at 652 nm. The obtained data were tted with the Michaelis-Menten equation and Lineweaver-Burk equation, as given below: where V 0 is the initial velocity of interaction of the Ag 3 PO 4 nanozymes with the substrate TMB, V max is the maximal velocity of the proposed reaction, [S] is the TMB substrate concentration and K m is the Michaelis-Menten constant.

Robustness with respect to reusability, pH, temperature and time
For reusability analysis, 80 mL (4.8 pH) acetate buffer, 2.4 mL (0.8 mg mL À1 ) Ag 3 PO 4 NPs and 4 mL (0.5 mM) TMB were mixed and incubated for 180 s before spectrophotometric analysis. For the next cycle, the sample was centrifuged at 16 000 rpm at 4 C and re-dispersed in 2.4 mL Milli-Q water; then, we followed the same experiment described above. This process was continued for six repeated cycles. For pH-based robustness, 50 mg of Ag 3 PO 4 NPs were incubated in each reaction system (pH 2-12) for 1 h. Aer incubation, the Ag 3 PO 4 NPs were centrifuged, Scheme 1 Schematic of silver phosphate nanoparticles synthesized by a simple co-precipitation route.   recording the absorbance of oxTMB at 652 nm every alternate day up to 26 days.

Colorimetric detection of chlorpyrifos pesticide
For colorimetric detection, all glassware was thoroughly cleaned with Milli-Q water and aquaregia. 200 ppm of chlorpyrifos stock solution was prepared in DMSO and stored for further use. 2 mL volumes of acetate buffer (pH 4.8) containing different concentrations of chlorpyrifos (20-200 ppm) were prepared by maintaining the volume. 60 mL (0.8 mg mL À1 ) of Ag 3 PO 4 NPs was added to all the glass vials; then, 100 mL of 0.5 mM TMB solution was agitated at room temperature. The obtained blue solution of oxTMB was analyzed by spectrophotometry.

Selectivity analysis
For selectivity analysis, seven different reaction systems were designed using 200 ppm of seven different pesticides (Benfuracarb, Endosulfan, Fenson, Carbofuran, Aldrin, Dieldrin and Chlorpyrifos) prepared in acetate buffer at pH 4.8. 60 mL (0.8 mg mL À1 ) Ag 3 PO 4 NPs were added to each solution of pesticides and incubated for 180 s. Further, 0.1 mL (0.5 mM) of TMB solution was distributed in each of the seven reaction systems to check the blue colour of oxTMB.

Real sample analysis
Soil samples were collected from two different agriculture sites (Pethapur Village and Chiloda Village), Gandhinagar, Gujarat. Large particles, roots, etc. were separated from the soil samples, and the samples were dried (38-40 C overnight), minced properly and ltered with clean cotton cloth to obtain homogeneous soil samples. The soil samples collected from Pethapur Village and Chiloda Village were labelled as S1 and S2, respectively. 10 g of each soil sample was agitated, washed at room temperature with 40 mL Milli-Q water, and ltered (Whatman No. 2 lter paper). The same process was repeated with hot (75-80 C) 40 mL Milli-Q water in continuation, and the settled soil was collected for drying. This washed and dried sample was washed again with 40 mL of DMSO; nally, the ltrate was collected and termed as S1L or S2L. Further, the standard addition method was employed for the colorimetric detection of chlorpyrifos by the Ag 3 PO 4 NPs catalyst.

Thermocol imprint method
A 4 Â 2 (l Â b) cm 2 rectangle of thermocol was imprinted with two alphabetic letters "A" beside each other. The imprinted letters were lled with 1 mg mL À1 Ag 3 PO 4 NPs; then, the catalyst was maintained inside the imprint overnight and stored as a kit for further use. The thermocol imprint was lled with 4.8 pH acetate buffer, chlorpyrifos and 0.5 mM TMB solution, whereas the other imprint was lled only with acetate buffer and 0.5 mM TMB solution. Aer 3 min, blue colour was observed in the absence of chlorpyrifos, and no colour was observed in the imprint to which chlorpyrifos was added.

Characterization
Powder X-ray diffraction (PXRD) patterns were recorded using a Panalytical X Pert Pro (Cu Ka, l ¼ 1.5406 A, 40 mA, 40 kV) with a step size of 0.03 at the rate of 0.6 per second. The crystallite sizes of the nanoparticles were calculated using the Scherrer equation: where s is the crystallite size, K is the shape factor, and l is the Xray wavelength. b is the corrected line broadening at half maximum intensity, and q is the Bragg angle at the peak position. The UV-Vis absorbance was measured with a Shimadzu UV-1800. Fourier transform infrared spectra were recorded with a PerkinElmer Spectrum 65 instrument. A eld emission scanning electron microscope (FE-SEM, Quanta 200 FEG) was used for the morphological analysis. A transmission electron microscope (TEM, Tecnai F20) was employed for the particle size analysis. Differential scanning calorimetry (DSC 6000, Perki-nElmer) was used for the thermal stability-based exothermic and endothermic processes. FT-Raman spectroscopy (multi RAM, standalone model) was used to determine the structural and vibrational properties in the 4000-50 cm À1 range with a ND: YAG laser source of 1064 nm. XPS (ESCA, Omnicorn Germany) was performed to determine the surface properties of the Ag 3 PO 4 NPs.

Results and discussion
The XRD pattern of the Ag 3 PO 4 NPs is shown in Fig. 1  and 275 nm (n-p*) near the UV light absorption range can be observed in Fig. S1a. † For the FT-IR analysis, $2 mg Ag 3 PO 4 NPs was mixed with 200 mg moistureless KBr, ground well and pressed with a hydraulic press to prepare a pellet. First, the KBr pellet was used for the background; then, the sample was analyzed in the range of 4000-400 cm À1 (Fig. S1b †) (DSC) curve shows the existence of a small endothermic peak at 334 C, which may be due to decomposition of the Ag 3 PO 4 NPs, as shown in Fig. 1b. The SEM image of the Ag 3 PO 4 NPs shows aggregated spherical particles with a size range of $100 to 200 nm, as shown in Fig. 2a. SEM elemental mapping micrographs clearly show the presence of O, P and Ag without any impurities (Fig. 2b-e).
The TEM image of the Ag 3 PO 4 NPs shows spherical particles in the range of 15-40 nm, as shown in Fig. 3(a). The high resolution TEM image of Ag 3 PO 4 NPs shows the crystal planes (101) and (020), corresponding to the cubic phase of Ag 3 PO 4 NPs (Fig. 3b). The electron diffraction pattern of the Ag 3 PO 4 NPs in Fig. 3c shows a bright spot corresponding to the planes (201) and (020). The Raman spectrum of the Ag 3 PO 4 NPs shows an inelastic, scattered intense peak at $910 cm À1 , attributed to the terminal oxygen vibrational stretching of PO 4 groups (Fig. 3d). The peak at 1051 cm À1 represents asymmetric stretching vibrations of O-P-O [PO 4 ], the peak at $708 cm À1 describes symmetric stretching of the O-P-O bond, the very weak peak at $559 cm À1 is associated with the asymmetric stretch of P-O-P vibrational bending, 33 the peak at $405 cm À1 corresponds to symmetric bending vibration modes related to [PO 4 ] clusters, and the peak at 101 cm À1 is due to symmetric vibrational bending of Ag-O bonds. 34 The XPS full survey scan of Ag 3 PO 4 is shown in Fig. 4a, in which the P 2p, Ag 3d and O 1s spin orbit core levels were detected. High-resolution XPS spectra of the P 2p, Ag 3d and O 1s spin orbit core levels are shown in Fig. 4b-d. Ag 3d has a core level spectrum with two binding energy peaks at 373.34 eV and 367.35 eV due to the electron orbitals of Ag 3d 3/2 and Ag 3d 5/2 , consistent with the oxidation state of Ag + in Ag 3 PO 4 . 35 Fig. 4c shows the high resolution XPS spectra for the P 2p spin orbit core levels; the two binding energy peaks originating at 132.6 and 131.7 eV correspond to the electron orbitals of P 2p 1/2 and P 2p 3/2 , which supports that phosphorous is in the P +5 oxidation state in Ag 3 PO 4 . 36

Optimization of TMB and steady state kinetic assay
For TMB optimization, various concentrations of TMB (0.05 to 0.5 mM) were added to 2 mL of acetate buffer (pH 4.8) containing 60 mL of a 0.8 mg mL À1 concentration of Ag 3 PO 4 . The UV/Vis results are shown in Fig. 5. As shown in Fig. 5a, as the concentration of TMB increases, the absorbance at 652 increases. The change in absorbance at 652 nm is shown in Fig. 5b, in which the graph plateaus at $0.5 mM. 0.5 mM TMB concentration is considered to be the optimal concentration for further experiments. For the steady state kinetic assay, TMB was used as a substrate and chromogenic material. Using the absorption coefficient of TMB (3 ¼ 39 000 M À1 cm À1 ), a Michaelis-Menten plot was obtained for increasing concentrations of TMB (Fig. 5c). The inset of Fig. 5c shows the linear response of TMB oxidation at all optimal conditions. A double reciprocal or Lineweaver-Burk plot was obtained and is given in Fig. 5d. The Michaelis-Menten constant (K m ) was calculated from the Michaelis-Menten plot and was found to be 0.15 mM.

Effects of Ag 3 PO 4 concentration, incubation pH, time and temperature
For the optimization of the concentration of Ag 3 PO 4 NPs, incubation pH, time and temperature, the reaction system was incubated with different concentrations of Ag 3 PO 4 NPs (0.1-1 mg mL À1 ), pH values (3.6-5.6), times (0-4.0 min.) and temperatures (20-55 C). Fig. 6a shows the effects of NPs concentration on TMB oxidation. As the NPs concentration increases, the absorbance at 652 increases; the graph plateaus at 0.8 mg mL À1 concentration, as shown in Fig. 6a. Hence, 0.8 mg mL À1 concentration of Ag 3 PO 4 was considered as the optimal concentration for further sensing applications. Fig. 6b-d show the effects of incubation pH, time and temperature on TMB oxidation, respectively. The maximum absorbance at 652 nm of oxTMB was recorded at pH 4.8, 3 min and 25 C. Hence, these parameters were set as the optimal conditions for sensing.

Mechanism of chlorpyrifos sensing
As shown in Scheme 2, when the Ag 3 PO 4 NPs come in proper orientation proximity with TMB, TMB acts as a substrate for the Ag 3 PO 4 nanozyme. Aer oxidation, TMB shows a blue colour, which can be tuned to optimal parameters. 37 Therefore, when chlorpyrifos pesticide comes in proximity with the Ag 3 PO 4 NPs, it is oxidized to form chlorpyrifos oxon and sulde ion, as shown in Scheme 2. These produced sulde ions feedback to the Ag 3 PO 4 NPs and block their activity. Similarly, W. Qin et al. synthesized Co 3 O 4 NPs for the sensing of sulphite in food and inhibited their colour formation; 38 also, R. Chen et al. created a hydrogen sulde gas sensor in the presence of silver NPs lm. 39 The sulde ion released in this reaction aer the oxidation of chlorpyrifos is attached to the active site of the Ag 3 PO 4 NPs, as shown in Scheme 2.

Robustness
Long-term durability of a nano-sensor is one of the most important aspects required for its satisfactory application. Indeed, the activity of colorimetric nano-sensors depends upon the colour signals generated without substrate and in the presence of the desired sensing materials. Thus, the deviation in colour intensity enables the quantication of the sensing material, and it should not deviate in response to common factors such as pH, temperature and time; also, the reusability of nano-sensors is important. The recyclability of the Ag 3 PO 4 NPs indicates the potential to reuse the same nanoparticles six times (Fig. 7a). A much less steep decrease in blue colour (oxTMB) was observed up to the sixth cycle. Other nanoparticles have also showed reusable properties, such as Fe 3+ -doped mesoporous carbon nanospheres. 40 Samples were incubated at different pH values from 2 to 12 for 1 h. The Ag 3 PO 4 NPs show stability under different harsh pH conditions (Fig. 7b). The catalytic activity is almost constant, and there is no recordable decrease in activity. The temperature stability of the Ag 3 PO 4 NPs was also recorded in the range of 20-80 C; as shown in Fig. 7c, no decrease in the reaction activity of the NPs was observed.
Similarly, the stability of the NPs was examined during storage from 2 to 26 days every alternative day (Fig. 7d). The storage time reaction stability was also found to be acceptable up to 26 days of continuous incubation. No considerable decrease in the catalytic activity of the NPs was observed. Thus, it can be concluded that the synthesized NPs are stable with respect to pH, temperature, and storage time and can be reused up to six times.

Colorimetric sensing of chlorpyrifos
Colorimetric sensing of chlorpyrifos was studied using TMB, which is a chromogenic substrate for Ag 3 PO 4 NPs. In the presence of Ag 3 PO 4 NPs, increasing concentrations of chlorpyrifos decreased the intensity of the peak of the blue colour (oxTMB) (Fig. 8a). 200 ppm chlorpyrifos shows a less intense peak at 650 nm. Further increasing the concentration of chlorpyrifos diminished the blue colour peak. Oxidized chlorpyrifos was converted into chlorpyrifos oxon and sulde ion, as shown in Scheme 2. Thus, the Ag 3 PO 4 NPs are conrmed to be oxidase mimetic nanozymes. In Scheme 2, one of the products, sulde ion, interacts with the Ag 3 PO 4 NPs; it inhibits the active sites and blocks the oxidizing activity to convert TMB to blue oxTMB as a negative feedback loop. The study of the reaction kinetics measured the reaction rate and affinity of the Ag 3 PO 4 nanoenzymes towards TMB (substrate).
Sulde ion sensing as a proof of concept experiment. We performed the S 2À ions sensing experiment in pre-optimized conditions as a proof of concept. For this experiment, we used Na 2 S with different concentrations (0 ppm to 200 ppm) in water and tested for S 2À ions in the reaction solution. Fig. 8e shows the UV/Vis. spectra with increasing concentrations of S 2À ions (Na 2 S), and Fig. 8f shows the change in absorbance at 652 nm. As the concentration of S 2À increases, the absorbance at 652 nm decreases gradually; this indicates that S 2À inhibits the oxidase mimicking activity of Ag 3 PO 4 . For the selectivity test, chlorpyrifos was tested with six other pesticides, namely Benfuracarb, Endosulfan, Fenson, Carbofuran, Aldrin and Dieldrin (their structures are shown in Fig. S2 †). A total of seven reaction systems were developed to evaluate potential interference with the Ag 3 PO 4 NPs in comparison to chlorpyrifos. In order to verify the feasibility of our approach for the detection of chlorpyrifos, the selectivity test results are shown in Fig. 9a and b. At l max ¼ 652 nm for oxTMB, the various pesticides other than chlorpyrifos showed comparatively high colour intensity. The efficacy of the substrate for any enzyme can be estimated by measuring the This journal is © The Royal Society of Chemistry 2020 K m value. Therefore, the K m value of the Ag 3 PO 4 NPs calculated for the TMB substrate is low, showing the higher prociency of the reaction toward the TMB substrate.
The K m value (Michaelis-Menten constant) represents the affinity of the catalytic efficiency of an enzyme for a substrate. Many researchers have developed oxidase mimetic nanoparticles, which are tabulated in Table 2. In this table, we can see that the standard enzyme HRP is most traditionally involved in experiments of oxidation based on amperometric biosensors for polyphenol determination. 41 HRP is used as a glucose biosensor, as established through electro-enzyme catalyst oxidation of glucose 42 and immobilizing HRP for oxidative polymerization of 2,6-dimethylphenol as a biocatalyst. 43 Thus, from Table 2, the K m value for HRP is about 0.434 mM, and the K m value for the Ag 3 PO 4 NPs is about 0.15 mM.
The developed analysis method, which is selective and sensitive for the standard chlorpyrifos, was applied for real sample analysis. Soil samples from Pethapur and Chiloda villages extracted in DMSO show absorbance at 652 nm, as shown in Fig. 9(c) and (d), respectively. The gradient decreases (Fig. 9e) in the absorbance of the standard chlorpyrifos with increasing concentrations of Pethapur and Chiloda DMSO soil extracts, which indicates that chlorpyrifos was sensed (Fig. 9f). The resulting amounts found in the soil extracts, recovery percentages, and RSD values are tabulated in Table 3.
We analysed the Ag 3 PO 4 catalyst aer reacting it with TMB through XPS for Ag valence identication, and the XPS results are shown in Fig. 10. These results have also been added with the manuscript and highlighted with redcolour. According to the XPS results of Ag 3 PO 4 , the OKLL peak can be clearly observed on the surface, showing a higher concentration of oxygen due to exposure to ambient atmospheric conditions. The total scan spectrum binding energy range of 0-1100 eV revealed that Ag, P and O elements coexist in the Ag 3 PO 4 nanoparticles. Noise peaks were observed aer the reaction. In Fig. 10(a), a full survey scan shows the same number of elements which were present before the reaction. In Fig. 10(b), the three peaks positioned at 373.52 eV, 368.00 eV and 367.48 eV in the high resolution spectrum of Ag 3d can be assigned to the electron orbitals of Ag 3d 3/2 and Ag 3d 5/2 of Ag + , respectively. Thus, aer the oxidation process of the Ag 3 PO 4 NPs, no remarkable change is observed. Therefore, silver is only present in the Ag + state before and aer the reaction. In Fig. 10(c), the peak with a binding energy of 132.51 eV is ascribed to P 2p of PO 4 3À . The binding energy de-convoluted peaks at 530.31 eV, 531.47 eV and 532.85 eV (Fig. 10d) were assigned to O 1s. Reactive oxygen species (ROS) were investigated by introducing iso-propyl alcohol (IPA) as an OHc scavenger and pbenzoquinone (BQ) as a superoxide radical ðO À 2 Þ scavenger. Fig. 11 shows the effects of the scavengers on the TMB oxidation process under all optimal conditions. Fig. 11a indicates the UV/ Vis. spectroscopic results of oxTMB in the presence and absence of IPA and BQ based on the absorbance at 652 nm, as shown in Fig. 11b. The oxidation of TMB was found to be $16.1% reduced in the presence of IPA (reaction system 3), while in the presence of BQ, a $40.64% reduction in TMB oxidation was recorded. As a result, much fewer OHc free radicals are being generated, but   comparatively higher amounts of ðO À 2 Þ are being generated in the reaction system through an in situ process; this helps to oxidize TMB and produce a blue product. 50 These results were in good agreement with our previous report on free radical validation processes. 51 These results have also been added to the manuscript and highlighted in red.  Thermocol alphabet imprinting sensing for chlorpyrifos. In Fig. 12a, the number of reaction systems was tested. The control experiment was performed in the absence of chlorpyrifos (set 1); set 2 contained water-washed aliquot + TMB, and set 3 contained hot water-washed aliquot + TMB, where the appearance of blue color shows that the expected impurities and 2 mg L À1 of chlorpyrifos were washed out by hot water washing. In DMSO, the soluble chlorpyrifos was extracted, and further spiking experiments were performed (set 4). A control experiment in the presence of standard chlorpyrifos was also performed (set 5). In this sensing method, the alphabet letter 'A' was imprinted twice on a thermocol sheet; both imprint volumes were lled with a solution of Ag 3 PO 4 NPs dissolved in water, and the water was evaporated. Only one of the imprints was lled with chlorpyrifos to discriminate the difference in the presence and absence of chlorpyrifos, and both imprints were lled with TMB. Pale blue colour was observed in the chlorpyrifos-free imprint, and colour inhibition was observed with chlorpyrifos, as shown in Fig. 12b.

Conclusion
The present work demonstrates a novel method for single-step colorimetric sensing of chlorpyrifos. Ag 3 PO 4 NPs were readily synthesized and characterized by different analytical tools, showing promising oxidase catalytic properties. The Ag 3 PO 4 NPs were optimized in terms of concentration, temperature, time and pH for superior catalytic activity. The present sensing method shows comparatively low K m values (the lower the K m value, the higher the sensitivity) for standard and real soil samples for chlorpyrifos detection; this indicates that the Ag 3 PO 4 nanozyme is more effective than the traditional HRP enzyme. The limit of detection (LOD) of standard chlorpyrifos was found to be about 9.97 ppm y 10 ppm for standard samples. For real soil sample analysis, the Ag 3 PO 4 NPs showed signicant results for the detection of chlorpyrifos at different village sites through a spiking process. This thermocol imprinting sensing method is applicable for commercial use of the catalyst by applying this facile and inexpensive approach for sensing applications and towards device fabrication.

Conflicts of interest
There are no conicts to declare.