A highly sensitive multi-catalytic sensing system for organophosphorus and organochlorine pesticides based on the peroxidase-like activity of ferric ions

Abstract

Developing rapid, efficient and highly sensitive sensing systems for organophosphorus (OPs) and organochlorine pesticides is important due to their potential damage to human health. Considering that Fe³⁺ ions were recently found to have much higher peroxidase-like activity than that of Fe₃O₄ magnetic nanoparticles, in this work, a novel and highly sensitive multi-catalytic sensing system has been successfully developed for OPs and organochlorine pesticides, on the basis of the color reaction of 3,3′,5,5′-tetramethyl benzidine (TMB) driven by Fe³⁺ ions, together with two enzymatic catalytic systems of acetylcholinesterase (AChE) and choline oxidase (CHO). Sub nM level limits of detection could be achieved for four tested OPs and organochlorine pesticides. Furthermore, several fruit/vegetable samples were successfully employed for evaluating this established sensing system.

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Introduction

Organophosphorus (OPs) pesticides are one of the most important pesticides, and are extensively used in the world.^1,2 It was reported that more than 100 kinds of OPs compounds have been or are being employed as pesticides since 1940's.³ Most of the widely used OPs belong to highly toxic pesticides, which could be easily released into the environment, resulting in serious ecological pollution and food safety problem.^4–6 OPs can inhibit the activity of acetylcholinesterase (AChE) via the formation of phosphorylated cholinesterase, lead to in vivo accumulation of acetylcholine. Excessive acetylcholine can cause serious impairment on nerve function and even death.⁷ Therefore, it is of importance to establish an efficient and sensitive detection method for OPs.

Numerous detection methods for OPs have been developed, based on various chemical or biological mechanisms.^8–12 Since 1960's, the emergence of chromatographic techniques^13–17 have greatly promoted the development of detecting OPs, thereby various chromatographic techniques including gas phase chromatography (GC), liquid phase chromatography (LC) and thin layer chromatography (TLC) have become important detection methods for OPs. Combining with mass spectrum, these methods can substantially improve their sensitivity and accuracy. However, these established analytical methods usually have several disadvantages, such as complicated operation procedures, time consuming, expensive instruments and requiring highly qualified technicians. Obviously, these methods are not suitable for the continuously, rapidly and on-line monitoring of OPs, especially for the emergency cases. Therefore, developing simple, facile and sensitive methods for detecting OPs is highly demanded. It should be noted that much more convenient detection methods based on electrochemical mechanisms and photoluminescence (PL)/UV-vis absorption spectrum techniques have been developed.^18–27

Artificial enzyme mimics have recently attracted extensive attentions,^28,29 because (1) they can serve as highly stable and low-cost alternatives to natural enzymes; (2) many kinds of artificial enzyme mimics have been found and created. Therefore, a wide range of applications such as serving as peroxidases or oxidases have also been explored.^30–36 Catalysts can repeatedly catalyse some certain reactions for many times to generate a large amount of products, especially photoluminescent or colorimetric compounds. Therefore, these catalytic reactions can be conveniently monitored by PL/UV-vis absorption spectra. A signal amplification could be achieved in these systems, which has been successfully employed in the fields of bio/chemosensing.^37–40 Numerous investigations focused on metal/metal oxides nanomaterials capable of acting as the peroxidases and oxidase mimics to catalyse a series of substrates.^41–45 However, in general two issues exist, (1) the observed peroxidase-like or oxidase-like activity were in public debated to originate from whether the nature of intact nanomaterials themselves or the surface bound and released metal ions; (2) intrinsic instability of these nanomaterials can susceptibly cause the surface oxidation/aggregation, resulting in the decrease and even loss of their catalytic activity. Recently, we found that Fe³⁺ ions could serve as the excellent peroxidase mimic towards the 3,3′,5,5′-tetramethyl benzidine (TMB)–H₂O₂ system.⁴⁶ Our investigations revealed that Fe³⁺ ions could exhibit much higher peroxidase-like activity than that of Fe₃O₄ nanoparticles when Fe concentrations were set to be exactly same. Therefore, if low concentration of catalysts (such as at sub μM level) can be employed, it will be believed to be capable of improving the sensitivity of the established catalytic sensing systems.

Herein, the peroxidase-like activity of Fe³⁺ ions towards the TMB–H₂O₂ system, and two enzymatic systems, that is, AChE catalyses the hydrolysis of S-acetylthiocholine (ATCh) to produce thiocholine (TCh), and choline oxidase (CHO) catalyses the oxidation of TCh to generate H₂O₂, were combined together to establish a multi-catalytic system. The resulting H₂O₂ could activate Fe³⁺ ions to catalyse the oxidation of TMB to produce a blue product. When OPs were introduced, the enzymatic activity of AChE can be inhibited by OPs due to the specific combination of OPs and AChE. The inhibition could further suppress the oxidation reaction of TMB catalysed by Fe³⁺ ions, due to producing less H₂O₂. Therefore, this change could indirectly indicate the amount of OPs. DDT, as one of organochlorine pesticides, could also inhibit the color reaction, similar to those of the tested OPs. The molecular structures of the tested OPs and DDT were showed in Scheme 1. The multi-catalytic mechanism could promise the high sensitivity of this sensing system towards OPs or organochlorine pesticides.

Scheme 1 The molecular structures of the tested OPs and DDT.

Experimental

Apparatus

UV-vis absorption spectra were recorded by a Thermo Evolution 300 UV-vis absorption spectrophotometer equipped with a temperature controller. pH measurements were made with a FE20-Five Eosy Plus pH meter.

Reagents

Fe(NO₃)₃·9H₂O (98.5%, AR), tri(hydroxymethyl) aminomethane (Tris, BR), HCl (36–38%, AR), NaOAc (99%, AR), HOAc (99.5%, AR), Co(NO₃)₃·6H₂O (99%), Cu(NO₃)₂·3H₂O (99–102%), AgNO₃ (99.95%), Ni(NO₃)₂·6H₂O (98.5%) and other organic compounds were obtained from GuoYao (Shanghai, China). 3,3′,5,5′-Tetramethyl benzidine (TMB, 99%), choline oxidase (CHO, from Alcaligenes sp.), acetylcholinesterase (AChE, from Electrophorus electricus), acetylthiocholine chloride (ATCh), dimethoate and 4,4′-DDT (5000 μg mL⁻¹ in methanol) were purchased from Sigma-Aldrich. Paraoxon and dichlorvos (DDVP, 1000 μg mL⁻¹ in methanol) were obtained from Aladdin. Ultrapure water was prepared by employing a Millipore system.

Procedures

Buffer solutions. 200 mM NaOAc–HOAc buffer solutions of pH 2.6, 3.8, 4.2, 4.6, 5.0 and 5.6, respectively, and 50 mM Tris–HCl buffer solution of pH 7.4 were prepared.

OPs or DDT stock solutions. OPs or DDT were dissolved in hexane to afford OPs stock solutions, which were stored in a refrigerator at 4 °C. Working solutions of OPs or DDT were freshly prepared in isopropanol by diluting corresponding OPs or DDT stock solution to achieve a series of concentrations.

The multi-catalytic system. The multi-catalytic system was established as follows: (1) 0.25 Unit activity of AChE, 0.25 Unit activity of CHO and 10 μL ATCh solutions were mixed with 200 μL 50 mM Tris–HCl buffer solution of pH 7.4, and the mixed solution was incubated at 37 °C for 15 min; (2) 675 μL 200 mM NaOAc–HOAc buffer solution of pH 4.2, 100 μL 5 mM TMB and 25 μL 0.2 mM Fe³⁺ ions solutions were added into the resulting solution of (1) to afford 1000 μL total volume. The mixed solution was instantly monitored by a Thermo Evolution 300 UV-vis absorption spectrophotometer equipped with a temperature controller at 37 °C, and absorbance at 652 nm wavelength was recorded with a certain time interval. Final concentrations of ATCh, AChE and CHO were 0.5 mM, 0.5 Unit mL⁻¹ and 0.5 Unit mL⁻¹, respectively. Error bars were obtained by measuring three parallel samples.

Sensing OPs or DDT. (1) 10 μL OPs or DDT working solutions were dissolved by 200 μL 50 mM Tris–HCl buffer solution of pH 7.4 containing 0.25 Unit activity of AChE, and then the resulting solution was incubated at 37 °C for 10 min; (2) 10 μL ATCh and 0.25 Unit activity of CHO solutions were then added into the resulting solution of (1), and the mixed solution was incubated at 37 °C for 15 min; (3) the resulting solution of (2) was then added into 800 μL 200 mM NaOAc–HOAc buffer solution of pH 4.2 containing 100 μL 5 mM TMB solution and 25 μL 0.2 mM Fe³⁺ ions solution. The mixed solution was incubated for another 15 min at 37 °C to allow color development. The absorbance at 652 nm wavelength of the resulting solution of (3) was measured. Final concentrations of ATCh, AChE and CHO in this sensing system were 0.5 mM, 0.5 Unit mL⁻¹ and 0.5 Unit mL⁻¹, respectively. Error bars were obtained by measuring three parallel samples.

Judging whether the practical vegetable/fruit samples were or not suspected samples. The procedure was operated as follows: (1) the fruit sample was peeled and then cut into ca. 0.2 cm × 0.2 cm pieces; the vegetable sample was directly cut into similar pieces; (2) the sample pieces were extracted by 5 mL 50 mM Tris–HCl buffer solution of pH 7.4 per 1 g sample; (3) 10 μL extracted solution was further dissolved in 200 μL 50 mM Tris–HCl buffer solution of pH 7.4 containing 0.25 Unit activity of AChE, and the mixed solution was incubated at 37 °C for 10 min; (4) 10 μL ATCh and 0.25 Unit activity of CHO were then added into the resulting solution of (3), and the mixed solution was incubated at 37 °C for 15 min; (5) the resulting solution of (4) was then added into 800 μL 200 mM NaOAc–HOAc buffer solution of pH 4.2 containing 100 μL 5 mM TMB solution and 25 μL 0.2 mM Fe³⁺ ions solution. The mixed solution was incubated for another 15 min at 37 °C to allow color development. The absorbance at 652 nm wavelength of the resulting solution of (5) was easily measured. Average values were obtained by measuring three parallel samples.

Recovery experiments of DDVP in spiked vegetable/fruit samples. Pear, Bok-choy and water spinach were chosen as the practical samples, and DDVP was chosen as a model OPs, to evaluate the feasibility of the established sensing method for OPs. The fruit sample was peeled and then cut into ca. 0.2 cm × 0.2 cm pieces, the vegetable sample was directly cut into similar pieces. The cut samples were extracted by 5 mL 50 mM Tris–HCl buffer solution of pH 7.4 per 1 g sample for 2 min oscillation. DDVP solution of known concentration was added into the extracted solution for 5 min standing. The extracted solution was filtered by 0.2 μm needle filter. 10 μL spiked solution was further diluted by 200 μL 50 mM Tris–HCl buffer solution of pH 7.4 containing 0.25 Unit activity of AChE, and the diluted solution was incubated at 37 °C for 10 min. 10 μL ATCh and 0.25 Unit activity of CHO were then added and further incubated at 37 °C for 15 min. The resulting solution was then added into 800 μL 200 mM NaOAc–HOAc buffer solution of pH 4.2 containing 100 μL 5 mM TMB and 25 μL 0.2 mM Fe³⁺ ions solutions. The mixed solution was incubated for another 15 min at 37 °C to allow color development. The absorbance at 652 nm wavelength of the resulting solution was measured. DDVP recovery in the spiked samples could be calculated according to the calibration curve. Average values were obtained by measuring three parallel samples.

Results and discussion

The color reaction of TMB–H₂O₂ system

Our recent investigations revealed that, Fe³⁺ ions possess extremely high peroxidase-like activity towards the TMB–H₂O₂ system.⁴⁶ Considering that other transition metal ions maybe have similar catalytic performance, several traditional transition metal ions including Ag⁺, Co²⁺, Ni²⁺ and Cu²⁺ ions were thus chosen to evaluate their peroxidase-like activity for catalysing the redox reaction of TMB–H₂O₂ system. Results indicated that, under similar experimental conditions, that is, 200 mM NaOAc–HOAc buffer solution of pH 4.2, 0.5 mM TMB, 4 mM H₂O₂ and 37 °C were used, the peroxidase-like activity of Fe³⁺ ions was much higher than those of other tested transition metal ions, even if their concentration were enhanced up to be 25-fold more than that of Fe³⁺ ions, color reaction was hardly recorded (Fig. 1a). This further supported that Fe³⁺ ions have distinctive and excellent peroxidase-like activity towards the TMB–H₂O₂ system. Therefore, Fe³⁺ ions were chosen as the peroxidase mimic to establish the multi-catalytic sensing system, taken together the two enzymatic systems of AChE and CHO.

Fig. 1 (a) Comparison of the peroxidase-like activity of Fe³⁺ ions and other tested transition metal ions including Ag⁺, Co²⁺, Ni²⁺, Cu²⁺ ions. [Fe³⁺ ions] = 2 μM, [other tested metal ions] = 50 μM; pH-dependent (b), temperature-dependent (c), and reaction time-dependent (d) peroxidase-like activity of Fe³⁺ ions. Other experimental conditions: [TMB] = 0.5 mM, [CHO] = 0.5 Unit mL⁻¹, [ATCh] = 0.5 mM, [AChE] = 0.5 Unit mL⁻¹, 50 mM Tris–HCl buffer solution of pH 7.4 (first and second catalytic reactions), [Fe³⁺ ions] = 5 μM except for (a), 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) except for (b), temperature of 37 °C except for (c), 15 min was chosen as the time for recording the absorbance except for (d).

Firstly, the experimental conditions of the redox reaction of TMB–H₂O₂ system catalysed by Fe³⁺ ions were optimized (third catalytic reaction). pH effect exhibited that the catalytic activity of Fe³⁺ ions decreased with enhancing pH when 200 mM NaOAc–HOAc buffer solution of various pH was employed to control pH of the system (Fig. 1b). Temperature effect revealed that, the catalytic activity of Fe³⁺ ions increased with enhancing temperature, when temperature was set as the range from 25 °C to 45 °C, however, further increasing temperature, the catalytic activity decreased (Fig. 1c). Furthermore, reaction time effect demonstrated that, with extending the reaction time window, absorbance at 652 nm wavelength increased (Fig. 1d). Therefore, 200 mM NaOAc–HOAc buffer solution of pH 4.2, temperature of 37 °C and time of 15 min were chosen in the Fe³⁺–TMB–H₂O₂ system for further experiments.

Optimizing experimental conditions of AChE and CHO catalytic reactions

The catalytic hydrolysis reaction of AChE towards ATCh as the substrate was investigated. This was the first catalytic reaction for constructing the multi-catalytic sensing system, due to producing TCh which could act as the substrate for CHO (second catalytic reaction). Considering the enzymatic stability of AChE and CHO, temperature of 37 °C and 50 mM Tris–HCl buffer solution of pH 7.4 were employed in the first and second catalytic reactions. With enhancing AChE concentration from 0.1 to 1 Unit mL⁻¹, absorbance at 652 nm wavelength gradually increased and then showed a platform region at the concentration range from 0.5 to 1.0 Unit mL⁻¹ (Fig. 2). Increasing absorbance was indicative of increasing the amount of color products. The effect of ATCh concentration also exhibited similar observations, that is, when ATCh concentration was enhanced from 30 μM to 500 μM, absorbance at 652 nm wavelength increased. Further enhancing ATCh concentration, absorbance showed somewhat decreasing (Fig. 3). Therefore, 0.5 Units mL⁻¹ AChE and 0.5 mM ATCh were chosen in this multi-catalytic sensing system for other experiments.

Fig. 2 The relationship between Abs@652 nm and measuring time of the multi-catalytic system under various AChE concentration (a). The relationship between AChE concentration and Abs@652 nm (b), when the reaction time was taken as 15 min. Experimental conditions: [TMB] = 0.5 mM; [Fe³⁺ ions] = 5 μM; [CHO] = 0.5 Unit mL⁻¹; [ATCh] = 0.5 mM; 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) and 50 mM Tris–HCl buffer solution of pH 7.4 (first and second catalytic reactions); temperature of 37 °C.

Fig. 3 The relationship between Abs@652 nm and measuring time of the multi-catalytic system under various ATCh concentration (a). The relationship between ATCh concentration and Abs@652 nm (b), when the reaction time was taken as 15 min. Experimental conditions: [TMB] = 0.5 mM; [Fe³⁺ ions] = 5 μM; [CHO] = 0.5 Unit mL⁻¹; [AChE] = 0.5 Unit mL⁻¹; 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) and 50 mM Tris–HCl buffer solution of pH 7.4 (first and second catalytic reactions); temperature of 37 °C.

Considering high selectivity of CHO as an oxidase for the oxidation reaction of TCh, the effect of CHO concentration was also investigated. It was found that, when CHO concentration was enhanced from 0.1 to 0.5 Unit mL⁻¹, absorbance at 652 nm wavelength gradually increased, indicative of producing much more color products; however, with further increasing CHO concentration, the catalytic activity was almost kept as a constant (Fig. 4). It was evident that the producing color intensity of the multi-catalytic sensing system was also dependent on the enzymatic activity of CHO. Therefore, the concentration of CHO was fixed as 0.5 Unit mL⁻¹ for other experiments.

Fig. 4 The relationship between Abs@652 nm and measuring time of the multi-catalytic system under various CHO concentration (a). The relationship between CHO concentration and Abs@652 nm (b), when the reaction time was taken as 15 min. Experimental conditions: [TMB] = 0.5 mM; [Fe³⁺ ions] = 5 μM; [AChE] = 0.5 Unit mL⁻¹; [ATCh] = 0.5 mM; 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) and 50 mM Tris–HCl buffer solution of pH 7.4 (first and second catalytic reactions); temperature of 37 °C.

OPs and organochlorine pesticides detection

Firstly, paraoxon, DDVP, and dimethoate were chosen as model OPs for exploring the feasibility of the established the multi-catalytic sensing system. In general, the color reaction for detecting OPs could be operated by three steps: (1) various concentration of OPs were incubated with AChE in 50 mM Tris–HCl buffer solution of pH 7.4 for 10 min at 37 °C; (2) the resulting solution of (1) was further mixed with the solutions of CHO and ATCh for 15 min at 37 °C; (3) the resulting solution of (2) was further incubated with Fe³⁺ ions and TMB solution for 15 min at 37 °C. After the operation procedures of three steps were conducted, the absorbance at 652 nm wavelength was instantly recorded by a UV-vis absorption spectrometer with a certain time interval.

OPs concentration-dependent response curves of absorbance at 652 nm wavelength revealed that, with increasing OPs concentration, apparent absorbance gradually decreased (Fig. 5, 7 and 9). Obviously, OPs could suppress this color reaction. It is well known that, AChE can catalyse the hydrolysis of ATCh to generate TCh (the first catalytic reaction), capable of acting as the substrate for CHO (the second catalytic reaction). The second catalytic reaction can produce H₂O₂. The binding of OPs and AChE can suppress the catalytic activity of AChE towards the hydrolysis of ATCh, which can further result in much less H₂O₂ than that of the case of in the absence of OPs. Therefore, the amount of introduced OPs can be indirectly indicated by the absorbance at 652 nm wavelength of resulting TMBox.

Fig. 5 The relationship curve between paraoxon concentration ranged from 2 nM to 50 μM and Abs@652 nm. Experimental conditions: [TMB] = 0.5 mM; [Fe³⁺ ions] = 5 μM; [AChE] = 0.5 Units mL⁻¹; [CHO] = 0.5 Units mL⁻¹; [ATCh] = 0.5 mM; 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) and 50 mM Tris–HCl buffer solution of pH 7.4 (first and second reactions); temperature of 37 °C; the value of Abs@652 nm was taken when the time of the color reaction was 15 min.

Fig. 6 The relationship between Abs@652 nm and log[paraoxon] (a) and the linear calibration plot between Abs@652 nm and log[paraoxon] (b). Experimental conditions: [TMB] = 0.5 mM; [Fe³⁺ ions] = 5 μM; [AChE] = 0.5 Unit mL⁻¹; [CHO] = 0.5 Unit mL⁻¹; [ATCh] = 0.5 mM; 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) and 50 mM Tris–HCl buffer solution of pH 7.4 (first and second reactions); temperature of 37 °C.

Fig. 7 The relationship curve between DDVP concentration ranged from 10 nM to 225 μM and Abs@652 nm. Experimental conditions: [TMB] = 0.5 mM; [Fe³⁺ ions] = 5 μM; [AChE] = 0.5 Unit mL⁻¹; [CHO] = 0.5 Unit mL⁻¹; [ATCh] = 0.5 mM; 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) and 50 mM Tris–HCl buffer solution of pH 7.4 (first and second catalytic reactions); temperature of 37 °C; the value of Abs@652 nm was taken when the time of the color reaction was 15 min.

Fig. 8 The relationship between Abs@652 nm and log[DDVP] (a) and the linear calibration plots between Abs@652 nm and log[DDVP] (b). Experimental conditions: [TMB] = 0.5 mM; [Fe³⁺ ions] = 5 μM; [AChE] = 0.5 Unit mL⁻¹; [CHO] = 0.5 Unit mL⁻¹; [ATCh] = 0.5 mM; 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) and 50 mM Tris–HCl buffer solution of pH 7.4 (first and second catalytic reactions); temperature of 37 °C.

Fig. 9 The relationship curve between dimethoate concentration ranged from 2 μM to 10 mM and Abs@652 nm. Experimental conditions: [TMB] = 0.5 mM; [Fe³⁺ ions] = 5 μM; [AChE] = 0.5 Unit mL⁻¹; [CHO] = 0.5 Unit mL⁻¹; [ATCh] = 0.5 mM; 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) and 50 mM Tris–HCl buffer solution of pH 7.4 (first and second catalytic reactions); temperature of 37 °C; the value of Abs@652 nm was taken when the time of the color reaction was 15 min.

Besides OPs, DDT, as one of banned organochlorine pesticides, is being illegally used. Therefore, DDT was employed for extending the scope of this sensing system. Similar to the OPs cases of paraoxon, DDVP and dimethoate, it was found that the absorbance at 652 nm wavelength of this sensing system was DDT concentration-dependent, too (Fig. 11). That is, with increasing DDT concentration to μM level, absorbance at 652 nm wavelength gradually decreased, indicated that DDT could effectively inhibit the enzymatic activity of AChE. However, on the basis of our knowledge, the inhibition of the catalytic activity of AChE by DDT has not been reported.

Although the relationships between absorbance at 652 nm wavelength and tested OPs or DDT concentrations showed non-linear curves, the linear range could be obtained when OPs or DDT concentrations were transferred into their corresponding logarithms (Fig. 6, 8, 10 and 12) with acceptable regression coefficients. Limit of detections (LODs) of these tested paraoxon, DDVP, dimethoate and DDT were estimated to be 0.15, 0.35, 11 and 0.85 nM, respectively. LODs in this work are lower than those of most of reported cases involving tested OPs or DDT, and the comparison was summarized in Table 1. These results supported that employing multi-catalytic reactions in the sensing system could substantially improve the sensitivity.

Fig. 10 The relationship between Abs@652 nm and log[dimethoate] (a) and the linear calibration plots between Abs@652 nm and log[dimethoate] (b). Experimental conditions: [TMB] = 0.5 mM; [Fe³⁺ ions] = 5 μM; [AChE] = 0.5 Unit mL⁻¹; [CHO] = 0.5 Unit mL⁻¹; [ATCh] = 0.5 mM; 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) and 50 mM Tris–HCl buffer solution of pH 7.4 (first and second catalytic reaction); temperature of 37 °C.

Fig. 11 The relationship curve between DDT concentration ranged from 10 nM to 80 μM and Abs@652 nm. Experimental conditions: [TMB] = 0.5 mM; [Fe³⁺ ions] = 5 μM; [AChE] = 0.5 Unit mL⁻¹; [CHO] = 0.5 Unit mL⁻¹; [ATCh] = 0.5 mM; 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) and 50 mM Tris–HCl buffer solution of pH 7.4 (first and second catalytic reactions); temperature of 37 °C; the value of Abs@652 nm was taken when the time of the color reaction was 15 min.

Fig. 12 The relationship between Abs@652 nm and log[DDT] (a) and the linear calibration plots between Abs@652 nm and log[DDT] (b). Experimental conditions: [TMB] = 0.5 mM; [Fe³⁺ ions] = 5 μM; [AChE] = 0.5 Unit mL⁻¹; [CHO] = 0.5 Unit mL⁻¹; [ATCh] = 0.5 mM; 200 mM NaOAc–HOAc buffer solution of pH 4.2 (third catalytic reaction) and 50 mM Tris–HCl buffer solution of pH 7.4 (first and second reactions); temperature of 37 °C.

Table 1 Comparison of the LODs of reported references and our present work

No.	Ref.	Methods	Sensing OPs or DDT	LOD
1	44	Colorimetry	Methyl-paraoxon; dimethoate	10 nM
				5 μM
2	27	Colorimetry	Paraoxon	200 nM
3	47	Colorimetry	DDVP	6.7 ppb
4	48	Colorimetry	DDT	27 ppb
5	49	Fluorometry	Paraoxon	10 nM
6	23	Fluorometry	DDVP	4.49 nM
7	50	Luminescence	DDVP	0.32 μM
8	51	Chromatography	DDT	0.32–0.51 μg L^−1
9	52	Fluorometry	Paraoxon	13.1 pM
10	22	Fluorometry	Paraoxon	8 nM
11	This work	Colorimetry	Paraoxon	0.15 nM
			DDVP	0.35 nM
			Dimethoate	11 nM
			DDT	0.85 nM

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In order to validate the feasibility of this established sensing system for OPs or DDT in practical samples, two group experiments were conducted, (1) judging whether the practical fruit/vegetable samples contain a detectable or no detectable amount of OPs/organochlorine pesticides, (2) the recovery experiments. Results were summarized in Tables 2 and 3.

Table 2 Results of judging whether the practical fruit/vegetable samples were or not suspected samples^a

Sample	Abs at 652 nm	Results
a In order to determinate that whether the sample is or not a suspected sample, it was assumed that, when the absorbance at 652 nm wavelength of a sample is lower 10% than that of control experiment, the sample could be judged as a “detectable” sample. If it is not, then it can be called as a “no detectable” sample. Measured Abs@652 nm was obtained by measuring three parallel samples to afford their average value.
Orange	0.469	Detectable
Bok-choy	0.562	Detectable
Pear	0.618	Detectable
Apple	0.481	Detectable
Water spinach	0.616	Detectable
Pleurotus eryngii	0.768	No detectable
Control experiment	0.798	Blank

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Table 3 Recovery experiments of DDVP in fruit/vegetable samples^a

Sample	Spiked/μM	Detected concentration/μM	Recovery/%	RSD
a The detected concentration was referred as the average value of the three parallel measurements. After the samples were soaked by 50 mM Tris–HCl buffer solution of pH 7.4, the resulting buffer solution was mixed with a certain volume of DDVP solution of a known concentration, and then the resulting solution was introduced into the sensing system.
Pear	7.2	7.39	102.6	5.4%
Bok-choy	7.2	7.22	100.3	7%
Water spinach	7.2	7.12	98.9	1.7%

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For judging whether the sample is or not a suspected sample, we assume that, when the absorbance at 652 nm wavelength of a sample is lower 10% than that of control blank experiment, the sample could be judged as a “detectable” sample. If it is not, then it can be called as a “no detectable” sample. Of course, allowable absorbance at 652 nm wavelength of the sample can also be lowered some, according to related regulations. Results revealed that five samples could be judged as “detectable” samples and one sample could be judged as “no detectable” sample according to our assumption (Table 2). However, for a practical sample obtained from market, this sensing system could only afford a preliminarily judge whether the sample is a suspected sample, yet could not determinate which kind of OPs or organochlorine pesticides and its content. Accurate detection should be conducted by other methods such as GC and LC. In despite of the drawback, this sensing system is suitable for employing as a powerful tool for preliminarily screening of suspected samples.

DDVP was chosen to further conduct the recovery experiments (Table 3). Results demonstrated that, for three fruit/vegetable samples including pear, Bok-choy and water spinach, the recovery of DDVP was found to locate at the range from 98.9% to 102.6%, when 7.2 μM DDVP solution was added into the soaked solution, with acceptable relative standard deviations (RSDs). This indicated good reliability of this sensing system.

Conclusion

In summary, a simple, facile and sensitive multi-catalytic sensing system has been successfully established based on the peroxidase-like activity of Fe³⁺ ions, taken together two enzymatic catalytic systems, AChE and CHO. OPs could inactivate AChE, resulting in no production of TCh, which further suppress the formation of H₂O₂. Therefore, the color reaction of the Fe³⁺–TMB–H₂O₂ system could be suppressed, indirectly indicative of the amount of OPs. DDT, one of organochlorine pesticides, showed similar suppression. High sensitivity of this sensing system could be achieved, because LODs of paraoxon, DDVP, dimethoate and DDT were estimated to be 0.15 nM, 0.35 nM, 11 nM and 0.85 nM, respectively. This supports that introducing multi-catalytic reaction could be substantially enhance sensitivity. Several fruit/vegetable samples have been successfully employed to check this established sensing system. Our sensing system showing several merits such as simplicity, rapid operation, and high sensitivity, is expected to be suitable for acting as a powerful tool for preliminarily screening of suspected samples.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant no. 21377124 and 21173268), by the project of new faculty of Huaqiao University (Grant no. 15BS202), and by the State Key Laboratory of NBC Protection for Civilian (Grant no. SKLNBC2013-04 and 2013-05).

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Footnote

† Y. X. and T. Y. contributed equally to this work.

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