Fabrication of Metal-Substituted Polyoxometalates for Colorimetric Detection of Dopamine and Ractopamine

A novel colorimetric detection method based on the peroxidase-like activity of metal-substituted polyoxometalates (POMs) of SiW9M3 (M = Co2+, Fe3+, Cu2+, Mn2+) has been established. POMs can catalyze oxidation of dopamine (DA) and ractopamine (RAC) by H2O2 in aqueous solutions. SiW9Co3-based POMs detect DA at concentrations as low as 5.38 × 10−6 mol·L−1 simply by observation of the color change from colorless to orange using the naked eye. RAC is detected by observing the change from colorless to slight red by SiW9Cu3 with a detection limit of 7.94 × 10−5 mol·L−1. This study shows that colorimetric DA and RAC detection using SiW9Co3 and SiW9Cu3 is highly selective and sensitive as well as visually observable.


Introduction
β-agonists are known as phenylethanolamines with different substituent groups on the aromatic ring and the terminal amino group. Usually, β-agonists are applied for the treatment of pulmonary disease and asthma. In recent years, β-agonists have been illegally used to promote animal growth and increase the percentage of lean meat in pig carcasses. However, the excessive addition of β-agonists in animal feed results in accumulation in human tissue after consumption of the defective meat, which can lead to acute or chronic poisoning. Therefore, there is a need to develop fast, easy, selective, and accurate methods for detecting β-agonists for protecting public health. Dopamine (DA) and ractopamine (RAC) are the two main β-agonists in the list of banned feed additives in China and European Union (EU). A series of approaches have been developed for detecting DA, such as electrochemical analysis [1], high performance liquid chromatography (HPLC) [2], and the chemiluminescence method [3]. Methods for the detection of RAC have also been established, including capillary electrophoresis [4], HPLC [5], enzyme-linked immunoassay (ELISA) [6], electrochemical detection [7], and colloidal gold [8]. However, these methods involve complicated sample treatment procedures and have poor reproductivity. In the past decades, colorimetric detection, as one convenient approach, has been explored for providing naked-eye readout signals with simple were purchased from Aladdin (Shanghai, China). RAC (97%) was provided by J&K Scientific LTD (Hong Kong, China).

Procedure for the Detection of RAC
POMs (8 mM) and H 2 O 2 (200 mM) were added into an aqueous solution of 1 mL. RAC (5 mM) was added into the above mixture, and then blended again. Reaction time was from 0 to 25 min. The sample solution was measured at 515 nm by a Shimadzu (Kyoto, Japan) UV-2700 UV-VIS spectrophotometer (200-600 nm).

Procedure for the Detection of DA
POMs (10 mM) and H 2 O 2 (100 mM) were added into an aqueous solution of 1 mL. DA (8 mM) was added into the above mixture, and then blended again. The mixed solution was laid aside for 0-15 min. The resulting solution was measured at 475 nm by a Shimadzu UV-2700 UV-VIS spectrophotometer (200-600 nm).

Characterization of Catalysts
FT-IR and UV-VIS were used to identify the different structural among SiW 11 M, SiW 10 M 2 , and SiW 9 M 3 in Table S1. These three kinds of POMs are all derivatives of Keggin POMs. The typical peaks of UV-VIS at 200 nm and 250 nm were attributed to the transition of O d -W and O b /O c →W, respectively, in the heteropolyacid anion. This was in accordance with the characteristic peaks of the Keggin heteropolyanions.
FT-IR results indicated that peaks in the range of 600-1100 cm −1 corresponded to the asymmetric vibrations of P-O a , W-O d , W-O b , and W-O c of the Keggin structure [22]. This confirmed the compounds all maintained the Keggin structure. However, the FT-IR spectrum of SiW 9 M 3 ( Figure S1) differed from those of SiW 11 M and SiW 10 M 2 due to the splitting of the peak at 600-800 cm −1 [21], and the particular results from the vibration of W-O c -W corresponded to the structure of SiW 9 M 3 .
XRD patterns were collected to characterize the solid structure of SiW 9 M 3 , SiW 9 , SiW 10 , SiW 11 , and SiW 12 in Figure S2. The obvious peaks of SiW 9 , SiW 10 , SiW 11 , and SiW 12 could be observed due to their distinctive crystal structures. As derivatives of Keggin structure, catalysts of SiW 9 M 3 also kept similar diffraction peaks to pure SiW 12 ( Figure S3a) [24,25] High-resolution spectra of different metal ions are shown in Figure S3b-e, which corresponded to the energy of Co 2+ , Cu 2+ , Fe 3+ , and Mn 2+ in the catalysts of SiW 9 M 3 .

Catalytic Activity of POM Catalysts
POMs are distinctive due to their fast and reversible multi-electron redox processes, even under wild conditions. The redox property of POMs was adjusted by the type and amount of metal ions to complete the reaction with different substrates. In our investigation, it has been confirmed that the For the detection of RAC, only the tri-substituted catalyst of SiW 9 Cu 3 affected the colorimetric detection and provided the obvious color change from colorless to slight red (Figure 1 top). Hence, the subsequent detections of RAC were all carried out by SiW 9 Cu 3 . For the detection of DA, the catalytic activity ( Figure 2) was in the order SiW 9 M 3 > SiW 10 M 2 > SiW 11 M. This indicated that the increasing number of transition metal ions led to the stronger redox property of POMs, which was beneficial to the colorimetric detection of DA. The effect of metal ions was in the order of Co > Mn > Cu > Fe, and SiW 9 Co 3 exhibited the highest catalytic performance from colorless to orange (Figure 1 bottom). Hence, the following detection studies of DA were all carried out by SiW 9 Co 3 . The above results confirmed that the type of transition metal ions had an effect on the catalytic performance and selectivity when different POM catalysts were used for the detection of DA. The obvious color change was easy for visual discrimination, and showed a superior performance to that of AuNPs. that the type and number of transition metal ions had a crucial effect on the catalytic results when the different POMs catalysts were used for the detection of DA and RAC. For the detection of RAC, only the tri-substituted catalyst of SiW9Cu3 affected the colorimetric detection and provided the obvious color change from colorless to slight red (Figure 1 top). Hence, the subsequent detections of RAC were all carried out by SiW9Cu3. For the detection of DA, the catalytic activity ( Figure 2) was in the order SiW9M3 > SiW10M2 > SiW11M. This indicated that the increasing number of transition metal ions led to the stronger redox property of POMs, which was beneficial to the colorimetric detection of DA. The effect of metal ions was in the order of Co > Mn > Cu > Fe, and SiW9Co3 exhibited the highest catalytic performance from colorless to orange (Figure 1 bottom). Hence, the following detection studies of DA were all carried out by SiW9Co3. The above results confirmed that the type of transition metal ions had an effect on the catalytic performance and selectivity when different POM catalysts were used for the detection of DA. The obvious color change was easy for visual discrimination, and showed a superior performance to that of AuNPs.

Detection of RAC
In this study, the effect of the amount of SiW9Cu3 (Figure 3a) was in the range of 20-110 µL. It was investigated at room temperature. The absorbance increased with the concentration of the catalyst, which illustrated that the POM detection system was highly specific for RAC. The that the type and number of transition metal ions had a crucial effect on the catalytic results when the different POMs catalysts were used for the detection of DA and RAC. For the detection of RAC, only the tri-substituted catalyst of SiW9Cu3 affected the colorimetric detection and provided the obvious color change from colorless to slight red (Figure 1 top). Hence, the subsequent detections of RAC were all carried out by SiW9Cu3. For the detection of DA, the catalytic activity ( Figure 2) was in the order SiW9M3 > SiW10M2 > SiW11M. This indicated that the increasing number of transition metal ions led to the stronger redox property of POMs, which was beneficial to the colorimetric detection of DA. The effect of metal ions was in the order of Co > Mn > Cu > Fe, and SiW9Co3 exhibited the highest catalytic performance from colorless to orange (Figure 1 bottom). Hence, the following detection studies of DA were all carried out by SiW9Co3. The above results confirmed that the type of transition metal ions had an effect on the catalytic performance and selectivity when different POM catalysts were used for the detection of DA. The obvious color change was easy for visual discrimination, and showed a superior performance to that of AuNPs.

Detection of RAC
In this study, the effect of the amount of SiW9Cu3 (Figure 3a) was in the range of 20-110 µL. It was investigated at room temperature. The absorbance increased with the concentration of the catalyst, which illustrated that the POM detection system was highly specific for RAC. The

Detection of RAC
In this study, the effect of the amount of SiW 9 Cu 3 (Figure 3a) was in the range of 20-110 µL. It was investigated at room temperature. The absorbance increased with the concentration of the catalyst, which illustrated that the POM detection system was highly specific for RAC. The performance at the concentration of 4.78 × 10 −4 mol·L −1 (80 µL) was the highest, and the absorbance of 90-110 µL was slightly decreased. Hence, 80 µL of catalyst was chosen for the naked-eye observation of RAC.
As a key factor, the oxidant content of H 2 O 2 has decisive influences on the catalysis of RAC. Results are shown in Figure 3b and increasing amounts of H 2 O 2 caused increasing absorption. When the concentration reached 2.39 × 10 −2 mol·L −1 (160 µL), the maximum absorption was obtained. However, with further increasing the amount of H 2 O 2 , there was no obvious improvement in absorbance.
performance at the concentration of 4.78 × 10 −4 mol·L −1 (80 µL) was the highest, and the absorbance of 90-110 µL was slightly decreased. Hence, 80 µL of catalyst was chosen for the naked-eye observation of RAC.
As a key factor, the oxidant content of H2O2 has decisive influences on the catalysis of RAC. Results are shown in Figure 3b and increasing amounts of H2O2 caused increasing absorption. When the concentration reached 2.39 × 10 −2 mol·L −1 (160 µL), the maximum absorption was obtained. However, with further increasing the amount of H2O2, there was no obvious improvement in absorbance. The absorption of samples was tested at room temperature every 5 min and the longest reaction time was 60 min (Figure 4). The obvious color change was observed from colorless to slight red at just under 5 min. After prolonging the response time, an increasing absorbance was obtained, but an apparent deepening of color was not observed. When reaction time was 25 min, the shape of absorption curve was significantly superior to the others. At an even longer time, the absorbance showed a slow increase and the curve shape kept constant. Thus, the optimum reaction time was determined as 25 min. The color of the solution was also stable even if the solution was laid aside for 60 min, which indicated that this system was suitable for the colorimetric detection of RAC with SiW9Cu3 in aqueous solution.  The absorption of samples was tested at room temperature every 5 min and the longest reaction time was 60 min (Figure 4). The obvious color change was observed from colorless to slight red at just under 5 min. After prolonging the response time, an increasing absorbance was obtained, but an apparent deepening of color was not observed. When reaction time was 25 min, the shape of absorption curve was significantly superior to the others. At an even longer time, the absorbance showed a slow increase and the curve shape kept constant. Thus, the optimum reaction time was determined as 25 min. The color of the solution was also stable even if the solution was laid aside for 60 min, which indicated that this system was suitable for the colorimetric detection of RAC with SiW 9 Cu 3 in aqueous solution.
Materials 2018, 11, x FOR PEER REVIEW 5 of 11 performance at the concentration of 4.78 × 10 −4 mol·L −1 (80 µL) was the highest, and the absorbance of 90-110 µL was slightly decreased. Hence, 80 µL of catalyst was chosen for the naked-eye observation of RAC. As a key factor, the oxidant content of H2O2 has decisive influences on the catalysis of RAC. Results are shown in Figure 3b and increasing amounts of H2O2 caused increasing absorption. When the concentration reached 2.39 × 10 −2 mol·L −1 (160 µL), the maximum absorption was obtained. However, with further increasing the amount of H2O2, there was no obvious improvement in absorbance. The absorption of samples was tested at room temperature every 5 min and the longest reaction time was 60 min (Figure 4). The obvious color change was observed from colorless to slight red at just under 5 min. After prolonging the response time, an increasing absorbance was obtained, but an apparent deepening of color was not observed. When reaction time was 25 min, the shape of absorption curve was significantly superior to the others. At an even longer time, the absorbance showed a slow increase and the curve shape kept constant. Thus, the optimum reaction time was determined as 25 min. The color of the solution was also stable even if the solution was laid aside for 60 min, which indicated that this system was suitable for the colorimetric detection of RAC with SiW9Cu3 in aqueous solution.   Figure 5 illustrated the amount of RAC had an effect on the colorimetric change. In the series of experiments, the amount of RAC was varied in the range of 60 to 160 µL and the solutions all showed a slight red color, which proved that the new system was suitable for qualitative detection of RAC with obvious color change. The maximum absorption was obtained at 100 µL of RAC. On further increasing the usage of RAC, the decreasing absorbance could be observed, which was attributed to the decreasing concentration of active catalyst sites and the shortage of oxidizing capacity of the system with increasing amounts of RAC. The linear range was in the range from 1.56 × 10 −4 to 3.73 × 10 −4 mol·L −1 and the limit of detection was 7.94 × 10 −5 mol·L −1 . Therefore, the optimum conditions could be considered as 80 µL SiW 9 Cu 3 , 100 µL RAC, and 160 µL H 2 O 2 , with a reaction time of 25 min. attributed to the decreasing concentration of active catalyst sites and the shortage of oxidizing capacity of the system with increasing amounts of RAC. The linear range was in the range from 1.56 × 10 −4 to 3.73 × 10 −4 mol·L −1 and the limit of detection was 7.94 × 10 −5 mol·L −1 . Therefore, the optimum conditions could be considered as 80 µL SiW9Cu3, 100 µL RAC, and 160 µL H2O2, with a reaction time of 25 min.

DA Detection
As β-agonists and the most important catecholamine neurotransmitters, DA has crucial effects in the human central nervous system under physiological conditions. Hence, the following experiments were all conducted in aqueous solutions, representing a wide range of application conditions in the biometric technology, medical, and pharmaceutical fields. Figure 6a illustrates the effect of the concentration of SiW9Co3 on the oxidation of DA at room temperature. When the usage of SiW9Co3 was in the range of 0-7.81 × 10 −4 mol·L −1 , the absorbance increased with the concentration of catalyst, which illustrated that the system was highly specific to DA. The concentration of 6.35 × 10 −4 mol·L −1 (80 µL) was considered as the optimum usage amount.
The influence of H2O2 (Figure 6b) was also detected at room temperature in the experiment. The absorption increased remarkably with the concentration of oxidant. The maximum absorption was observed at 4.76 × 10 −3 mol·L −1 (60 µL). Then, the results were slightly decreased by increasing the concentration of H2O2. This indicated that the amount of H2O2 was crucial in the experiment.
The appropriate reaction time is also one of the necessary reaction conditions for the colorimetric detection of DA. The results of absorbance ( Figure 7) increased with the prolonged reaction time and the optimum time was 10 min. However, with further increasing reaction time, the color of solution was changed from orange to dark brown, which was less observable using the naked eye for readouts.
It was confirmed that SiW9Co II 3 was oxidized by H2O2 and then the product of SiW9Co III mCo II 3-m reacted with DA by the reaction of catalytic oxidation [26,27]. As a result, the orange aminochrome (AC) was formed, which was detected at 475 nm with UV-VIS spectrometer, and could be applied for colorimetric detection. Figure 1 (bottom photographs) demonstrates the colorimetric process for DA with SiW9Co3 and an obvious color change could be observed from colorless to orange by the

DA Detection
As β-agonists and the most important catecholamine neurotransmitters, DA has crucial effects in the human central nervous system under physiological conditions. Hence, the following experiments were all conducted in aqueous solutions, representing a wide range of application conditions in the biometric technology, medical, and pharmaceutical fields. Figure 6a illustrates the effect of the concentration of SiW 9 Co 3 on the oxidation of DA at room temperature. When the usage of SiW 9 Co 3 was in the range of 0-7.81 × 10 −4 mol·L −1 , the absorbance increased with the concentration of catalyst, which illustrated that the system was highly specific to DA. The concentration of 6.35 × 10 −4 mol·L −1 (80 µL) was considered as the optimum usage amount.
The influence of H 2 O 2 (Figure 6b) was also detected at room temperature in the experiment. The absorption increased remarkably with the concentration of oxidant. The maximum absorption was observed at 4.76 × 10 −3 mol·L −1 (60 µL). Then, the results were slightly decreased by increasing the concentration of H 2 O 2 . This indicated that the amount of H 2 O 2 was crucial in the experiment.
The appropriate reaction time is also one of the necessary reaction conditions for the colorimetric detection of DA. The results of absorbance ( Figure 7) increased with the prolonged reaction time and the optimum time was 10 min. However, with further increasing reaction time, the color of solution was changed from orange to dark brown, which was less observable using the naked eye for readouts.
It was confirmed that SiW 9 Co II 3 was oxidized by H 2 O 2 and then the product of SiW 9 Co III m Co II 3-m reacted with DA by the reaction of catalytic oxidation [26,27]. As a result, the orange aminochrome (AC) was formed, which was detected at 475 nm with UV-VIS spectrometer, and could be applied for colorimetric detection. Figure 1 (bottom photographs) demonstrates the colorimetric process for naked eye within an appropriate reaction time. However, a longer reaction time was not beneficial to the color observation because the AC can regroup as 5,6-dihydroxyindole (DHI) which was further oxidized to indole-5,6-quinone (IQ). Finally dark brown neuromelanin was generated.  The concentration of DA could be detected by SiW9Co3 under the optimum reaction condition. As shown in Figure 8, the calibration plot for absorbance at 475 nm against catalyst activity of DA was detected with a linear range from 1.08 × 10 −4 to 5.38 × 10 −6 mol·L −1 . The linear relationship indicated that the detection was kinetically controlled by DA and thus the reporting system could be used for the DA activity assay.  naked eye within an appropriate reaction time. However, a longer reaction time was not beneficial to the color observation because the AC can regroup as 5,6-dihydroxyindole (DHI) which was further oxidized to indole-5,6-quinone (IQ). Finally dark brown neuromelanin was generated.  The concentration of DA could be detected by SiW9Co3 under the optimum reaction condition. As shown in Figure 8, the calibration plot for absorbance at 475 nm against catalyst activity of DA was detected with a linear range from 1.08 × 10 −4 to 5.38 × 10 −6 mol·L −1 . The linear relationship indicated that the detection was kinetically controlled by DA and thus the reporting system could be used for the DA activity assay.  The concentration of DA could be detected by SiW 9 Co 3 under the optimum reaction condition. As shown in Figure 8, the calibration plot for absorbance at 475 nm against catalyst activity of DA was detected with a linear range from 1.08 × 10 −4 to 5.38 × 10 −6 mol·L −1 . The linear relationship indicated that the detection was kinetically controlled by DA and thus the reporting system could be used for the DA activity assay. naked eye within an appropriate reaction time. However, a longer reaction time was not beneficial to the color observation because the AC can regroup as 5,6-dihydroxyindole (DHI) which was further oxidized to indole-5,6-quinone (IQ). Finally dark brown neuromelanin was generated.  The concentration of DA could be detected by SiW9Co3 under the optimum reaction condition. As shown in Figure 8, the calibration plot for absorbance at 475 nm against catalyst activity of DA was detected with a linear range from 1.08 × 10 −4 to 5.38 × 10 −6 mol·L −1 . The linear relationship indicated that the detection was kinetically controlled by DA and thus the reporting system could be used for the DA activity assay. According to CODEX, the highest limitation of RAC is 2.96 × 10 −8 mol·L −1 (10 ppb). DA is also one of the main β-agonists. Thus, studies [28][29][30][31][32][33][34] have been put forward (Table 1). In recent years, electrochemical biosensors [15][16][17] based on POMs have made a rapid progress in the detection of DA. Compared with the other results in the colorimetric detection of DA and RAC, SiW 9 Co 3 is shown to represent a rapid simple detective system for DA with the addition of only H 2 O 2 . In recent years, few studies have reported on the use of POMs for the colorimetric detection of RAC. A new detection system of RAC was put forward in this study that was simple, easily observable using the naked eye, and stable over time.

Interference Detection
The amino group was effective in the structure of DA and RAC for colorimetric detection [35]. In order to validate the reliability of the proposed method, the influence of the other molecules containing the amino group should be discussed in the sample detection. Four chemicals, including glycine, alanine, glucose, and urea, were selected as interfering substances to evaluate the selectivity of catalysts, and results are presented in Figure 9. It was evident that interfering substances did not lead to a higher absorbance, which revealed that the system was highly selective for DA and RAC. Therefore, it was clear that SiW 9 Co 3 and SiW 9 Cu 3 were specific for the colorimetric detection of DA and RAC, respectively. According to CODEX, the highest limitation of RAC is 2.96 × 10 −8 mol·L −1 (10 ppb). DA is also one of the main β-agonists. Thus, studies [28][29][30][31][32][33][34] have been put forward (Table 1). In recent years, electrochemical biosensors [15][16][17] based on POMs have made a rapid progress in the detection of DA. Compared with the other results in the colorimetric detection of DA and RAC, SiW9Co3 is shown to represent a rapid simple detective system for DA with the addition of only H2O2. In recent years, few studies have reported on the use of POMs for the colorimetric detection of RAC. A new detection system of RAC was put forward in this study that was simple, easily observable using the naked eye, and stable over time.

Interference Detection
The amino group was effective in the structure of DA and RAC for colorimetric detection [35]. In order to validate the reliability of the proposed method, the influence of the other molecules containing the amino group should be discussed in the sample detection. Four chemicals, including glycine, alanine, glucose, and urea, were selected as interfering substances to evaluate the selectivity of catalysts, and results are presented in Figure 9. It was evident that interfering substances did not lead to a higher absorbance, which revealed that the system was highly selective for DA and RAC. Therefore, it was clear that SiW9Co3 and SiW9Cu3 were specific for the colorimetric detection of DA and RAC, respectively.

Conclusions
Novel detection systems for DA and RAC have been established with POMs with highly visible color change for the first time. SiW 9 Co 3 exhibited a color change from colorless to orange with H 2 O 2 for the detection of DA. SiW 9 Cu 3 produced a color change from colorless to slight red for the detection of RAC with H 2 O 2 . SiW 9 Co 3 and SiW 9 Cu 3 both led to a more convenient method for observation using the naked eye only. Therefore, these catalysts have great application potential for the detection of DA and RAC in aqueous solutions, and the corresponding detection system is simple, rapid, selective, and sensitive to DA and RAC.