Facile Synthesis of Porous Hexapod Ag@AgCl Dual Catalysts for In Situ SERS Monitoring of 4-Nitrothiophenol Reduction

: Controllable morphological metal catalytic materials have always been a focus in research. In the previous work, hexapod AgCl was successfully synthesized. In this paper, hexapod Ag@AgCl microstructures with diverse Ag contents are prepared through NaBH 4 reduction. They are characterized by scanning electron microscopy (SEM) and the element distribution is proved by an energy dispersive X-ray spectrometer (EDS). They are porous dendritic microstructures with a large specific surface area and a rough surface, which display high catalytic performance and surface-enhanced Raman spectroscopy (SERS) activity. Furthermore, the hexapod Ag@AgCl microstructure is devoted as a dual catalyst to monitor the reduction of 4-nitrothiophenol (4-NTP) to 4-aminothiophenol (4-ATP) in situ using SERS. Ultraviolet–visible (UV–Vis) spectroscopy was employed to evaluate the catalytic performance of the hexapod Ag@AgCl microstructures. The consequences show that the velocity of reaction became faster with a corresponding increase in silver content and the reaction mechanism was cleared. In addition to preparing a good catalyst, this work also promotes the combination of micro-nano materials and spectroscopy technology. The solution had an obvious absorption peak at 520 nm. When the Ag cube was mixed with 4-NTP, the absorption peak position changed between 395 nm to 445 nm. Then NaBH 4 was added to start the reaction and the absorption peak dropped by about a quarter in 20 min. This control experiment showed that the catalytic performance of Ag silver nanoparticles was weaker than that of sample 1, which had the worst catalytic performance in the samples.


Introduction
In recent years, micro-nano metal materials with controllable morphologies have been a hot topic discussed by researchers. Due to their special physical and chemical properties, they have potential applications in catalysis [1,2], sensors [3,4], photothermal therapy [5][6][7] and surface-enhanced Raman spectroscopy (SERS) [8,9]. Theoretical research has shown that the physical and chemical properties of metallic materials depend to a great extent on their shape and size [10]. Thus, a large number of well-defined metal structures have been prepared, such as concaves [11,12], cubes [13][14][15], stars [16,17] and rods [18]. Among various types of the structures, Ag-based materials attracted wide attention, since they are outstanding in the reduction of 4-NTP to 4-ATP. 4-NTP is a common water pollutant, which is usually difficult to degrade under natural conditions. On the contrary, 4-ATP, which is the corresponding reduction product, has shown great value in various fields, including as a desiccant and in photographic development [19,20]. Meanwhile, in the chemical industry, 4-ATP is a raw material for the production of drugs [21]. Therefore, this reaction has attracted a lot of research as a standard reaction. Sebastian et al. synthesized Au/Pt/Au core/shell nano-raspberries to monitor Pt-catalyzed In order to expand and strengthen its function, it was reduced to an Ag@AgCl microstructure by NaBH4 solution. By controlling the amount of NaBH4 added, different reduction levels of Ag@AgCl microstructures were obtained, which have different Ag contents. The Ag@AgCl microstructures maintained their original shape and had a rough surface, which could be a good SERS substrate. Moreover, it was easy to find the Ag@AgCl microstructures under the Raman microscope. Thus, the process of finding SERS hotspots was simplified. For the convenience of the subsequent description, we will label the obtained Ag@AgCl samples according to the different molar ratios (R) of Ag and AgCl (learned from energy dispersive X-ray spectrometer (EDS), will be discussed later), as sample 1 (pure AgCl, R = 0:1), sample 2 (R = 1:2), sample 3 (R = 4:1) and sample 4 (pure Ag, R = 1:0). After reduction, the morphology of the samples remained unchanged and the SEM images of the four samples are shown in Figure S1 (see details in Supplementary Material (SM)). Figure 2A-D shows SEM images of the surface details of the above four samples. In the process, irregular Ag particles began to form on the surface and the entire structure gradually become porous. Due to the porous structure, the reactants were more likely to penetrate into the catalyst, resulting in more effective contact. In addition, the porous structure had a large specific surface area, which provided good active sites and SERS hotspots. In order to expand and strengthen its function, it was reduced to an Ag@AgCl microstructure by NaBH 4 solution. By controlling the amount of NaBH 4 added, different reduction levels of Ag@AgCl microstructures were obtained, which have different Ag contents. The Ag@AgCl microstructures maintained their original shape and had a rough surface, which could be a good SERS substrate. Moreover, it was easy to find the Ag@AgCl microstructures under the Raman microscope. Thus, the process of finding SERS hotspots was simplified. For the convenience of the subsequent description, we will label the obtained Ag@AgCl samples according to the different molar ratios (R) of Ag and AgCl (learned from energy dispersive X-ray spectrometer (EDS), will be discussed later), as sample 1 (pure AgCl, R = 0:1), sample 2 (R = 1:2), sample 3 (R = 4:1) and sample 4 (pure Ag, R = 1:0). After reduction, the morphology of the samples remained unchanged and the SEM images of the four samples are shown in Figure S1 (see details in Supplementary Material (SM)). Figure 2A-D shows SEM images of the surface details of the above four samples. In the process, irregular Ag particles began to form on the surface and the entire structure gradually become porous. Due to the porous structure, the reactants were more likely to penetrate into the catalyst, resulting in more effective contact. In addition, the porous structure had a large specific surface area, which provided good active sites and SERS hotspots. The EDS spectra of sample 4 is shown in Figure 3 and the others are shown in Figures S3-S5. Figure 3B,D confirms the distribution of Ag and Cl in sample 4. The EDS elemental mapping of Ag@AgCl, indicating the Ag and Cl elemental distribution, is shown in Figure S2. In Figure 3B, the spectrum of Cl shows peaks at 0.3 and 2.6 keV, while the peak at 3.2 keV was attributed to Ag. The content of Ag reached 98.74% (wt%), and only a small amount of Cl (1.26%) remained, suggesting  The EDS spectra of sample 4 is shown in Figure 3 and the others are shown in Figures S3-S5. Figure 3B,D confirms the distribution of Ag and Cl in sample 4. The EDS elemental mapping of Ag@AgCl, indicating the Ag and Cl elemental distribution, is shown in Figure S2. In Figure 3B, the spectrum of Cl shows peaks at 0.3 and 2.6 keV, while the peak at 3.2 keV was attributed to Ag. The content of Ag reached 98.74% (wt%), and only a small amount of Cl (1.26%) remained, suggesting that almost all AgCl was reduced. According to this, the molar ratio of Ag and AgCl in Ag@AgCl was calculated and the results of other samples can also be obtained by a similar method. It is worth noting that in the EDS mapping of pure AgCl in Figure S3, the Ag content reached 51.6% (atom.%), which is slightly higher than the theoretical value of 50%. This is probably because the electron beam reduced a small part of AgCl when the SEM image was taken. The EDS spectra of sample 4 is shown in Figure 3 and the others are shown in Figures S3-S5. Figure 3B,D confirms the distribution of Ag and Cl in sample 4. The EDS elemental mapping of Ag@AgCl, indicating the Ag and Cl elemental distribution, is shown in Figure S2. In Figure 3B, the spectrum of Cl shows peaks at 0.3 and 2.6 keV, while the peak at 3.2 keV was attributed to Ag. The content of Ag reached 98.74% (wt%), and only a small amount of Cl (1.26%) remained, suggesting that almost all AgCl was reduced. According to this, the molar ratio of Ag and AgCl in Ag@AgCl was calculated and the results of other samples can also be obtained by a similar method. It is worth noting that in the EDS mapping of pure AgCl in Figure S3, the Ag content reached 51.6% (atom.%), which is slightly higher than the theoretical value of 50%. This is probably because the electron beam reduced a small part of AgCl when the SEM image was taken.

In Situ Monitoring of Catalytic Reactions by UV-Vis and Catalytic Performance
The catalytic activity of Ag@AgCl microstructures was investigated through the reduction of 4-NTP at room temperature, monitored by UV-Vis spectra. The UV-Vis spectra of the mixed solution of 4-NTP and NaBH4 at different times are shown in Figure 4. It is found that the absorption peak was shown at about 410 nm and the spectra hardly changed as 1 h passed in the absence of catalyst. Therefore, we speculate that the reduction reaction did not occur. After adding four Ag@AgCl samples as catalysts, we attained their UV-Vis spectra, as shown in Figure 5A,C,E,G. From samples 1 to 4, the position of absorption peak did not change and the rate of the reduction reaction gradually became faster. In the presence of sample 1, 4-NTP was only reduced by about one third in 20 min and there was no obvious peak at 270 nm simultaneously. This means that only a small amount of 4-ATP was produced. When using samples 2 and 3 as catalysts, it can be found from Figure 5C,E that the peak at 410 nm decreased faster and the peak at 270 nm increased significantly, which illustrates the rapid conversion of 4-NTP to 4-ATP and the improvement of the catalytic effect. As sample 4 was used, 4-NTP was almost completely reduced in 10 min. Sample 4 showed the best catalytic effect.
there was no obvious peak at 270 nm simultaneously. This means that only a small amount of 4-ATP was produced. When using samples 2 and 3 as catalysts, it can be found from Figure 5C,E that the peak at 410 nm decreased faster and the peak at 270 nm increased significantly, which illustrates the rapid conversion of 4-NTP to 4-ATP and the improvement of the catalytic effect. As sample 4 was used, 4-NTP was almost completely reduced in 10 min. Sample 4 showed the best catalytic effect. To better illustrate the catalytic effect of the Ag@AgCl microstructure, we used a 75 nm Ag nanocube as a comparison. The Ag cube was synthesized by the reported method [33] and its relevant characterization is shown in Figure S6, which implies the uniformity and stability of the Ag cube. We firstly measured the UV-Vis absorption spectra of the Ag cubes, as shown in Figure S7A. The solution had an obvious absorption peak at 520 nm. When the Ag cube was mixed with 4-NTP, the absorption peak position changed between 395 nm to 445 nm. Then NaBH4 was added to start the reaction and the absorption peak dropped by about a quarter in 20 min. This control experiment showed that the catalytic performance of Ag silver nanoparticles was weaker than that of sample 1, which had the worst catalytic performance in the samples.
In order to clearly describe the change in the reaction rate, the reaction kinetics were applied. Pseudo first-order kinetics fit the catalytic reduction rate. As shown in Figure 5B,D, F and H, a linear correlation of ln (A0/A) with time was ascertained and the corresponding line equation was fitted, in To better illustrate the catalytic effect of the Ag@AgCl microstructure, we used a 75 nm Ag nanocube as a comparison. The Ag cube was synthesized by the reported method [33] and its relevant characterization is shown in Figure S6, which implies the uniformity and stability of the Ag cube. We firstly measured the UV-Vis absorption spectra of the Ag cubes, as shown in Figure S7A. The solution had an obvious absorption peak at 520 nm. When the Ag cube was mixed with 4-NTP, the absorption peak position changed between 395 nm to 445 nm. Then NaBH 4 was added to start the reaction and the absorption peak dropped by about a quarter in 20 min. This control experiment showed that the catalytic performance of Ag silver nanoparticles was weaker than that of sample 1, which had the worst catalytic performance in the samples.
In order to clearly describe the change in the reaction rate, the reaction kinetics were applied. Pseudo first-order kinetics fit the catalytic reduction rate. As shown in Figure 5B,D,F,H, a linear correlation of ln (A 0 /A) with time was ascertained and the corresponding line equation was fitted, in which A is the absorbance at a certain moment and A 0 is the initial absorbance of 4-NTP. Some unreasonable data points were deleted during the fitting process. The relevant parameters of the fitted straight line are shown in Figure S8 and the deviation values used by the error bars when fitting straight lines are shown in Table S1. We speculate that the experimental error may be mainly due to the system error of the instrument, the time difference during multiple measurements and the error of the baseline. The Pearson's coefficients of the four fitted straight lines are 0.98902, 0.99731, 0.98586, 0.99861, which show good linear relationships and correlations between the data points. This shows that the fitting process and results are trustworthy and can be used in the next calculation of reaction rate constants. According to the theory of first order reaction, the slope of the ln (A 0 /A) vs. time is the reaction rate constant. The values of the reaction rate constant are calculated to be 0.02035 ± 0, 0.03103 ± 0.001, 0.08781 ± 0.006 and 0.18251 ± 0.004 min −1 . This shows that the reaction rate is accelerated and the catalytic performance of samples 1 to 4 is gradually enhanced. The sample 4 (pure Ag) catalyst demonstrated the fastest reduction rate among all the catalysts. However, this does not mean that the other three catalysts are of no value, but that we can choose the appropriate catalyst according to our needs to achieve the reaction rate we desire. The mechanism of this reaction can be understood with Ag@AgCl as an intermediate for transferring the hydrogen between NaBH4 and 4-NTP. The hydrogen comes from the reaction of NaBH4 and water. Thus, the reaction of the 4-NTP reduction is explained as the Ag porous structure easily absorbing the hydrogen generated in the solution and releasing the hydrogen in the reduction reaction [34]. Therefore, as the reduction degree of AgCl increases, the porosity increases and a larger amount of hydrogen can be adsorbed, so the reaction rate increases. As for the catalytic function of pure AgCl, we speculate that under the ultraviolet or visible light, Ag@AgCl will be regarded as an effective medium for transferring electrons, transporting the electrons provided by The mechanism of this reaction can be understood with Ag@AgCl as an intermediate for transferring the hydrogen between NaBH 4 and 4-NTP. The hydrogen comes from the reaction of NaBH 4 and water. Thus, the reaction of the 4-NTP reduction is explained as the Ag porous structure easily absorbing the hydrogen generated in the solution and releasing the hydrogen in the reduction reaction [34]. Therefore, as the reduction degree of AgCl increases, the porosity increases and a larger amount of hydrogen can be adsorbed, so the reaction rate increases. As for the catalytic function of pure AgCl, we speculate that under the ultraviolet or visible light, Ag@AgCl will be regarded as an effective medium for transferring electrons, transporting the electrons provided by BH − 4 to the nitro group. Therefore, the kinetic barrier will be reduced, thereby promoting the occurrence of reduction reactions [35].

In Situ Monitoring of Catalytic Reactions with SERS
Since the Ag@AgCl microstructure has SERS activity, we also used Raman spectroscopy to monitor the reduction process of 4-NTP in situ. For the purpose of avoiding possible side reactions and for a better display of the entire process, we used a 0.25 mW 633 nm low-power laser. In order to confirm that the low-power laser had no effect on the reaction, we first conducted a control experiment. Fifty microliters of 4-NTP solution and 3 mg sample 4 were mixed for testing. After continuous irradiation with a 633 nm laser with a power of 0.25 mW for 20 min, the spectra of 4-NTP did not change, as shown in Figure S9A, which proved that the low-power laser had no effect on the reaction pathway of 4-NTP reduction. Figure 6 shows the SERS spectra of 4-NTP at different time points after putting in the reducing agent NaBH 4 and Ag@AgCl. The spectra show that the reduction process of 4-NTP adsorption on Ag@AgCl is mainly divided into two steps. Under the action of NaBH 4 , 4-NTP is first reduced to trans-p, p'-dimercaptoazobenzene (DMAB), and then trans-DMAB is converted to 4-ATP. According to previous research, the individual peaks of 4-NTP at 1572, 1336 and 1108 cm −1 matched well with the C-C stretching modes (νCC), NO 2 symmetric stretching (νNO 2 ), and C-N (νCN) bending, respectively [36]. When t = 0 min, characteristic vibrations at 1572, 1336 and 1108 cm −1 could be clearly identified. At t = 2 min, three novel peaks arose at 1429, 1388 and 1142 cm −1 , which were attributed to νNN + βCH, νNN + νCN and βCH + νCN of trans-DMAB, respectively [37]. This meant the progress of the reaction and the generation of trans-DMAB. When proceeding to t = 4 min, peaks of 4-NTP at 1336 and 1108 cm −1 vanished, which demonstrated the decrease in 4-NTP. As the reaction continued, the three peaks of trans-DMAB continuously increased in intensity and a new peak at 1595 cm −1 appeared, which marked the start of the conversion of DMAB to 4-ATP. At t = 14 min, the peaks of trans-DMAB and 4-NTP all died away, which illustrated the completion of the entire reduction reaction.
Catalysts 2020, 10, x FOR PEER REVIEW 8 of 13 group. Therefore, the kinetic barrier will be reduced, thereby promoting the occurrence of reduction reactions [35].

In Situ Monitoring of Catalytic Reactions with SERS
Since the Ag@AgCl microstructure has SERS activity, we also used Raman spectroscopy to monitor the reduction process of 4-NTP in situ. For the purpose of avoiding possible side reactions and for a better display of the entire process, we used a 0.25 mW 633 nm low-power laser. In order to confirm that the low-power laser had no effect on the reaction, we first conducted a control experiment. Fifty microliters of 4-NTP solution and 3 mg sample 4 were mixed for testing. After continuous irradiation with a 633 nm laser with a power of 0.25 mW for 20 min, the spectra of 4-NTP did not change, as shown in Figure S9 A, which proved that the low-power laser had no effect on the reaction pathway of 4-NTP reduction. Figure 6 shows the SERS spectra of 4-NTP at different time points after putting in the reducing agent NaBH4 and Ag@AgCl. The spectra show that the reduction process of 4-NTP adsorption on Ag@AgCl is mainly divided into two steps. Under the action of NaBH4, 4-NTP is first reduced to trans-p, p'-dimercaptoazobenzene (DMAB), and then trans-DMAB is converted to 4-ATP. According to previous research, the individual peaks of 4-NTP at 1572, 1336 and 1108 cm −1 matched well with the C-C stretching modes (νCC), NO2 symmetric stretching (νNO2), and C-N (νCN) bending, respectively [36]. When t = 0 min, characteristic vibrations at 1572, 1336 and 1108 cm −1 could be clearly identified. At t = 2 min, three novel peaks arose at 1429, 1388 and 1142 cm −1 , which were attributed to νNN + βCH, νNN + νCN and βCH + νCN of trans-DMAB, respectively [37]. This meant the progress of the reaction and the generation of trans-DMAB. When proceeding to t = 4 min, peaks of 4-NTP at 1336 and 1108 cm −1 vanished, which demonstrated the decrease in 4-NTP. As the reaction continued, the three peaks of trans-DMAB continuously increased in intensity and a new peak at 1595 cm −1 appeared, which marked the start of the conversion of DMAB to 4-ATP. At t = 14 min, the peaks of trans-DMAB and 4-NTP all died away, which illustrated the completion of the entire reduction reaction. It is worth noting that, compared with Raman in situ monitoring, there was no characteristic absorption peak of trans-DMAB during the UV-Vis monitoring. We speculate that the reaction occurred on the surface of the catalyst and the intermediate was strongly adsorbed on the surface. It is worth noting that, compared with Raman in situ monitoring, there was no characteristic absorption peak of trans-DMAB during the UV-Vis monitoring. We speculate that the reaction occurred on the surface of the catalyst and the intermediate was strongly adsorbed on the surface. This matches with Matthias Ballauff's research [38]. We also took out the catalyst when the reaction proceeded to 5 min for testing. The strong trans-DMAB spectrum was found on its surface, as shown in Figure S9B. The laser is usually irradiated on the surface of the catalyst, so 4-DMAB can be detected by Raman spectroscopy. Meanwhile, UV-Vis spectroscopy tests the species in solution, so the intermediate is difficult to detect. Therefore, the reaction process is summarized in Figure 7. All in all, by employing the Ag@AgCl dual functional material, the monitoring of the reduction of 4-NTP to 4-ATP was achieved.
All glassware were washed in a bath of freshly prepared aqua regia (HCl:HNO 3 = 3:1) and rinsed thoroughly twice in deionized water and ethanol, respectively.

Characterization and Instruments
The scanning electron microscopy (SEM) images were obtained on a Hitachi SU-70 electron microscope (3.0 kV, Hitachi, Tokyo, Japan) to characterize the morphology and size of the hexapod Ag@AgCl microstructures. SEM-EDS mapping illustrated the element distribution in the samples and provided their corresponding absorption spectra.
A Cary 60 Scan UV-Vis spectrophotometer (Agilent Technologies Inc., State of California, United States) was applied to collect absorption spectra. It has excellent capabilities of fast data acquisition. The scan rate was 24,000 nm/min, which can scan the entire wavelength range (190-1100 nm) in less than 3 s. The spectral resolution was less than 1.5 nm and wavelength reproducibility was ±0.1 nm. It used a stable xenon flash lamp and the working power was about 13 W.
A confocal Raman spectrometer (Horiba HR Evolution 800, Tokyo, Japan) was used to record Raman spectra. The spectral resolution was less than 0.35 cm −1 and spectral repeatability was less than 0.03 cm −1 . So, it had high accuracy. Meanwhile, it was equipped with three wavelength lasers of 532, 633 and 785 nm and the incident laser power was adjustable (maximum 10 mW). This provided great convenience and flexibility for the measurement of samples. The spectral analysis software package LabSpec 6 was used to analyze the obtained spectrum, providing powerful data processing functions.
The above instruments and equipment ensured that we quickly obtained reliable results.

Preparation of Hexapod Ag@AgCl Microstructures
We adopted reported methods to synthesize hexapod Ag@AgCl microstructures [29]. For a typical synthesis of AgCl crystals, 300 µL of AgNO 3 solution (0.1 M) and 0.8 mL of DDA were added to 20 mL of EG and mixed well at room temperature. The solution was placed in a 190 • C oil bath for 30 min with agitation. The flask was stewed until it cooled down to room temperature. The resulting product was first centrifuged at 10,000 r/min for 15 min to remove the supernatant. The precipitate (AgCl) was washed twice with water and ethanol, respectively.
One hundred milligrams of dried hexapod AgCl crystals were dispersed in 8 mL of deionized water. Then 0.02 M NaBH 4 solution was added to the dispersion and it was reacted at 300 r/min for 30 min to reduce AgCl to prepare Ag@AgCl microstructures. Changing the volume of NaBH 4 adjusted the reduction degree of AgCl to obtain Ag@AgCl with different ratios of Ag to AgCl.

In Situ Detection of Catalytic Reactions
The conversion of 4-NTP to 4-ATP was used to evaluate the catalytic performance of the prepared Ag@AgCl microstructures. All experiments were conducted at room temperature and normal pressure.
In situ monitoring of the reaction by UV-Vis. The UV-Vis spectrometer used a dual light path mode when collecting spectra. The scanning range was 200 to 800 nm and the scanning speed was 24,000 nm/min. Firstly, the UV-Vis absorption spectrum of 4-NTP mixed with the NaBH 4 solution was obtained. Then, 10 mg Ag@AgCl powder were dispersed in 10 mL water uniformly. One hundred microliters of the above solution, 1.9 mL of 1 mM 4-NTP and 1 mL of 0.1 M NaBH 4 were added into the measuring cell. The absorption spectrum was recorded every minute and the measurement was repeated three times. Throughout the experiment, the measuring cell was always placed in the dark sample chamber of the spectrometer. The above experiment was conducted with Ag@AgCl with different Ag contents (samples 1-4).
In situ monitoring of the reaction by SERS. The Raman spectrometer was calibrated by a silicon wafer at 520 cm −1 and the laser beam was focused through a 50 × objective. Ten microliters of the Ag@AgCl suspension, 30 µL 0.1 mM 4-NTP and 10 µL of 0.1 M NaBH 4 were dropped on a glass slide with a small liquid pool. Raman spectra were collected every minute for the above reaction solution. A 633-nm excitation laser was used to irradiate the mixture and the laser power was kept at about 0.25 mW. A total accumulation time of 10 s was employed and the grating of 600 gr/mm was used. The above reaction solution was subjected to a surface-enhanced Raman test at different times.

Conclusions
In summary, Ag@AgCl microstructures were successfully prepared by NaBH 4 reduction. By adjusting the volume of the NaBH 4 solution, Ag@AgCl microstructures with different molar ratios of Ag and AgCl were obtained. The material had the advantages of a 3D structure and a large specific surface area, and was a bifunction catalyst with SERS activity. We applied it to monitoring the conversion of 4-NTP to 4-ATP in situ by SERS. UV-Vis spectroscopy was used to evaluate its catalytic performance. This work not only developed a method for synthesizing 3D Ag structures, but also offers new ideas for the application of micro-nano materials in spectroscopy.  Table S1: The deviation values used by the error bars when fitting straight lines for lines in Figure 5B

Conflicts of Interest:
The authors declare no conflict of interest.