Ferrocene-Doped Polystyrene Nanoenzyme and DNAzyme Cocatalytic SERS Quantitative Assay of Ultratrace Pb2+

A new, stable and high-catalytic activity ferrocene-doped polystyrene nanosphere (PNFer) sol was prepared by the hydrogel procedure and characterized by electron microscopy and molecular spectroscopy. Results show that the nanosol exhibits excellent catalysis of the new indicator nanoreaction between AgNO3 and sodium formate to generate nanosilver with strong surface-enhanced Raman scattering (SERS), resonance Rayleigh scattering (RRS) and surface plasmon resonance absorption (Abs) trimode molecular spectral signals. This new nanocatalytic amplification trimode indicator reaction was coupled with the G-quadruplex DNAzyme catalytic amplification of Pb2+ aptamer to fabricate a new SERS quantitative/RRS/Abs assay platform for the determination of ultratrace amounts of Pb2+. The Pb2+ content in water samples was analyzed with satisfactory results.


Introduction
Lead (Pb) is a toxic heavy metal; it has caused great concern due to its damage to the kidneys and nervous system. The measurement of trace Pb 2+ is becoming more and more common and urgent in the food safety, environmental, clinical and toxicology fields. At present, methods for the determination of Pb include dithizone colorimetry, flame atomic absorption spectrometry, graphite furnace atomic absorption spectrometry, hydride generation atomic fluorescence spectrometry, catalytic polarography and so on [1,2]. The atomic spectral methods use large-scale instruments with high costs. Classical dithizone colorimetry demonstrates low sensitivity and some toxic reagents are used, which poses a threat to the health of inspectors. Recently, some new methods for Pb have been attractive to analysts. One of the most important methods is the aptamer (Apt) assay, which is simple with good selectivity. Based on the interaction between single-stranded DNA (ssDNA) and nanogold (AuNPs), a turn-on plasmonic absorption assay for the detection of 8.0 nM Pb 2+ was fabricated [3], and the sensitivity was not high. Acoupled DNAzymeactivated hybridization chain reaction with bio-barcodes and a surface-enhanced Raman scattering (SERS) were developed for assay of Pb 2+ with a dynamic range of 1.0 × 10 -13 M to 1.0 × 10 −7 M [4]. However, the SERS signal was not linear to the concentration. In addition, the process was complicated. Thus, it is necessary to develop a simple and sensitive Apt method for Pb 2+ -based DNAzyme and nanozyme catalytic amplification. To our best knowledge, there are no reports about the preparation of a stable and high-catalytic activity ferrocene (Fer)-doped polystyrene nanosphere (PN Fer ) sol, the PN Fer -AgNO 3 -sodium formate (SF) nanosilver indicator reaction, or the coupled DNAzyme catalytic reaction to co-amplify the SERS/resonance Rayleigh scattering (RRS)/absorption (Abs) trimode signals for determination of ultratrace Pb 2+ .
DNA enzymes (DNase) were synthesized simply by Apts, with the advantages of specificity, low costs and good stability. The method has been used in analytical chemistry,
A Pb 2+ aptamer with sequence of 5'-3' GGT TGG TGT GGT TGG (Sangong Bioengineering Co., Ltd., Shanghai, China); Styrene (CP, Guangxi Long Science Co., Ltd., Nanning, China); Chloroauric acid (AR, Shanghai Mike Lin Biochemical Co., Ltd., Shanghai, After 45 min in a 45 • C water bath, the solution turned dark blue. Then, the solution was freeze-dried to obtain a TMBox solid. Next, 0.0031 g TMBox was dissolved in 5 mL secondary water to obtain 0.62 mg/mL TMBox. In the experiment, it was diluted and used step by step. The reagents used were analytically pure, and the experimental water was boiled twice before use. Preparation of polystyrene nanospheres: Preparation of polystyrene nanospheres (PN) was as follows, 5, 10, 15, and 20 mL styrene, respectively, were added into a 500 mL three-necked flask containing 20 mL water, and passed through nitrogen to exclude air. Following this, 0.1 g SDS was added and heated at 70 • C for 1 h under stirring. Next, 0.1 g potassium persulfate and 0.1 g NaHCO 3 were added, diluted to 100 mL with water, reacted for 10 h to complete the reaction, and let cool to room temperature. The obtained white emulsion was centrifuged at a low speed to remove the unreacted styrene on the upper layer, dialyzed with a 500 Da dialysis membrane for 12 h to remove inorganic salts, and finally the obtained polystyrene nanosphere (PN) solution was vacuum-dried to a constant weight. A white powdery PN was obtained, which was numbered PN [1][2][3][4] according to the quality of the styrene. Finally, 0.10 g PN 1-4 was dissolved in 100 mL water to prepare a 1 g/L solution, and diluted step by step during use.
Preparation of ferrocene-doped polystyrene nanozyme: For PN Fer1-3 preparation, 20, 50, 100 mg Fer, 15 mL styrene, and 0.1 g SDS were added, respectively, into a 500 mL threenecked flask containing 20 mL deionized water, and passed through nitrogen to remove air. It was heated at 70 • C for 1 h under stirring. Next, 0.1 g potassium persulfate and 0.1 g NaHCO 3 were added, diluted to 100 mL with water, reacted for 10 h to complete the reaction, and let cool to room temperature. The obtained white emulsion was centrifuged at a low speed to remove the unreacted styrene on the upper layer, dialyzed with a 500 Da dialysis membrane for 12 h to remove inorganic salts, and finally the obtained ferrocenedoped polystyrene nanosphere (PN Fer ) solution was dried under vacuum to constant weight. A light yellow powdered PN was obtained, which was numbered PN Fer1 , PN Fer2 , or PN Fer3 according to the different mass ratio of ferrocene to styrene. Finally, 0.10 g PN Fer was dissolved in 100 mL water to prepare a 1 g/L solution, and diluted step by step during use.

Procedure for RRS and SERS Detection of Pb 2+
In a 5 mL graduated tube with stopper, 100 µL 100 mg/L polystyrene nanoparticle solution, 150 µL pH 4.4 NaAc-HAc buffer (88.8 mM in NaAc concentration), 120 µL 0.1 µM Apt, 90 µL 0.1 µM heme and a certain concentration of Pb 2+ solution were added, then mixed well. After 15 min, 100 µL 0.01 M AgNO 3 and 80 µL 1.0 M formate were added, diluted to 1.5 mL and shaken well, placed in a water bath at 85 • C for 10 min, and cooled with ice water to terminate the reaction. The solution was placed in a quartz four-way cuvette, at 350 V, excited slit = emission slit = 5 nm, and a fluorescence spectrophotometer was used to record the RRS signal at 370 nm (I 370nm ). With no Pb 2+ as a blank, the RRS peak at 370 nm (I 370nm ) 0 was measured and ∆I = I 370nm − (I 370nm ) 0 was calculated. Then 50 µL of 10 µM VBB was added to the reaction solution, the Raman signal recorded at 1618 cm −1 under the power of 2.0 mW and a slit of 25.0 nm, and the value of ∆I 1618 cm −1 = I 1618 cm −1 − (I 1618 cm −1 ) 0 obtained.

Analysis Principle
At 70 • C, potassium persulfate was used as the initiator and sodium lauryl sulfate sodium as the surfactant to initiate the polymerization reaction of styrene to form polystyrene nanospheres (PN). When Fer was added, the PN Fer was obtained ( Figure 1). Under room temperature, AgNO 3 -SF is unreactive. When nanospheres were added as a catalyst, the reaction can be completed within a few minutes. When Apt was added, because Apt has a better affinity with the nanospheres, Apt will coat the surface of the nanospheres to reduce its catalytic activity, and will therefore produce low SERS/RRS/Abs signal. When Pb 2+ was added, Apt will specifically bind to Pb 2+ , and at the same time, it will combine with the existing heme (HM) to form DNase. Both DNase and the released nanospheres have a catalytic effect on the AgNO 3 -SF indicator reaction to enhance the trimode signals of SERS/RRS/Abs. Thus, a highly sensitive, low-cost trimode assay platform was proposed for the detection of Pb 2+ .

Characterization of PNFer and Nanocatalytic Analysis System
Figure 2a-d are electron microscope images of PN3, PNFer3, PNFer3-Apt-Pb 2+ -AgNO3-SF and PNFer3-Apt-AgNO3-SF systems. It can be seen that the PN3 average particle size was larger than that of PNFer3, indicating that after PN doping with the electron-rich molecule Fer, the particle size became small and the particle size distribution was uniform. This

Characterization of PN Fer and Nanocatalytic Analysis System
Figure 2a-d are electron microscope images of PN 3 , PN Fer3 , PN Fer3 -Apt-Pb 2+ -AgNO 3 -SF and PN Fer3 -Apt-AgNO 3 -SF systems. It can be seen that the PN 3 average particle size was larger than that of PN Fer3 , indicating that after PN doping with the electron-rich molecule Fer, the particle size became small and the particle size distribution was uniform. This may be related to the increase in electron density and intermolecular forces. The energy spectrum ( Figure 2b) shows that there are three peaks at 0.46, 6.4 and 7.1 keV, ascribed to the Fe element of Fer. There are less spherical AgNPs in the blank system due to the inhibition of Apt, and the average size is 42 nm (Figure 2c). Upon addition of Pb 2+ , the Apt reaction turned on the catalytic AgNP nanoreaction to form a large number of AgNPs with an average size of 25 nm (Figure 2d). Both energy spectra demonstrated that there was a peak at 3.0 keV ascribed to the Ag element (Figure 2c,d).
Nanomaterials 2022, 12, x FOR PEER REVIEW 6 of 15 caused by CH at 2859 and 2926 cm −1 are 3023 cm −1 . The nearby characteristic peaks were caused by the =CH stretching vibration. At the same time, the medium-strong peak at 1598 cm −1 indicates that =CH exists in aromatic substances. The two characteristic peaks at 1450 and 1495 cm −1 directly show that the material was synthesized from styrene, and the stretching vibration peak of C=C does not appear at 1640 cm −1 , indicating that styrene has polymerized to form polystyrene. The characteristic peak at 3075 cm −1 was the stretching vibration peak caused by CH in Fer, and the characteristic peak at 1101 cm −1 was caused by the σ bond formed by the metal ion and the phenyl compound, indicating that the doped Fer has a certain effect. Figure 4b shows that in the range of 2θ = 15-25°, there are a wide range of structural belts composed of disordered carbon elements in the two materials. The FeC structure formed by Fer and polystyrene appeared in the range of 2θ = 31° and 44° in PNFer3, indicating that the doping of Fer was successful.

Molecular spectra and X-ray diffraction (XRD) characterization of PN 3 and PN Fer3
: RRS is a simple and sensitive technique for studying nanoparticles, and was chosen for use. Figure 3a,b show that the 20 mg/L PN 3 and 20 mg/L PN Fer3 exhibit the biggest RRS peak at 320 nm and 360 nm respectively. As the PN 3 and PN Fer3 concentration increases, the RRS signal increases linearly, due to the particle numbers increased. Figure 3c shows that there are no UV absorption peaks for both materials. Figure 3d shows the emission spectra of nanomaterials at an excited wavelength of 250 nm, none of the both shows any fluorescence peak, the two peaks are ascribed to Rayleigh scattering. The SERS spectra of PN 3 and PN Fer3 materials were studied, and no SERS signal appeared. The nanomaterial was prepared according to the experimental method, was vacuum-freeze-dried to obtain the solid samples, and was recorded with a Fourier transform infrared spectrometer. Figure 4a shows the infrared spectra of PN 3 and PN Fer3 . Among them, 760 cm −1 is the vibration peak caused by CH that is not in the plane of styrene, and the stretching vibration peaks caused by CH at 2859 and 2926 cm −1 are 3023 cm −1 . The nearby characteristic peaks were caused by the =CH stretching vibration. At the same time, the medium-strong peak at 1598 cm −1 indicates that =CH exists in aromatic substances. The two characteristic peaks at 1450 and 1495 cm −1 directly show that the material was synthesized from styrene, and the stretching vibration peak of C=C does not appear at 1640 cm −1 , indicating that styrene has polymerized to form polystyrene. The characteristic peak at 3075 cm −1 was the stretching vibration peak caused by CH in Fer, and the characteristic peak at 1101 cm −1 was caused by the σ bond formed by the metal ion and the phenyl compound, indicating that the doped Fer has a certain effect. Figure 4b shows that in the range of 2θ = 15-25 • , there are a wide range of structural belts composed of disordered carbon elements in the two materials. The FeC structure formed by Fer and polystyrene appeared in the range of 2θ = 31 • and 44 • in PN Fer3 , indicating that the doping of Fer was successful.

SERS, RRS and Abs Spectra of Nanocatalysis System
The effects of the four probes (VBB, VB4R, TMB OX , and Rh6G) on the system were studied (Figure S1a-d). With an increased Pb 2+ concentration, the SERS peaks increased due to the formation of more SERS active AgNPs. It can be seen that the system has higher signal intensity, less interference of spurious peaks and better sensitivity upon addition of VBB, so the VBB molecular probe was selected for use. The Pb 2+ -Apt-heme-PN 3 -AgNO 3formate-VBB system shows strong peak changes at the Raman shifts of 1167, 1202, 1398, and 1614 cm −1 . The SERS effect at 1617 cm −1 was ascribing to the vibration of C=C in the benzene ring frame. Figure S1e shows that PN 3 has a good catalytic performance for the AgNO 3 -SF reaction, and the SERS signal was positively correlated with the PN 3 concentration. Figure S1f shows that with the increase in Apt, its inhibitory effect in the Apt-PN 3 -AgNO 3 -formate system gradually decreases due to the formation of fewer AgNPs. Figure S1g shows that with the increase in heme content, the catalytic capacity of the DNase produced increases, but the catalytic performance was limited. Figure 5a shows that as regards the heme-Apt-Pb 2+ -PN 3 -AgNO 3 -formate system, under the action of two enzymes, the reaction system has a good detection ability for Pb 2+ . In the comparison of the catalytic performance of the heme-Apt-Pb 2+ -AgNO 3 -formate detection system (Figure 5b,c), the PN Fer3 signal was the strongest and was chosen for the assay.
Results ( Figure S2a) show that after adding PN 3 , the RRS signal of the system was much higher than the blank without PN 3 , which proves that PN 3 has a catalytic effect on the AgNO 3formate system. The RRS signal has a positive correlation with the PN 3 concentration. After selecting the appropriate PN 3 concentration and adding Apt ( Figure S2b), because Apt coats on the surface of PN 3 , its catalytic performance was reduced. Therefore, as Apt increases, the lower the RRS signal produced due to the formation of fewer nanosilvers. When Pb 2+ was added, the Pb 2+ -Apt complex detaches from the surface of PN 3 and combines with heme to form a DNase. With the increase in heme, DNase increases, the catalytic ability becomes stronger, and the RRS signal increases linearly ( Figure S2c). Figure 6a shows that when Pb 2+ increases, the Pb 2+ -Apt complex detaches from the PN 3 surface, releases more Nanomaterials 2022, 12, 1243 7 of 13 PN 3 , and the catalysis is enhanced. The more DNase formed, the stronger the catalytic effect on the system, and the RRS signal increases linearly with the concentration of Pb 2+ . Figure 6b,c are the RRS spectra of PN 3 /PN Fer3 and DNase for the detection of Pb 2+ . In contrast, the PN Fer3 was the most sensitive, and was chosen for use.      AgNO3-formate system gradually decreases due to the formation of fewer AgNPs. Figure  S1g shows that with the increase in heme content, the catalytic capacity of the DNase produced increases, but the catalytic performance was limited. Figure 5a shows that as regards the heme-Apt-Pb 2+ -PN3-AgNO3-formate system, under the action of two enzymes, the reaction system has a good detection ability for Pb 2+ . In the comparison of the catalytic performance of the heme-Apt-Pb 2+ -AgNO3-formate detection system (Figure 5b,c), the PNFer3 signal was the strongest and was chosen for the assay.  Results ( Figure S2a) show that after adding PN3, the RRS signal of the system was much higher than the blank without PN3, which proves that PN3 has a catalytic effect on the AgNO3-formate system. The RRS signal has a positive correlation with the PN3 concentration. After selecting the appropriate PN3 concentration and adding Apt ( Figure  S2b), because Apt coats on the surface of PN3, its catalytic performance was reduced. Therefore, as Apt increases, the lower the RRS signal produced due to the formation of fewer nanosilvers. When Pb 2+ was added, the Pb 2+ -Apt complex detaches from the surface of PN3 and combines with heme to form a DNase. With the increase in heme, DNase increases, the catalytic ability becomes stronger, and the RRS signal increases linearly (Figure S2c). Figure 6a shows that when Pb 2+ increases, the Pb 2+ -Apt complex detaches from the PN3 surface, releases more PN3, and the catalysis is enhanced. The more DNase formed, the stronger the catalytic effect on the system, and the RRS signal increases linearly with the concentration of Pb 2+ . Figure 6b,c are the RRS spectra of PN3/PNFer3 and DNase for the detection of Pb 2+ . In contrast, the PNFer3 was the most sensitive, and was chosen for use.  Figure S3a shows that the absorption peak at 430 nm increased with the PN3 catalyst concentration increasing, and the yellow gradually deepened. The corresponding UV absorption signal also increases linearly with the increase in PN3 concentration due to the formation of more nanosilvers, which also shows that PN3 material has excellent catalytic ability. The peak was ascribed to the surface plasmon resonance absorption peak of na-  Figure S3a shows that the absorption peak at 430 nm increased with the PN 3 catalyst concentration increasing, and the yellow gradually deepened. The corresponding UV absorption signal also increases linearly with the increase in PN 3 concentration due to the formation of more nanosilvers, which also shows that PN 3 material has excellent catalytic ability. The peak was ascribed to the surface plasmon resonance absorption peak of nanosilvers. Figure S3b shows that the acetic acid-sodium acetate buffer with ph = 4.4 is the best condition. Figure S3c shows that as the Apt increases, the more PN 3 is coated by Apt and the stronger the inhibition ability for the AgNO 3 -formate reaction, and it shows that the UV absorption signal decreases linearly with the concentration of Apt. Figure S3d shows that as the concentration of HM increases, the DNase produced gradually increases and the catalytic ability increases. As the concentration of Pb 2+ increases, the released PN 3 increases, the DNase generated gradually increases, and the catalytic ability increases due to the formation of more nanosilvers (Figure 7a). Figure 7b,c show that PN Fer3 has the strongest Abs signal in the determination of Pb 2+ and has a higher correlation. Comparing the linear equations, it is found that the PN Fer3 has the best catalytic performance, and the result is the same as the RRS result. and the catalytic ability increases. As the concentration of Pb 2+ increases, the released PN3 increases, the DNase generated gradually increases, and the catalytic ability increases due to the formation of more nanosilvers (Figure 7a). Figure 7b,c show that PNFer3 has the strongest Abs signal in the determination of Pb 2+ and has a higher correlation. Comparing the linear equations, it is found that the PNFer3 has the best catalytic performance, and the result is the same as the RRS result.

Catalytic Reaction Mechanism of Nanoenzymes and DNase
Formate and silver nitrate can generate porous silver [28], but this reaction requires a microwave reaction vessel to keep pressure and temperature. Under the experimental conditions, it is difficult for the reaction between AgNO3 and SF to take place. After adding the catalyst, a fast reaction can be realized, AgNO3 is reduced to AgNPs, and SF is oxidized to CO2. Table S1 shows a comparison of the linear equations for catalysis of several nanomaterials. It can be seen that PNFer3 has the strongest catalytic performance, with the highest slope. The catalysis of PNFer3 with slope of 236.6 was larger than the sum (109.7 + 9.81 = 119.51) of PN3 (109.7) and Fer (9.81), in the SERS method. This was related to the coupling of PN3 and Fer electrons.

Catalytic Reaction Mechanism of Nanoenzymes and DNase
Formate and silver nitrate can generate porous silver [28], but this reaction requires a microwave reaction vessel to keep pressure and temperature. Under the experimental conditions, it is difficult for the reaction between AgNO 3 and SF to take place. After adding the catalyst, a fast reaction can be realized, AgNO 3 is reduced to AgNPs, and SF is oxidized to CO 2 . Table S1 shows a comparison of the linear equations for catalysis of several nanomaterials. It can be seen that PN Fer3 has the strongest catalytic performance, with the highest slope. The catalysis of PN Fer3 with slope of 236.6 was larger than the sum (109.7 + 9.81 = 119.51) of PN 3 (109.7) and Fer (9.81), in the SERS method. This was related to the coupling of PN 3 and Fer electrons. Figure 8 shows a schematic diagram of the catalytic mechanism of PN Fer3 to enhance the signal. In the absence of nanocatalyst, the nanoreaction speed is slow, the product is small, and the RRS/SERS/Abs signal is low. PNs have a small particle size, large specific surface area, and can be stably and uniformly dispersed in aqueous solution. The surface of PNs provides reaction sites for the AgNO 3 and SF reaction system. Fer is rich in electrons and has good thermal and chemical stability [29]. Due to poor water solubility, it is rarely used in aqueous solution. The use of polystyrene nanospheres doped with ferrocene can effectively solve this problem. The excellent electronic conductivity of ferrocene enhances the catalytic performance of PNs. After optimizing the proportion of doped-ferrocene, high-efficiency catalytic performance PN Fer3 can be achieved. The main reason is that the nanosurface electrons and the electrons of Fer were coupled organically to enhance the electron density. Those electrons accelerate Ag + -formate redox electron transfer to enhance the nanoreaction. of PNs provides reaction sites for the AgNO3 and SF reaction system. Fer is rich in electrons and has good thermal and chemical stability [29]. Due to poor water solubility, it is rarely used in aqueous solution. The use of polystyrene nanospheres doped with ferrocene can effectively solve this problem. The excellent electronic conductivity of ferrocene enhances the catalytic performance of PNs. After optimizing the proportion of dopedferrocene, high-efficiency catalytic performance PNFer3 can be achieved. The main reason is that the nanosurface electrons and the electrons of Fer were coupled organically to enhance the electron density. Those electrons accelerate Ag + -formate redox electron transfer to enhance the nanoreaction.

Conditional Selection
As the concentration of PNFer3 increased, the RRS signal gradually increased due to the formation of more nanosilvers ( Figure S4a). When the concentration of PNFer3 exceeded 6.67 mg/L, the RRS signal of the solution decreased sharply due to nanosilver aggregations. Therefore, the concentration of 6.67 mg/L PN3 in this experiment was chosen. When the NaAc-HAc buffer concentration calculated with NaAc is 8.88 mM (pH = 4.4), the RRS signal change value of the system is the largest, so the NaAc-HAc buffer condition was selected ( Figure S4b,c). As the concentration of Apt increases, the system produced a stronger RRS signal ( Figure S4d). However, when the Apt concentration was greater than 8 nM, too much Apt will encapsulate PNFer3, causing the amount of catalyst in the system to decrease, and the RRS signal will decrease. So, 8 nM Apt was selected in this experiment. For the same reason, when the concentration of HM is low, it has a positive effect on the system, and when its concentration is greater than 6 nM, it will affect the catalytic performance and cause the signal to decrease ( Figure S4e). Therefore, the concentration of HM was selected as 6 nM. As shown in Figure S4f, the reaction temperature should be maintained above 75 °C. If the temperature is too low, the reaction cannot be started. If the temperature is too high, the enhanced blank that caused the analytical signal will decrease. At 85 °C, the analytical signal is the strongest. A reaction time of 15 min was optimal ( Figure S4g). The above conditions were selected for use.

Conditional Selection
As the concentration of PN Fer3 increased, the RRS signal gradually increased due to the formation of more nanosilvers ( Figure S4a). When the concentration of PN Fer3 exceeded 6.67 mg/L, the RRS signal of the solution decreased sharply due to nanosilver aggregations. Therefore, the concentration of 6.67 mg/L PN 3 in this experiment was chosen. When the NaAc-HAc buffer concentration calculated with NaAc is 8.88 mM (pH = 4.4), the RRS signal change value of the system is the largest, so the NaAc-HAc buffer condition was selected ( Figure S4b,c). As the concentration of Apt increases, the system produced a stronger RRS signal ( Figure S4d). However, when the Apt concentration was greater than 8 nM, too much Apt will encapsulate PN Fer3 , causing the amount of catalyst in the system to decrease, and the RRS signal will decrease. So, 8 nM Apt was selected in this experiment. For the same reason, when the concentration of HM is low, it has a positive effect on the system, and when its concentration is greater than 6 nM, it will affect the catalytic performance and cause the signal to decrease ( Figure S4e). Therefore, the concentration of HM was selected as 6 nM. As shown in Figure S4f, the reaction temperature should be maintained above 75 • C. If the temperature is too low, the reaction cannot be started. If the temperature is too high, the enhanced blank that caused the analytical signal will decrease. At 85 • C, the analytical signal is the strongest. A reaction time of 15 min was optimal ( Figure S4g). The above conditions were selected for use.

Work Curve
Under the chosen experimental conditions, the working curve was plotted with different concentrations of the analyte and the corresponding signal intensity changes. The analysis characteristics are shown in Table 1. Among the working curves for Pb 2+ determination, the PN Fer3 SERS analytical system was more sensitive than the RRS and Abs, with a detection limit (DL) of 0.03 nM. In addition, the linear range was widest. Compared with the RRS and Abs methods, the blank of the SERS system was very low due to the large AgNPs with low SERS activity. Thus, the PN Fer3 catalytic SERS analysis system was used to measure Pb 2+ . Obviously, the two methods were simpler than the SERS, without molecular probes of VBB. Of course, the Abs method had the lowest cost. Compared with the reported methods (Table S2) [30][31][32][33][34][35][36][37][38], this method is one of the more sensitive methods.

Influence of Coexisting Substances
According to the experimental method, the relative error was within ±10%, and the influence of coexisting substances on the detection of 1 nM Pb 2+ (Table S3) was investigated. The results show that some common ions have little effect on the determination of Pb 2+ , indicating that this method has better selectivity.

Sample Analysis
The samples, two of farmland water, two of lake water, and one of industrial wastewater, were taken with sampling bottles. Each 10 mL water sample was filtered with filter paper to remove suspended solids, then centrifuged at 12,000 rpm/g for 10 min, and finally filtered with a 0.45 µm microporous filter membrane. It was diluted to obtain the sample solution. The SERS method of the PN Fer3 -Pb 2+ -Apt-HM-AgNO 3 -SF system was used to determine Pb 2+ content five times. Then, a certain amount of Pb 2+ was added to the sample to obtain the recovery ( Table 2). These results were agreement with that of atomic absorption spectrometry, the relative standard deviation (RSD) was between 3.7% and 7.8%, and the recovery rate was between 91.2% and 108.7%. Those indicate that the SERS assay was accurate and reliable.

Conclusions
A stable and high-catalytic activity ferrocene-doped polystyrene nanosphere sol catalyst was prepared. It was found that it can effectively catalyze the AgNO 3 -SF reaction to generate nanosilver with strong SERS/RRS/Abs trimode signals. The Apt of analyte lead ion was added to inhibit the catalytic ability. When the analyte lead ion exists, Apt combined with lead ions to generate G-quadruplex DNA enzyme in the presence of heme, and released ferrocene-doped polystyrene nanospheres. This DNA enzyme can also catalyze the AgNO 3 -SF reaction to generate nanosilver. Thus, a new di-enzyme trimode analysis method was established for detecting trace lead ions.
Supplementary Materials: The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/nano12081243/s1 Figure S1: SERS spectra of nanocatalytic analysis system with different molecular probes; Figure S2: RRS spectra of nanocatalysis and analysis system; Figure S3: UV absorption spectra of nanocatalytic and analysis system; Figure S4: Optimization of reaction conditions; Table S1: Comparison of the nanocatalysis of AgNO 3 -SF reaction by means of the slope; Table S2: Comparison of methods reported to detect Pb 2+ ; Table S3: The influence of interfering substances on the determination of Pb 2+ .