Doped N/Ag Carbon Dot Catalytic Amplification SERS Strategy for Acetamiprid Coupled Aptamer with 3,3′-Dimethylbiphenyl-4,4′-diamine Oxidizing Reaction

The as-prepared co-doped N/Ag carbon dot (CDNAg) has strong catalysis of H2O2 oxidation of 3,3′-dimethylbiphenyl-4,4′-diamine (DBD). It forms an oxidation product (DBDox) with surface-enhanced Raman scattering (SERS) activity at 1605 cm−1 in the silver nanosol substrate, and a CDNAg catalytic amplification with SERS analytical platform can be structured based on aptamer (Apt) with the DBD oxidizing reaction. For example, the aptamer (Apt) of acetamiprid (ACT) can be adsorbed on the surface of CDNAg, resulting in inhibited catalytic activity, the reduced generation of DBDox, and a weakened SERS intensity. When the target molecule ACT was added, it formed a stable Apt-ACT complex and free CDNAg that restored catalytic activity and linearly enhanced the SERS signal. Based on this, we proposed a new quantitative SERS analysis method for the determination of 0.01–1.5 μg ACT with a detection limit of 0.006 μg/L.


Introduction
Exploring highly selective, sensitive, simple, and rapid contaminant analysis methods is one of the vital development directions of environmental analytical chemistry. Highly selective and sensitive assays rely on highly selective analytical reactions and highly sensitive detection techniques. Nucleic acid aptamers (Apt) are oligonucleotide fragments that have specific recognition of and high affinity with the target. It has applications in genomics, food safety, medical diagnosis, and biomedicine [1][2][3][4][5][6][7][8][9][10][11][12][13]. Surface-enhanced Raman scattering (SERS) is a sensitive molecular spectral technique with real-time in-situ detection; less sample is needed, there is little damage to the sample, and it is simple and fast [5][6][7][8][9][10]. Although many SERS studies have been reported, most of them were qualitative due to poor reproducibility. Reproducibility not only affects qualitative analysis but is a principle of quantitative analysis, which limits its development. To overcome the shortage, some reproducible preparations of clean and highly Ag and Au SERS active substrates have been reported in Reference [14]. Bassi et al. [15] prepared recyclable SERS active glass chips. The gold nanostars were grafted on functionalized glasses by means of electrostatic interactions and then they were coated with a silica layer of controllable thickness. The SERS activity was examined in terms of reproducibility using rhodamine 6G molecular probes. As an analytical technique, the highly sensitive and selective electrochemical methods [28][29][30][31][32][33][34][35][36][37]. However, such methods are expensive, complex to operate or pretreat, have low sensitivity and are time-consuming. Therefore, it is necessary to develop a rapid, sensitive, and selective method for the determination of trace ACT pesticide residues. Jin et al. [33] reported a sensor for the detection of ACT in vegetables based on a photocatalytic degradation compound. In this paper, the Apt reaction [25] was coupled with the SERS catalytic reaction of 3,3 -dimethylbiphenyl-4,4 -diamine (DBD) oxidized product (DBD ox ), and a novel, rapid and sensitive SERS method for the detection of ACT was established.

Instruments
A model of DXR smart Raman spectrometer (Thermo, Waltham, MA, USA), with a laser wavelength of 633 nm, power of 3.5 mW, slit of 50 µm and acquisition time of 5 s, a model of a 3K-15 high-speed refrigerated centrifuge (Sigma Co., Darmstadt, Germany), a model of a 79-1 magnetic stirrer with heating (Zhongda Instrumental Plant, Nanjing, China), a model of an HH-S2 electric hot water bath (Earth Automation Instrument Plant, Jintan, China), a model of a WX-6000 microwave digestion instrument (Preekem Scientific Instruments Co., Ltd., Shanghai, China), a model of a two spectrum Fourier transform infrared spectrometer and its supporting tableting device (Bojin Elmer Co., Ltd., Shanghai, China), a model of an FD-1C-50 vacuum drying freezer (Hangzhou Jutong Electronics Co., Ltd., Hangzhou, China), and a model of a S-4800 field emission scanning electron microscope (Hitachi High-Technologies Corporation, Japan/Oxford company, Oxford, UK) were used.

Reagents
The aptamer sequence (Apt): 5 -CTGAC ACCAT ATTAT GAAGA -3 (Shanghai Sangon Biotech Co., Ltd., Shanghai, China), acetamiprid (ACT) (98.5% purity, Shanghai Shenggong Biotechnology Co., Ltd.); imidacloprid (purity 98.5%, Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China); atrazine (purity 98%, Shanghai Aladdin Biochemical Technology Co., Ltd.); carbendazim (purity 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.); and chlorpyrifos (purity 99%, Shanghai Aladdin Biochemical Technology Co., Ltd.) were used. A 10 mmol/L AgNO 3 solution, 0.1 mol/L sodium citrate solution, 30% H 2 O 2 solution, 0.1 mol/L NaBH 4 solution, 0.2 mol/L pH 3.6 HAc-NaAc buffer solution, 1.0 mol/L HCl solution, and a 0.25 mol/L NaOH solution were prepared. A 30 mg/L ACT standard solution was prepared as follows: In a breaker, 7.5 mg ACT were dissolved in 20 mL anhydrous ethanol. It was transferred into a 250 mL volumetric flask and diluted to the mark with water. A 0.5 mmol/L DBD solution was prepared as follows: First, 11 mg DBD were dissolved and transferred to a 100 mL volumetric flask. This was diluted to the mark with the volume ratio of 6:4 ethanol:water. The nanosilver sol (AgNPs) was prepared as follows [38]: Water, 44 mL, was added to a conical flask. Then, 2 mL 10 mM AgNO 3 , 2.0 mL 100 mM trisodium citrate, 600 µL 30% H 2 O 2 , and 600 µL 0.1 M NaBH 4 were added into the water successively. After the color turned blue, with rapidly stirring, the mixture was immediately shifted to the air light wave stove. After irradiation for 10 min at 250 • C, the color went from blue to orange-red. The mixture was cooled naturally to room temperature. It was then diluted to 50 mL. The concentration of the nanosilver sol was 0.4 mM Ag, calculated by AgNO 3 concentration. All reagents were of analytical grade and the water was double distilled.
Preparation of CD NAg was as follows: one gram of glucose, 0.8 g of urea and 0, 200, 500, and 700 µL 0.01 mol/L AgNO 3 were sonicated in 30 mL water. Then, it was transferred to a high-pressure reaction vessel with a polytetrafluoroethylene substrate, sealed and irradiated in a microwave oven for 10 min with a power of 640 W. After the irradiation, it was cooled to room temperature to obtain a pale yellow solution, which was calculated by adding the total amount of carbon to obtain a solution of C = 13 mg/mL CD N , CD NAg1 , CD NAg2 and CD NAg3 , respectively.

Procedure
A suitable amount of 1.0 µg/L ACT standard solution, 25 µL of 1.55 µmol/L Apt, and 400 µL 1.7 mg/L CD, 60 µL 2 mmol/L H 2 O 2 , 75µL 0.5 mmol/L DBD, 100 µL 1 mmol/L pH 3.6 HAc-NaAc solution were added to a 5 mL test tube and then diluted to 1 mL with water and mixed well. The tube was placed in a bath at 50 • C for 20 min. The reaction was stopped by tap water cooling. A 400 µL 0.4 mmol/L AgNPs solution was added and diluted to 1.5 mL with water. The mixture was transferred to a quartz cell and its SERS spectra were recorded. The SERS peak intensity I at 1605 cm −1 was measured, the blank (I 0 ) without ACT was recorded, and the ∆I = I − I 0 was calculated.

Analytical Principle
In the pH 3.6 HAc-NaAc buffer solution, the CD had a strong catalytic effect on the reaction of H 2 O 2 -DBD to form DBD oxidation products (DBD ox ). When the Apt was present, it adsorbed on the CD surface, resulting in a weakening of the catalytic action of CD. After the addition of the target molecule ACT, it specifically bound to the Apt and, with the CD released, the catalytic activity was restored. With the increase of ACT, there was more desorption of CD, a faster catalyzed H 2 O 2 -DBD reaction, and a greater the concentration of DBD ox formed. After the addition of the AgNP nanosol substrate, the SERS signal enhanced linearly ( Figure 1). Coupling this catalytic amplification reaction with the Apt reaction, a new SERS quantitative analytical method could be established for detecting ultratrace ACT.

Procedure
A suitable amount of 1.0 μg/L ACT standard solution, 25 μL of 1.55 μmol/L Apt, and 400 μL 1.7 mg/L CD, 60 μL 2 mmol/L H2O2, 75μL 0.5 mmol/L DBD, 100 μL 1 mmol/L pH 3.6 HAc-NaAc solution were added to a 5 mL test tube and then diluted to 1 mL with water and mixed well. The tube was placed in a bath at 50 °C for 20 min. The reaction was stopped by tap water cooling. A 400 μL 0.4 mmol/L AgNPs solution was added and diluted to 1.5 mL with water. The mixture was transferred to a quartz cell and its SERS spectra were recorded. The SERS peak intensity I at 1605 cm −1 was measured, the blank (I0) without ACT was recorded, and the ΔI = I − I0 was calculated.

Analytical Principle
In the pH 3.6 HAc-NaAc buffer solution, the CD had a strong catalytic effect on the reaction of H2O2-DBD to form DBD oxidation products (DBDox). When the Apt was present, it adsorbed on the CD surface, resulting in a weakening of the catalytic action of CD. After the addition of the target molecule ACT, it specifically bound to the Apt and, with the CD released, the catalytic activity was restored. With the increase of ACT, there was more desorption of CD, a faster catalyzed H2O2-DBD reaction, and a greater the concentration of DBDox formed. After the addition of the AgNP nanosol substrate, the SERS signal enhanced linearly ( Figure 1). Coupling this catalytic amplification reaction with the Apt reaction, a new SERS quantitative analytical method could be established for detecting ultratrace ACT.

SERS Spectra
In a pH 3.6 HAc-NaAc buffer solution, the H2O2-DBD system was difficult to react in the 60 °C water bath. In the presence of nano-catalysts such as CDN and CDNAg1-3, the oxidation of DBD by H2O2 was catalyzed and the oxidation product of DBDox had the strongest SERS activity for the CDNAg2 catalytic system. The SERS substrates, such as AgNP, gold nanoparticles and CDNAg sols, were examined. The AgNP was the most sensitive and was chosen for use. After the addition of AgNP sol as a SERS substrate, the CDNAg2 catalytic system exhibited a peak at 1189 cm −1 , ascribing to the stretching vibration of C-N, at 1335 cm −1 , ascribing to the bending vibration of C-H, and at 1402 cm −1 , owing to the bending vibration of C=C. The strongest peak was at 1605 cm −1 , ascribing to the tensile vibration of C=N and C=C-C=C ( Figure 2a). When the Apt was added it was coated on the surface of the carbon dot, thereby inhibiting the catalytic activity of the CD, and the SERS signal attenuated (Figure 2b). With the addition of ACT, the combination of ACT and Apt specifically released CDs, which restored its catalytic activity. With the increase of the ACT concentration, the release of CDs increased. The SERS peak at 1605 cm −1 increased linearly, due to more DBDox products being generated (Figure 2c), and was chosen for use.

SERS Spectra
In a pH 3.6 HAc-NaAc buffer solution, the H 2 O 2 -DBD system was difficult to react in the 60 • C water bath. In the presence of nano-catalysts such as CD N and CD NAg1-3 , the oxidation of DBD by H 2 O 2 was catalyzed and the oxidation product of DBD ox had the strongest SERS activity for the CD NAg2 catalytic system. The SERS substrates, such as AgNP, gold nanoparticles and CD NAg sols, were examined. The AgNP was the most sensitive and was chosen for use. After the addition of AgNP sol as a SERS substrate, the CD NAg2 catalytic system exhibited a peak at 1189 cm −1 , ascribing to the stretching vibration of C-N, at 1335 cm −1 , ascribing to the bending vibration of C-H, and at 1402 cm −1 , owing to the bending vibration of C=C. The strongest peak was at 1605 cm −1 , ascribing to the tensile vibration of C=N and C=C-C=C (Figure 2a). When the Apt was added it was coated on the surface of the carbon dot, thereby inhibiting the catalytic activity of the CD, and the SERS signal attenuated (Figure 2b). With the addition of ACT, the combination of ACT and Apt specifically released CDs, which restored its catalytic activity. With the increase of the ACT concentration, the release of CDs increased. The SERS peak at 1605 cm −1 increased linearly, due to more DBD ox products being generated (Figure 2c), and was chosen for use.

Nanocatalysis and Aptamer Inhibition
Under the selected conditions H 2 O 2 and DBD systems were difficult to carry out. As the CD catalyst concentration increased, the catalytic ability enhanced, the DBD ox increased, and the SERS signal increased linearly in the AgNP nanosol substrate. When Apt was added, the CDs were entrapped by the Apt, resulting in inhibition of the CDs catalysis, and the SERS peak intensity decreased linearly (Table 1). In this article, the slope of the linear relationship was used to measure the catalysis and inhibition with simplicity and rapidity. The CD NAg2 was strongest and the strongest inhibition was Apt-CD NAg2 . In the experiment, citric acid was used as the carbon source, urea was involved in the reaction, and a nitrogen source and AgNO 3 as the Ag source were provided to produce a nitrogen/silver co-doped CD. The CD was composed of various molecules that contain amide chains and many N-Ag atoms on the surface. Due to the fact that N and Ag atoms are electron donating atoms, the CD with a proper amount of nitrogen and silver atoms containing a lone pair of electrons could enhance the π bond conjugation. The CD was a catalyst to speed up the redox electron-transfer of DBD and H 2 O 2 to form DBD ox with SERS activity. Nitrogen/silver-doped CDs showed high catalysis due to their greater number of surface electrons. This accelerated the redox electron transfer, which is a new type of CD catalyst with promising applications for the amplification of analytical signal.

Electron Microscopy (EM) and Infrared Spectra
According to the experimental method, 1.5 mL of the reaction solution were centrifuged in a 2 mL centrifuge tube for 10 min (70 × 100 rpm), the supernatant was discarded, and the volume was adjusted to 1.5 mL with water and sonicated for 15 min. We repeated the above centrifugation step twice and added 1.5 mL water for scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The spherical AgNPs were observed by SEM and TEM to have an average size of 45 nm (Figure 3a) and 50 nm (Figure 3b), and exhibited a surface plasmon resonance absorption peak at 400 nm. For the Apt-ACT-CD NAg2 -H 2 O 2 -DBD-AgNPs system, when there was no ACT in the system, Apt encapsulated the carbon dots, inhibiting the CD N -catalyzed H 2 O 2 -DBD reaction and forming fewer DBD ox probes, which caused weak AgNPs aggregations with an average particle size of 50 nm (Figure 3b). With the increase of ACT concentration, CD N was gradually released and the catalytic effect was enhanced to form more DBD ox probes, which caused strong AgNPs aggregations with an average particle size of 60 nm (Figure 3c). In theory, the sizes of the AgNPs, the blank, and the analytical systems are the same. The size difference was ascribed to the different conditions of the three systems. The prepared CD N were diluted and dropped onto a silicon wafer for transmission electron microscopy scanning. The average particle diameter was about 20 nm (Figure 3d). The energy spectra (Figure 3e) indicated that there were Ag atoms doped in the CD because the peak at 3.0 keV was ascribed to Ag. For the CD infrared spectra (Figure 3f) eight peaks were observed, at 3908, 3785, 3430, and 3202 cm −1 ascribing to O-H stretching vibrations, 1716 cm −1 ascribing to C=O stretching vibration, 1666 cm −1 ascribing to C=C conjugate structure stretching vibrations, 1384 cm −1 ascribing to an N=N stretching motion, 1077 cm −1 ascribing to C-H in-plane bending vibrations, and at 642 cm −1 ascribing to N-C=O bending vibrations.

Optimization of the Analytical Conditions
The effect of the concentration of H 2 O 2 , DBD, CD NAg2 , pH, Apt and AgNPs, the reaction temperature and the time on the SERS signal of the system were investigated, respectively (Figure 4). The SERS signal was the strongest when 0.08 mmol/L H 2 O 2 , 0.025 mmol/L DBD, 6.67 µg/L CD NAg2 , pH 3.6 HAc-NaAc buffer solution, 0.026 µmol/L Apt and 0.11 mmol/L AgNPs solution was added. Thus, those concentrations were chosen for use, respectively. When the reaction temperature was 50 • C for 20 min, the SERS was the strongest, so these conditions were selected.

Optimization of the Analytical Conditions
The effect of the concentration of H2O2, DBD, CDNAg2, pH, Apt and AgNPs, the reaction temperature and the time on the SERS signal of the system were investigated, respectively ( Figure  4). The SERS signal was the strongest when 0.08 mmol/L H2O2, 0.025 mmol/L DBD, 6.67 μg/L CDNAg2, pH 3.6 HAc-NaAc buffer solution, 0.026 μmol/L Apt and 0.11 mmol/L AgNPs solution was added. Thus, those concentrations were chosen for use, respectively. selected.

Working Curve
Under the selected conditions, the relationship between the ACT concentration (C) and its corresponding ∆I was obtained ( Figure 5, Table 3). In the four systems, the slope of the CD NAg2 system was the largest due to CD NAg2 having the strongest catalysis. Therefore, the system was chosen for the assay of ACT in real samples. This SERS method is simpler and more sensitive than the reported spectral method for determining ACT [28][29][30][31][32][33][34][35][36][37] (Table 4).

Working Curve
Under the selected conditions, the relationship between the ACT concentration (C) and its corresponding ΔI was obtained ( Figure 5, Table 3). In the four systems, the slope of the CDNAg2 system was the largest due to CDNAg2 having the strongest catalysis. Therefore, the system was chosen for the assay of ACT in real samples. This SERS method is simpler and more sensitive than the reported spectral method for determining ACT [28][29][30][31][32][33][34][35][36][37] (Table 4).

Fluorescence
The aptasensor for ACT based on the inner filter effect between nanogold and CD.

Sample Analysis
Pakchoi, cucumber and tomato were purchased from Guilin Agricultural Market and 50 g samples were weighed. The grinding bowl was ground thoroughly and 1 mL of 99.5% acetone was added. The mixture was filtered with filter paper and centrifuged at 1000 RPM for 5 min. The supernatant was collected and stored in a refrigerator at 4 °C for use. We followed the procedure for SERS detection and tested the recovery. Table 5 indicates that the recovery was 95.7-99.4% and the relative standard deviation was 3.5-5.6%. Table 5. Determination results of ACT in water samples.

Fluorescence
The aptasensor for ACT based on the inner filter effect between nanogold and CD.

Sample Analysis
Pakchoi, cucumber and tomato were purchased from Guilin Agricultural Market and 50 g samples were weighed. The grinding bowl was ground thoroughly and 1 mL of 99.5% acetone was added. The mixture was filtered with filter paper and centrifuged at 1000 RPM for 5 min. The supernatant was collected and stored in a refrigerator at 4 • C for use. We followed the procedure for SERS detection and tested the recovery. Table 5 indicates that the recovery was 95.7-99.4% and the relative standard deviation was 3.5-5.6%.

Conclusions
In this paper, a nitrogen/silver co-doped carbon dot with high stability and high catalytic activity was synthesized by microwave method. Based on the catalytic effect of the carbon dot on the H 2 O 2 -DBD SERS reaction, combined with the specific reaction of ACT-Apt, a new SERS quantitative analysis method for ACT was constructed. It has the advantages of simple operation, high sensitivity and good selectivity.