A Sensitive Gold Nanoplasmonic SERS Quantitative Analysis Method for Sulfate in Serum Using Fullerene as Catalyst

Fullerene exhibited strong catalysis of the redox reaction between HAuCl4 and trisodium citrate to form gold nanoplasmon with a strong surface-enhanced Raman scattering (SERS) effect at 1615 cm−1 in the presence of Vitoria blue B molecule probes. When fullerene increased, the SERS peak enhanced linearly due to formation of more AuNPs as substrate. Upon addition of Ba2+, Ba2+ ions adsorb on the fullerene surface to inhibit the catalysis of fullerene that caused the SERS peak decreasing. Analyte SO42− combined with Ba2+ to form stable BaSO4 precipitate to release free fullerene that the catalysis recovered, and the SERS intensity increased linearly. Thus, a new SERS quantitative analysis method was established for the detection of sulfate in serum samples, with a linear range of 0.03–3.4 μM.


Introduction
Surface plasmon polaritons (SP) are the collective oscillatory behavior of free electrons on metal surfaces when light wave incidentd on the interface between the metal and medium. When surface plasmon is localized on the surface of metal nanoparticles with particle sizes far less than the wavelength of incident light, localized surface plasmon resonance (SPR) is the resonant oscillation of conduction electrons at the nanosurface that are excited by the light [1]. Combined with its good biocompatibility and the mature functional modification of biological molecules surface, the SPR effect has given excellent physical and chemical properties to noble metal nanomaterials such as gold, silver and copper. Recently, it has become a promising technology for nanosensing, bioimaging, analytical separation and biomedical research [1,2]. Among various nanomaterials, gold nanoparticles are widely used to construct visual sensors due to their unique SPR optical properties [2]. The nanoparticle resonance scattering effect is one of the important applications of nanoplasmon in analytical chemistry. It includes elastic resonance Rayleigh scattering (RRS) and inelastic surface-enhanced Raman scattering (SERS). RRS spectroscopy is simple, easy and sensitive, and has been used for the analysis of trace proteins, nucleic acids and heavy metals [3]. Short-chain DNA has been generated from DNA enzymes by lead ion-catalytic cleavage, which protects the gold nanoparticles from aggregation by NaCl. Non-aggregated gold nanoparticles also have a catalytic effect on HAuCl 4 -H 2 O 2 reaction, and trace amounts of lead ion can be detected by RRS. However, a high concentration of NaCl was used as aggregating agent, and large-size gold nanoparticles such as gold nanoflower were not suitable for the system [4]. SERS is one of the most direct applications of nanoplasmon [5][6][7]. This powerful molecular spectral technique is based on the enhancement of inelastic scattering of plasma-excited and surface-adsorbed molecules upon irradiation of the nanostructured surface plasmons. It is one of the few available molecular detection techniques [7,8]. SERS detection probes can be constructed by modifying Raman reporter molecules and target capture molecules on the surface of noble metal nanoparticles. The high specificity and high sensitivity of DNA, proteins and other molecules can be detected by the specific effect of target capture molecules [9]. The formation of further enhanced localized "hot spots" by interparticle coupling further enhances the Raman enhancement factor. Wang et al. [10] used double-modified gold nanoparticles and an antigen-antibody mediated self-assembly sandwich structure formed by interaction between particles to form a "hot spot" to achieve high-specificity multiplex detection of three cytokines in complex biological systems. In recent years, SERS quantitative analysis has greatly improved [11][12][13][14][15], especially in the preparation of sensitive and reproducible nanosol substrates. Liang et al. [11] used H 2 O 2 , NaBH 4 and citric acid as a reducing agent to prepare silver nanorods/reduced graphene oxide (AgNR/rGO) nanosol substrate with good stability to detect 8-1500 nM iodide ion. Yang et al. [12] reported silver nanoparticles as a substrate to determine SCN −1 in milk powder by the SERS method, with a linear range of 2-191.0 mg/L SCN −1 . Luo et al. [13] prepared triangular nanosilver using graphene oxide as catalyst, and SERS quantitative analysis of 0.7-72 nM nitrite. Zhang et al. [14] reported a SERS method for sulfur dioxide in food, with the lowest detectable concentration of 1 mg kg −1 , based on the S atom Raman peak at 630 cm −1 . Shang et al. [15] used AgNO 3 as the precursor to prepare stable silver nanochain (AgNC) sol as a SERS substrate and analyzed 0.0125-0.3 µM sodium hexametaphosphate. However, for anions such as sulfate, whose Raman-scattering section is small, the sensitivity of direct detection is too low to be detected. To the best of our knowledge, there are no SERS quantitative analysis methods for trace sulfates. Therefore, it is of great importance to develop a new SERS quantitative assay for small anions such as sulfate based on the fullerene catalytic generation of nanoplasmon.
Fullerene (C 60 ) is a very important carbon nanomaterial that is applied in the field of solar energy-conversion materials, catalysis and analytical science [16][17][18]. C 60 is a hydrophobic carbon nano-material, its ability to dissolve in water is very low, and it easily accumulates in water, which limits its application. For this reason, researchers usually modify the surface of C 60 to enhance its water solubility so as to obtain better applications. Lanzellotto et al. [19] used fullerene as a bridge, through the fullerene surface alcohol-connected electrode-AuNP and laccase (TvL) to construct an electrochemical biosensor for detection of 0.03-0.30 mM tea polyphenols in beer. Lu et al. [20] inserted fullerol into mercaptoporphyrins and monolayer polyaniline films to prepare electrochemical molecular probes to detect 0.029-10,000 nM benzene. Hang et al. [21] covalently bonded fullerol-rich hydroxyls to thioglycolic acid and PO 4 3− of DNA to construct a DNA molecular probe to detect Sulfate ions play a very important role in life and environmental science. After entering the environment, these will pollute it and cause harm to the human body [25]. Therefore, selective and highly sensitive detection of sulfate ions in biological and environmental samples is of great significance. At present, several methods, including ion chromatography (IC), chemiluminescence, spectrophotometry and atomic absorption spectrometry, have been reported for the determination of sulfate ions [26][27][28][29][30]. Among them, IC is a good method, but its sensitivity is low. Therefore, it is important to develop highly sensitive and selective methods for sulfate ions using the new technology of SERS and the new material of fullerene nanocatalyst. In this article, a new and sensitive SERS quantitative analysis method was developed for the determination of sulfate, coupling the BaSO 4 reaction and C 60 catalytic reaction of HAuCl 4 -trisodium citrate.

Analytical Principle
At 60 • C, the AuNP reaction between HAuCl 4 and trisodium citrate (TSC) is very slow, and C 60 exhibits strong catalysis of the AuNP reaction. Ba 2+ ions adsorb on C 60 surface to inhibit the AuNP reaction. Upon addition of SO 4 2− , stable BaSO 4 precipitates form to escape free and for C 60 catalysis recovery, causing the SERS peak to increase due to the formation of more nanosol substrate of AuNPs when molecular probes of Victoria blue B (VBB) was added. On these grounds, a new SERS quantitative analysis was established for the detection of trace sulfate ( Figure 1). of sulfate ions [26][27][28][29][30]. Among them, IC is a good method, but its sensitivity is low. Therefore, it is important to develop highly sensitive and selective methods for sulfate ions using the new technology of SERS and the new material of fullerene nanocatalyst. In this article, a new and sensitive SERS quantitative analysis method was developed for the determination of sulfate, coupling the BaSO4 reaction and C60 catalytic reaction of HAuCl4-trisodium citrate.

Analytical Principle
At 60 °C, the AuNP reaction between HAuCl4 and trisodium citrate (TSC) is very slow, and C60 exhibits strong catalysis of the AuNP reaction. Ba 2+ ions adsorb on C60 surface to inhibit the AuNP reaction. Upon addition of SO4 2− , stable BaSO4 precipitates form to escape free and for C60 catalysis recovery, causing the SERS peak to increase due to the formation of more nanosol substrate of AuNPs when molecular probes of Victoria blue B (VBB) was added. On these grounds, a new SERS quantitative analysis was established for the detection of trace sulfate (Figure 1).

Surface-Enhanced Raman Scattering (SERS) Spectra
C60 and C60OH analytical systems were examined by the SERS technique using VBB as molecular probes. There are three strong SERS peaks at 1202 cm −1 , 1394 cm −1 and 1615 cm −1 . When SO4 2− concentration increased, SERS signals increased greatly due to the formation of more AuNPs. For the two analytical systems (Figure 2), the C60 system is the most sensitive and the peak at 1615 cm −1 is strongest. Thus, the C60 system with a SERS peak at 1615 cm −1 was chosen to detect SO4 2− .

Surface-Enhanced Raman Scattering (SERS) Spectra
C 60 and C 60 OH analytical systems were examined by the SERS technique using VBB as molecular probes. There are three strong SERS peaks at 1202 cm −1 , 1394 cm −1 and 1615 cm −1 . When SO 4 2− concentration increased, SERS signals increased greatly due to the formation of more AuNPs. For the two analytical systems (Figure 2), the C 60 system is the most sensitive and the peak at 1615 cm −1 is strongest. Thus, the C 60 system with a SERS peak at 1615 cm −1 was chosen to detect SO 4 2− .

Transmission Electron Microscopy (TEM)
Transmission electron microscopy (TEM) of HAuCl4-TSC-C60-Na2SO4-BaCl2-VBB analytical system was undertaken. In the absence of Na2SO4, the AuNP reaction is very slow and formed few spherical AuNPs of an average size of 20 nm (Figure 3). When Na2SO4 was added, more AuNPs of an average size of 15 nm formed due to recovering C60 catalytic activity that caused the SERS signal to be enhanced.

Optimization of Analytical Conditions
According to the procedure, the following parameters were optimized: concentration of HAuCl4, TSC, C60, C60OH, BaCl2 and VBB, reaction temperature and time. Respective data and figures are given in Figure 4. The following conditions were found to give the best results: 4.2 μM HAuCl4, 170 μM TSC, 0.33 mg/L C60, 9.99 ng/L C60OH, 53 μM BaCl2 and 0.33 μM VBB, and a reaction temperature of 60 °C for 20 min.

Transmission Electron Microscopy (TEM)
Transmission electron microscopy (TEM) of HAuCl 4 -TSC-C 60 -Na 2 SO 4 -BaCl 2 -VBB analytical system was undertaken. In the absence of Na 2 SO 4 , the AuNP reaction is very slow and formed few spherical AuNPs of an average size of 20 nm (Figure 3). When Na 2 SO 4 was added, more AuNPs of an average size of 15 nm formed due to recovering C 60 catalytic activity that caused the SERS signal to be enhanced.

Transmission Electron Microscopy (TEM)
Transmission electron microscopy (TEM) of HAuCl4-TSC-C60-Na2SO4-BaCl2-VBB analytical system was undertaken. In the absence of Na2SO4, the AuNP reaction is very slow and formed few spherical AuNPs of an average size of 20 nm (Figure 3). When Na2SO4 was added, more AuNPs of an average size of 15 nm formed due to recovering C60 catalytic activity that caused the SERS signal to be enhanced.

Optimization of Analytical Conditions
According to the procedure, the following parameters were optimized: concentration of HAuCl4, TSC, C60, C60OH, BaCl2 and VBB, reaction temperature and time. Respective data and figures are given in Figure 4. The following conditions were found to give the best results: 4.2 μM HAuCl4, 170 μM TSC, 0.33 mg/L C60, 9.99 ng/L C60OH, 53 μM BaCl2 and 0.33 μM VBB, and a reaction temperature of 60 °C for 20 min.

Optimization of Analytical Conditions
According to the procedure, the following parameters were optimized: concentration of HAuCl 4 , TSC, C 60 , C 60 OH, BaCl 2 and VBB, reaction temperature and time. Respective data and figures are given in Figure 4. The following conditions were found to give the best results: 4.2 µM HAuCl 4 , 170 µM TSC, 0.33 mg/L C 60 , 9.99 ng/L C 60 OH, 53 µM BaCl 2 and 0.33 µM VBB, and a reaction temperature of 60 • C for 20 min.

Working Curve
For the C60 analytical system, SERS intensity was linear to the SO4 2− concentration in the 0.03-2.31 μM linear range (LR), with a regression equation of ΔI = 550.3C + 23.3, coefficient of 0.9474  (Figure 5b). The C 60 is more sensitive than the C 60 OH, and was selected for the detection of sulfate. In short, this article was utilized advance nanoplasmonic SERS technique and nanoreaction to develop a highly sensitive SERS quantitative analysis method for sulfate ions that firstly used VBB as label-free molecular probe and C 60 as nanocatalyst for AuNP reaction between HAuCl 4 and trisodium citrate. In a comparison of reported methods [26][27][28][29][30] for sulfate determination (Table 1), this new SERS quantitative analysis method for sulfate is one of most sensitive methods, with less serum sample used.
Nanomaterials 2018, 8, x FOR PEER REVIEW 6 of 10 and detection limit (DL) of 0.01 μM (Figure 5a). For the C60-OH system, the SERS intensity was linear to the SO4 2− concentration in the 0.06-2.31 μM range, with a regression equation of ΔI = 447.9C + 24.7, coefficient of 0.9635, and DL of 0.03 μM (Figure 5b). The C60 is more sensitive than the C60OH, and was selected for the detection of sulfate. In short, this article was utilized advance nanoplasmonic SERS technique and nanoreaction to develop a highly sensitive SERS quantitative analysis method for sulfate ions that firstly used VBB as label-free molecular probe and C60 as nanocatalyst for AuNP reaction between HAuCl4 and trisodium citrate. In a comparison of reported methods [26][27][28][29][30] for sulfate determination (Table 1), this new SERS quantitative analysis method for sulfate is one of most sensitive methods, with less serum sample used.

Influence of Interfering Ions
The interference of 16 coexisting substances on the determination of 0.66 μM SO4 2− was investigated according to the procedure. Results (Table 2) show that common substances did not interfere with the determination. This indicated that the SERS method has good selectivity.

Influence of Interfering Ions
The interference of 16 coexisting substances on the determination of 0.66 µM SO 4 2− was investigated according to the procedure. Results (Table 2) show that common substances did not interfere with the determination. This indicated that the SERS method has good selectivity.

Analysis of Samples
Total sulfur in whole blood included inorganic sulfate ions and various non-inorganic sulfates [30]. This proposed SERS method was applied to the determination of inorganic sulfates in human blood serum samples collected from three healthy people. Trichloroacetic acid was added in a 0.10 mL serum and 9 mL water to remove proteins by centrifugation at 7000 r/min for 10 min, and was diluted to 10 mL to obtain the sample solution. Sulfate content was determined five times to obtain a single value according to the procedure outlined in Section 3.3 below, with relative standard deviation (RSD) of 1.9-4.2%. 0.300 µg/mL sulfate was added in three samples respectively, and determined the sulfate concentration. Then, recovery was calculated and was between 95.0% and 99.3% (Table 3). According to a dilution time of 100 and average values, the content of sulfate in serum was between 36.9 µg/mL and 41.9 µg/mL.

Apparatus
The following were used: DXR model smart Raman spectrometer (Thermo, Waltham, MA, USA) with laser wavelength of 633 nm, power of 3.

Reagents
2.9 mM HAuCl 4 (National Pharmaceutical Group Chemical Reagents Company, Shanghai, China, http://www.reagent.com.cn); 10 µM VBB (Shanghai Reagent Three Factory, Shanghai, China) stock solution; 1.0 mM BaCl 2 (Hunan Reagent Factory, Changsha, China); 1.00 mM Na 2 SO 4 (Xilong Science Co., Ltd., Shantou, China); and 3.4 mM trisodium citrate (Xilong Chemical Plant, Shantou, China) were prepared. Fullerene solution C 60 : A 0.02 g C 60 was dissolved in 20 mL toluene by ultrasonic waves to obtain a bright purple solution. Then, 100 mL water was added and placed in an ultrasonic instrument to volatilize all toluene and so prepare a deep yellow suspension with concentration of 0.2 g/L C 60 . Fullerol (C 60 OH) solution: an accurately weighed 0.2 g C 60 OH was dissolved in 100 mL water to obtain a concentration of 2 g/L C 60 OH.

Procedure
In a 5 mL test tube, a suitable amount of Na 2 SO 4 , 80 µL 1 mM BaCl 2 and 25 µL 20 mg/L C 60 were added and mixed well. Then, 100 µL 0.1% HAuCl 4 and 75 µL 3.4 mM TSC solution were added and diluted to 1.5 mL. The mixture was heated to 60 • C in a water bath for 20 min, cooled with ice-water, and 50 µL 10 µM VBB was added. The Raman spectrum was recorded by a scanning Raman spectrometer. The SERS intensity at 1615 cm −1 ( I 1615 cm −1 ) and blank value ( I 1615 cm −1 ) 0 without sulfate were measured. The ∆I = I 1615 cm −1 − ( I 1615 cm −1 ) 0 value was calculated.

Conclusions
C 60 exhibited a strong catalysis of reduction of HAuCl 4 by trisodium citrate to form high SERS-active AuNPs. Ba(II) ions can combine with C 60 to produce Ba-C 60 complexes to inhibit the nanocatalysis. Upon the addition of sulfate ions, stable BaSO 4 precipitate formed to release C 60 , which activated the catalytic effect of C 60 and enhanced the SERS peak linearly. Thus, a new SERS quantitative analysis method was established for the determination of trace sulfate in serum samples, with simplicity, high sensitivity and selectivity, and less serum sample consumption.