In the experiment, the spectrometer was a T64000 grating confocal micro-Raman spectrometer from HORIBA. Spectra-Physics’ Stabilite 2017 Ar ion gas laser was used as the excitation beam source and its excitation line is 514.5 nm. The laser power of the sample surface was approximately 9.64 mW. OLYMPUS’ s BX41 (N_A=0.25) 10× microscope. Fipronil (Purity≥ 98%) was purchased from Aladdin Reagent Company (China). Other reagents such as acetone, ethanol and silver nitrate were of analytical grade and were purchased from Beijing Chemical Company. Water used in this experiment was ultrapure water (18.2 MΩ ).

DFT can intuitively reflect molecular vibration information and was the most commonly used method in quantum chemical calculations. In this paper, the Raman spectrum of fipronil was calculated by G09W quantum chemical software package. The molecular configuration was calculated by Gauss View 5.0 at B3LYP/ 6-311G++(d, p) level. The keyword was opt freq=raman b3lyp/6-311++g(d, p).

The Ag/Cu nano-substrate was prepared according to the method of Xiaohong Jiang^[13]. The 2 cm× 1 cm copper piece was ultrasonically cleaned with acetone, ethanol and ultrapure water for 10 min, then immersed in dilute nitric acid for 1 min to remove the surface oxide. Rinsed the treated copper foil with ultrapure water and dried it at room temperature. The dried copper sheet was immersed in the mixed solution containing 0.02 mol· L^-1 SnCl₂ and 0.02 mol· L^-1 HCl for 1 min, taken out, dried again at room temperature, and then immerse the dried copper sheet in the 0.01 mol· L^-1 AgNO₃ solution containing PVP [m(AgNO₃):m(PVP)=2:1] for 1 min. Finally, the copper piece was placed in the solution containing Sn²⁺ and then immersed, taken out, rinsed with ultrapure water, and dried at room temperature. The 10^-3 mol· L^-1 fipronil solution was prepared with acetone as a solvent, and the Ag/Cu nano-substrate was immersed in the prepared sample solution for 30 min at room temperature, taken out, and dried at room temperature in a vacuum oven. Measured the spectrum directly after completion.

The calculation result had no imaginary frequency, and the stable structure of the fipronil molecule was obtained. As Fig.2 shows, the ring composed of 1C, 2C, 3C, 4C, 5C, 6C and the ring composed of 15N, 16C, 17C, 18C, 19N are not on a plane. The dihedral angle of the plane of the two rings is 86.432 71° . The triangular pyramid structure composed of 13Cl, 14Cl, 15N, 9C and 10-12F can be considered as a substitution of a benzene ring.

Fig.2

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	Fig.2 The optimized molecular structure of fipronil

Fig.3(a) shows the NRS of the fipronil, and Fig.3(b) shows the calculated Raman spectrum of fipronil. The calculation results were observed with Gauss View 5.0. The assignments of theoretical calculations and experimentally determined vibration peaks were finished with the help of Gauss View 5.0 and were listed in Table 1^{[13, 14, 15]}.

Fig.3

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	Fig.3 Experimental normal Raman spectrum of fipronil (a) and Theoretical Raman spectrum of fipronil (b)

Table 1

Table 1

Table 1 The assignments of the calculated vibrational frequencies of fipronil (cm-1) at B3LYP/ 6-311G++(d, p) level NRS/cm-1 DFT/cm-1 Assignment 211 218 τ (21H-22H) 254 273 ρ (18C-29C)τ (25C-26F) 291 ρ (9C-11F) 308 311 δ (10F-11F)ω (21H-22H) 350 348 τ (15N-22H) 375 ω (16C-17C) 407 401 ω (7H-8H) 443 447 τ (23S-24O) 458 δ (21H-22H) 463 460 ρ (1C-5C-6C) 511 509 ρ (ph) br(15N-16C-17C-18C-19N) 529 525 ω (7H-8H)σ (10F-12F) 557 σ (26F-28F) 607 606 τ (7H-8H) 614 ρ (16C-17C) 646 644 τ (ph)ρ (18C-29C) 712 708 δ (9C-10F-11F-12F) 726 724 τ (16C-17C-18C) 800 806 ρ (20N-21H) 820 815 ν (2C-4C) 867 867 ν (6C)τ (21H) 889 900 ρ (7H-8H) 1 062 τ (17C-19N) 1 065 1 066 δ s(25C-26F-27F-28F)ν s(23S-24O) 1 101 1 103 ν (24O) 1 123 1 127 τ (6C-9C)ρ (20N-21H-22H) 1 139 1 134 ν (17C) 1 153 1 154 τ (9C) 1 174 1 175 ν (17C)τ (15N-19N) 1 293 1 294 ν (9C)τ (7H-8H) 1 323 1 318 br(pi)ν (9C) 1 402 1 396 ν (18C-15N) 1 432 1 419 τ (7H-8H) 1 516 1 530 ν (3C-16C) 1 564 1 579 ν (16C) τ (30N-29C) 1 639 1 635 τ (1C-5C) 1 662 σ (21H-22H) ν : stretching vibration; σ : scissor vibration; ρ : in-plane swing vibration; ω : out-of-plane swing vibration; τ : torsional vibration; δ : deformation vibration; br: breath vibration; s: symmetry; ph: benzene ring

Table 1 The assignments of the calculated vibrational frequencies of fipronil (cm-1) at B3LYP/ 6-311G++(d, p) level

Comparing the calculated values of DFT with the measured values of NRS, it has been found that there are little differences between them, but most of them are the same^[16]. Some peaks appear in the calculations but are not found in the measured spectrum, mainly because the theoretical calculation is simply simulation of pure theoretical vibrations of individual molecule. But in the experiment, what has been measured is the solid powder, and there are mutual influence factors such as the intermolecular force and the interaction between the groups; therefore, the theoretical calculation itself has a certain error anyway. In the comparison, we can also see the intensity of the experimental measured value of the solid powder is much higher than the theoretical calculation. But the theoretical calculation still provides a lot of important information for the basic information of the molecule.

Fig.4

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	Fig.4 SERS of fipronil (a) and NRS of fipronil (b)