Role of 2‒13C Isotopic Glyphosate Adsorption on Silver Nanoparticles Based on Ninhydrin Reaction: A Study Based on Surface—Enhanced Raman Spectroscopy

Glyphosate is one of the most commonly used and non-selective herbicides in agriculture, which may directly pollute the environment and threaten human health. A simple and effective approach to its detection is thus quite necessary. Surface-enhanced Raman scattering (SERS) spectroscopy was shown to be a very effective method to approach the problem. However, sensitivity in SERS experiments is quite low, caused by different orientation/conformation of the adsorbed molecules on the metal surface, which limit its detection by using SERS. In this paper, 2‒13C‒glyphosate (hereafter: 13–GLP) was designed as a model molecule for theoretical and experimental studies of the molecule structure. Vibrational modes were assigned based on the modeling results obtained at the B3LYP/6-311++G** level by density functional theory (DFT) calculations, which were performed to predict the FT‒IR and Raman spectra. Band downshifts were caused by 13C atom isotopic substitution with mass changed. Moreover, SERS spectra of 13–GLP by combining ninhydrin reaction on Ag NPs were obtained. Isotopic Raman shifts are helpful in identifying the components of each Raman band through vibrations across the molecular system. They are coupled by probe molecules and thus bind to the substrates, indirectly offering the opportunity to promote interactions with Ag NPs and reduce the complex equilibrium between different orientation/conformation of glyphosate molecules on the metal surface.


Introduction
Glyphosate (C 3 H 8 NO 5 P, CAS: 1071-83-6) is one of the most commonly used and non-selective herbicides in agriculture, which may directly pollute the environment and threaten human health. Glyphosate was first synthesized by the Monsanto company and marketed under the name "Roundup", and then became well known as a common herbicide to control weeds. Based on its chemical properties, glyphosate is expected to be adsorbed by the soil; due to its widespread use, it directly pollutes the environment and contaminate foods. The detection of glyphosate residues water, food, and feed stuff is an essential step in regulating and monitoring the level. The majority of these methods are based on either gas chromatography (GC) or high-performance liquid chromatograph (HPLC) coupled to a variety of detectors, mainly mass spectrometric detectors. Most of the methods are highly sensitive, Nanomaterials 2020, 10, 2539 2 of 11 however, in most cases tedious since they include numerous steps for the purification and derivatization of the compound.
Surface-enhanced Raman scattering (SERS) has been widely used as a powerful tool for ultrasensitive chemical analysis, as this technique is sensitive enough to detect glyphosate molecule [1]. Taking glyphosate as an example, although numerous efforts have been made, disagreements are still unresolved regarding the vibration assignments, and a complex equilibrium between different orientation/conformation of glyphosate molecules on the metal surface further prevents an explicit understanding of the glyphosate-detection process at a molecular level by SERS. We reported a simple and sensitive method for the determination of glyphosate by combining ninhydrin reaction and SERS spectroscopy [2]. The product (purple color dye, PD) of the ninhydrin reaction is found to SERS-active and directly correlate with the glyphosate concentration. However, vibration assignments and absorption were not completely clear.
The SERS detection method based on isotope labeling can achieve the purpose of defining vibration mode precisely. Isotope labeling has been reported in the detection of glyphosate residues and its transformation products based on NMR, MS, HPLC, GC, and GC-MS [3,4]. The SERS detection method based on isotope labeling has its own advantages. The vibration frequency of different isotopes is different, and there will be great differences. In terms of the attribution of characteristic peaks, the molecular vibration of isotope labeling was reported partly, however, the theoretical and practical spectral differences still need to be compared with DFT calculation with clearly defined assignments [5].
In this paper, in order to make more clearly defined assignments to each atom, we determined 2-13 C-glyphosate (13-GLP) as a model molecule for the theoretical and experimental studies of the molecule structure. Isotopic effects on Raman/FT-IR vibrational frequency and intensity due to the change of reduced masses and the vibrational coupling were verified by density functional theory (DFT) simulations and its corresponding isotopic SERS measurements. Density functional theory (DFT) calculations were performed to predict the Fourier Transform Infrared (FT-IR) and Raman spectra for the molecule. In order to reduce the complex equilibrium between different orientation/conformation of glyphosate molecules on the metal surface, a glyphosate detection method coupled by probe molecules with accurate vibration assignments thus bind to the substrates indirectly offered the opportunity to promote interactions with Ag NPs.

Instruments
Raman spectra were recorded on a Jobin Yvon/HORIBA LabRam ARAMIS Raman spectrometer (Tokyo, Japan) equipped with an integral BX 41 confocal microscope (Tokyo, Japan). Radiation from an air-cooled internal HeNe laser (632.8 nm) and an external cavity diode laser (785 nm) were used as the excitation source. The FT-IR spectra of ninhydrin were recorded as KBr disks at room temperature by a Bruker IFS-66V FT-IR spectrometer (Karlsruhe, Germany), equipped with a DTGS detector (Karlsruhe, Germany) at a resolution of 4 cm −1 .

Preparation of Silver Nanoparticles
Ag NPs were prepared according to the Lee and Meisel method, starting from silver nitrate (36 mg AgNO 3 added into 200 mL H 2 O) and using sodium citrate (1% ω/V, 4 mL) as reducing agent [6]. After heating for 40 min at 85 • C, a grey-green colloid formed and was naturally cooled at room temperature.

Theoretical Method
All geometries were optimized using the B3LYP exchange-correlation functional, which is a hybrid of Becke's three-parameter exchange, and the Lee-Yang-Parr correlation functionals [7][8][9]. The 6-311++G(d,p) basis set was used for the H, C, O, N atoms. All calculations were carried out using the Gaussian 09 software program (Wallingford, CT, USA) [10]. The molecular electrostatic potential (MEP) was obtained by the Gaussian 09 software program (Wallingford, CT, USA) [11].

Characterization of the Ag Nanoparticles (NPs)
Ag NPs are most commonly used SERS-active substrates in fundamental and applied sciences. The spheroidal Ag NPs used in this study have a maximum absorption around 430 nm with an average diameter of 60 nm, which are consistent with the results reported in the literature ( Figure S1) [12].

Molecular Geometry
The optimized geometry of 13-GLP or 12-GLP is shown in Figure 1a. In Figure 1a, 13 C at C7 position. The corresponding structural parameters of bond lengths, bond angles, and dihedral angles are shown in Table 1. The atom numerical labels in the following discussion refer to Figure 1a. The MEP mapping of the 13-GLP or 12-GLP is presented in Figure 1b. MEP mapping provides a visual method to understand the relative polarity of a molecule. Notably, it is a very useful procedure to study the relationship between molecular structures and their physiochemical properties. In Figure 1b, negative charges (electrophilic regions) are represented in red, while positive charges (nucleophilic regions) are represented in green; the MEP increases in the order of red < orange < yellow < green < blue. The total electron density and MEP on the complex surfaces were generated at the B3LYP/6-311++G(d,p) level of theory. 2-13 C-glyphosate and glyphosate are only one isotopic atom apart, thus their structural parameters are exactly the same, as shown in Table S1 (Supplementary Materials). As we know, there is no difference in space configuration between isotope labeled molecules and protomolecules, and results in this report consistent with the results reported in the literature.    Table 1 and Table S2 (Supplementary Materials). Optimized geometry of 13-GLP and 12-GLP are the same, however, the vibrations calculated are different in some bonds.
The absence of imaginary frequencies in the DFT calculations indicates that the calculated 13-GLP and 12-GLP structures are minima on their respective potential energy surfaces. To fit the theoretical to the experimental wavenumbers, a scaling factor of 0.980 was employed to optimize the computed data under 1800 cm −1 , and 0.9621 for wavenumbers higher than 1800 cm −1 . The theoretical Raman spectra of the 13-GLP and 12-GLP calculated (scaled) at the B3LYP/6-311++G(d,p) level of theory is presented in Figure 2A(a,b), while the experimental Raman spectra in the solid state is presented in Figure 2B(a,b).
Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.    (Table S2), Table 1 and Figure 2A(a). Results of the calculated frequencies of 12-GLP (Figure 2A(b)) were consistent with the results reported by Holanda et al. [13]. The frequencies of 13-GLP (Figure 2A(a)) in the DFT calculations indicate that most of the vibrational modes in calculated frequencies of 13-GLP agreed well with the theoretically predicted frequencies of 12-GLP. However, the isotope labeled atom changed the Raman intensity of some bonds and changed frequencies. Raman bands at 849, 1027, 1130, 1350 cm −1 for theoretical 13-GLP Raman spectrum appear at 856, 1036, 1137, 1362 cm −1 for theoretical 12-GLP assigned to C2N5C7C10 skeleton, C2N5C7 symmetric stretching, C2N5C7 asymmetric stretching, C7H 2 wagging + C10C7 stretching, respectively. It is noteworthy that Raman band at 1426 and 1460 cm −1 of 13-GLP appear at 1426 and 1464 cm −1 of 12-GLP assigned to δ(CH 2 ) from δ(C7H 2 ) or δ(C2H 2 ). Large shifts are also observed at wavenumbers higher than 1800 cm −1 , Raman bands at 2792, 2992 cm −1 for theoretical 13-GLP Raman spectrum appear at 2799, 3001 cm −1 for theoretical 12-GLP assigned to C7H8 stretching, C7H9 stretching. However, C7H 2 twisting has the same Raman spectrum appear at 1237 cm −1 in both 13-GLP and 12-GLP. As we know, in similar structures, the smaller the atomic mass of the chemical bond has a higher infrared absorption frequency. The isotopic substitution is expected to cause downshifts for those bands deriving from modes which involve the 13 C atom. Thus, a downshift for band involves 13 C atom isotopic substitution.

Experimental Raman Spectra of 13-GLP and 12-GLP
The observed vibration modes are listed in Table S2 (Supplementary Materials), with a tentative assignment, which is mainly based on previous work on glyphosate and related molecules [13][14][15][16]. Results of the frequencies of 12-GLP and 13-GLP ( Figure 2B(a,b)) were consistent with the results reported [5]. The normal Raman modes of 13-GLP in the solid state are observed at 206, 291, 305, 320,  342, 456, 485, 511, 577, 639, 773, 800, 857, 917, 927, 987, 1026, 1036, 1069, 1136, 1197, 1255, 1283, 1338,  1425, 1432, 1460, 1482, 1564, 1729, 2956, 2967, 2991, and 3011 cm −1 . Most of vibrational modes observed in the solid-state Raman spectrum of 13-GLP agreed well with frequencies of 12-GLP. Summary observed normal Raman scattering vibration modes of glyphosate 12-GLP and 13-GLP are listed in Table 1. However, the downshifts for some bands cause by 13 C atom isotopic substitution can be assigned more accurately. As with the results of theoretical vibration, some Raman bands downshift were caused by the isotopic substitution. Raman bands at 504, 639, 928, 1069, 1350, and 1460 cm −1 for 13-GLP Raman spectrum in solid state appear at 511, 648, 933, 1082, and 1465 cm −1 for 12-GLP assigned to O12H bending and C7H 2 bending, C2N5H twisting and C7N5H twisting, and C7H 2 rocking, C7H 2 bending. Calculated theoretical Raman spectrum of 12-GLP/13-GLP is different from experimental Raman spectrum, the relative intensities are not precisely predicted for all the Raman bands. However, it could show structural parameters of 12-GLP/13-GLP, as they are still fairly useful for the assignments of the normal modes in the Raman spectrum. Raman spectrum will be influenced by excitation wavelength, and this may explain these discrepancies. The shapes of theoretical spectrum can well conform to experiment, but the wavenumbers are underestimated by about 15 cm −1 .
It is a remarkable fact that Raman shift of CH 2 bending downshift from 1465 cm −1 for 12-GLP to 1460 cm −1 for 13-GLP, another Raman shift is still 1482 cm −1 , however, in the calculated frequencies of CH 2 bending, one Raman shift is 1426, another downshift from 1464 cm −1 to 1460 cm −1 .
Isotopic substitution (an atom is replaced by an isotope of larger mass) leads to a decrease in the wavenumber of all modes that involve the movements of the 13 C atoms (harmonic oscillator: a reduces mass, µ, increases and vibrational energy level). According to the baseline of vibrational spectrum ν = 1 2πc k µ shows that the larger the mass, the smaller the wavenumber. Therefore, isotopic substitution has opened the door to studying more complex systems. Thus, we assigned 1460 cm −1 for 13-GLP in normal Raman spectrum in solid state to C7H 2 bending, and 1482 cm −1 to C2H 2 bending. Similarly, we assigned 928 cm −1 for 13-GLP in normal Raman spectrum in solid state to C7H 2 rocking.
In conclusion, spectra shift by isotopic can be assigned vibrational modes more accurately because it does not change the symmetry of molecular structure and force constants between chemical bonds. The vibrational frequency of isotope substitution is shifted due to the difference of mass. In this paper, bands downshifts cause by 13 C atom isotopic substitution to 12 C.

Raman and SERS Spectra of PD POroduct from 12-GLP/13-GLP
Glyphosate Raman and SERS spectra have been subject of different experimental and theoretical studies, we also obtained SERS spectra of 13-GLP absorbed on Ag NPs. However, the observed SERS pattern in terms of a complex equilibrium between different orientation/conformation of the glyphosate molecules on the metal surface shows that sensitivity in SERS experiments is quite low [5].
Pesticide molecules without SERS-active functional groups could be coupled by some probe molecules to achieve a better signal [17]. In previous work, we reported a glyphosate detection method based on ninhydrin reaction and surface−enhanced Raman scattering spectroscopy (SERS) to overcome this disadvantage. The PD product of the ninhydrin reaction is found to SERS-active and directly correlate with the glyphosate concentration. Meanwhile, experimental information about the structure changes of glyphosate and its PD product absorbed on Ag nanoparticles still needs further detection. Normal Raman and SERS spectra of PD product from 12-GLP/13-GLP with vibrational assignments are shown in Figure 4 and Table S3. They are clearly distinguishable with two relatively stronger peaks around 662 and 791 cm −1 in each spectroscopy. Figure 4a,c compares the Raman spectra of PD product from 13-GLP and 12-GLP are very similar, yet two band at 662 and 791 cm −1 display intensity changed due to mass changes caused by the 13 C atom isotopic substitution. The band at 538 cm −1 shift to 521/524 cm −1 when absorbed on Ag NPs in SERS spectra. A strong peak appears at 690/689 cm −1 in SERS spectra that is not present in experimental Raman spectra ( Figure 4). The peak at 872/873 cm −1 assigned to Mo-O from Na 2 MoO 4 which is a catalyst forms a weak π-complex with aromatic rings of the ninhydrin component in the PD product. This peak in PD product from 13-GLP is much higher with wider full width of half maximum than from 12-GLP.
This reaction is different from the classic amino acid color reaction with ninhydrin, in which no decarboxylation or dealdehyding reaction takes place. The molecular structure of PD product includes two moieties, N5 atom from glyphoate combines with ninhydrin, forming a new C−N bond and linking glyphoate and ninhydrin. Ninhydrin molecular is C 2 symmetry, however, in PD product changed to C 1 symmetry. The peaks at 657 and 786/784 cm −1 are assigned to the out-of-plane bending vibration of C=O from ninhydrin molecular. The peak 689 and 524/521 cm −1 are related to the vibration of the benzene ring carbon skeleton. This suggests that PD product interacts with the Ag atoms via the oxygen atoms of the ninhydrin moiety. According to the surface selection rule, we infer the PD product molecules adsorbed on the surface had a perpendicular orientation. 13 C atom isotopic substitution to 12 C change the mass, thus PD product coupled MoO 4 2− with a weak π-complex slightly changes the adsorption orientation on Ag NPs. Glyphosate Raman and SERS spectra have been the subject of different experimental and theoretical studies but sensitivity in SERS experiments is quite low due to small Raman cross-section and complex equilibrium between different orientation/conformation of the adsorbed molecules on the metal surface.
-COO− group does not participate in the interaction of the glyphosate with Ag NPs. Imidazole ring in PD product interacts with the Ag NPs surface, adopting almost erect state orientation relative to it. None of these bands can be attributed to the vibrations of glyphosate, therefore these moieties do not affect the SERS spectrum of glyphosate on the surface of Ag NPs. Thus, the combination of high Raman scattering probe with isotopic labeling provides avenues for accurate and quantitative SERS. We believe such a method will open a new promising area for isotopic SERS to investigate the mechanism of surface photoinduced reactions through tracking the fingerprint information changes of the species on metal nanostructures.

Quantitative Analysis of 13-GLP
We examined the SERS spectra of the PD products from 13-GLP at different concentrations shown in Figure 5

Conclusions
Glyphosate Raman and SERS spectra have been the subject of different experimental and theoretical studies, however, sensitivity in SERS experiments is still quite low due to the complex equilibrium between different orientation/conformation of the adsorbed molecules on the metal surface. 2-13 C isotopically substituted derivative is used to confirm the vibration modes in Raman and FT-IR spectra. The isotopic substitution is expected to cause downshifts deriving from modes which involve the 13 C atom, and PO symmetric stretching and PC stretching with no bond to 13 C atom. This downshift caused by isotopic substitution is also present in DFT calculations, though there is no difference in space configuration between isotope labeled molecules and protomolecules. We further reported the SERS spectra of 13-GLP by combining ninhydrin reaction on Ag NPs coupled by probe molecules and thus bind to the substrates. This indirectly offers the opportunity to promote interactions with Ag NPs and reduce the complex equilibrium between different orientation/conformation of glyphosate molecules on the metal surface with a LOD of 4.38 × 10 −7 mol·L −1 and LOQ of 5.0 × 10 −7 mol·L −1 . 2-13 C isotopically substituted derivative is used to confirm the vibration modes in Raman and FT-IR spectra more conveniently with higher sensitivity, better reproducibility, and lower relative standard deviation. This work will open a new promising area for isotopic SERS to investigate the mechanism of surface photoinduced reactions through tracking the fingerprint information changes of the species on metal nanostructures.

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