Guanhua Zhang,Chunxia Ma,Qing He,Hongjing Dong,Li Cui,Lili Li,Lingyu Li,Yan Wang,Xiao Wang
An efficient Pt@MXene platform for the analysis of small-molecule natural products
Materials property,Biomaterials,Nanomaterials
Summary Small-molecule (m/z<500) natural products have rich biological activity and significant application value thus need to be effectively detected. Surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) has become a powerful detection tool for small-molecule analysis. However, more efficient substrates need to be developed to improve the efficiency of SALDI MS. Thus, platinum nanoparticle-decorated Ti3C2 MXene (Pt@MXene) was synthesized in this study as an ideal substrate for SALDI MS in positive ion mode and exhibited excellent performance for the high-throughput detection of small molecules. Compared with using MXene, GO, and CHCA matrix, a stronger signal peak intensity and wider molecular coverage was obtained using Pt@MXene in the detection of small-molecule natural products, with a lower background, excellent salt and protein tolerance, good repeatability, and high detection sensitivity. The Pt@MXene substrate was also successfully used to quantify target molecules in medicinal plants. The proposed method has potentially wide application.
An efficient Pt@MXene platform for the analysis of small-molecule natural products

Small-molecule (m/z<500) natural products have rich biological activity and significant application value thus need to be effectively detected. Surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) has become a powerful detection tool for small-molecule analysis. However, more efficient substrates need to be developed to improve the efficiency of SALDI MS. Thus, platinum nanoparticle-decorated Ti3C2 MXene (Pt@MXene) was synthesized in this study as an ideal substrate for SALDI MS in positive ion mode and exhibited excellent performance for the high-throughput detection of small molecules. Compared with using MXene, GO, and CHCA matrix, a stronger signal peak intensity and wider molecular coverage was obtained using Pt@MXene in the detection of small-molecule natural products, with a lower background, excellent salt and protein tolerance, good repeatability, and high detection sensitivity. The Pt@MXene substrate was also successfully used to quantify target molecules in medicinal plants. The proposed method has potentially wide application.

Small-molecule (m/z<500) natural products in plants, including amino acids, carbohydrates, phytohormones, organic acids, and alkaloids, perform several important functions for the viability of organisms. These products also exhibit rich physiological and pharmacological activities and are widely used in the fields of medicine, chemistry, and food. The metabolic pathways, varieties, and physiological characteristics of different plants may lead to differences in the species and content levels of small-molecule natural products., Consequently, comprehensive, accurate, and reliable detection of small-molecule natural products is imperative and can contribute to metabolomic research, species identification, and elucidation of the growth mechanism of plants. Currently, the analysis of small-molecule natural products is typically performed using thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and liquid chromatography‒mass spectrometry (LC‒MS). These methods usually have good analytical capabilities. However, these methods also have significant shortcomings, including complex sample preparation, low throughput, the need for additional derivatization procedures, and large solvent consumption, and can therefore be slow, laborious, and expensive.,, Hence, a rapid and comprehensive small-molecule natural product analysis technology is urgently needed. Surface-assisted laser desorption/ionization (SALDI) mass spectrometry (MS) has emerged in the past decade as a powerful technology for the detection of small-molecule natural products. In SALDI MS, an optical-thermal conversion process enables energy transfer from the substrate to the analyte deposited on its surface, facilitating desorption/ionization of analyte molecules. Similar to classical matrix-assisted laser desorption/ionization (MALDI) MS, SALDI MS only requires replacing the traditional organic matrices into nanomaterials or structured inorganics as substrates. Generally, inorganic substrates of SALDI MS can exhibit superior UV laser absorption, better efficiency energy transfer and higher physical/chemical stability than conventional matrices. The samples exhibited various desorption/ionization efficiency because of differences in the substrates. Some materials with surface modification have been also employed to select the adsorption of small molecules in LDI MS analysis. Therefore, SALDI MS eliminates some limitations of MALDI MS. Examples of these limitations are that organic matrices, such as 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA), undergo non-uniform co-crystallization with a sample, resulting in poor repeatability, and the matrix background interferes with the detection of small-molecule natural products. In some cases, organic matrix effects can hinder the ionization of small-molecule analytes., In addition, SALDI MS can enhanced detection sensitivity by tens of times compared to conventional MALDI MS, allowing microanalysis (∼pmol, ∼fmol) for natural products in bio-samples. Thus, a large number of nanomaterial substrates have been developed for laser desorption/ionization (LDI) MS, such as graphene, TiO2 nanoparticles (NPs), metal NPs, and MXenes. Among these substrates, MXenes are emerging two-dimensional nanomaterials with excellent hydrophilicity, high ultraviolet‒visible (UV‒Vis) absorption, good charge mobility, and extensive application prospects for SALDI MS. The chemical formula of MXenes is Mn+1XnTx (n = 1–3). M, X, and T represent transition metals (such as Ti, V, and Mo), C and/or N, and surface termination groups (such as-O,-F, and-OH), respectively. Ti3C2 MXene (denoted as MXene) is a representative MXenes that has received extensive attention. Abundant functional groups on the MXene surface can promote the adsorption of small-molecule natural products and the enrichment and transference of Na+ or K+. Previous studies have shown that MXene can facilitate more effective SALDI MS detection than graphene oxide (GO), carbon nanotubes (CNTs), and organic matrices (DHB and CHCA)., More importantly, MXene has a high surface area and surface defects/vacancies and can be used to support other heterogeneous materials to enhance SALDI MS performance. Precious-metal (Ag, Au, Pt) NPs have unique localized surface plasma resonance characteristics and outstanding optical-thermal conversion efficiency and are considered to be one of the most attractive nanomaterials for SALDI MS substrates. However, Au or Ag NPs produce cluster ions that may interfere with detection in SALDI MS., Pt NPs are a stable substrate that exhibits a clean background and high desorption/ionization efficiency in SALDI MS., Pt NPs-decorated MXene (Pt@MXene) exhibits superior electrical conductivity and optical-thermal conversion properties than MXene. In addition, Pt@MXene has highly biosafety both in vitro and in vivo, ensuring that no adverse effects are caused during use., As a result of these good properties, Pt@MXene platforms have been mainly used in nanocatalysts, electrochemistry, and biomedicine.,, However, the performance of Pt@MXene as a substrate for SALDI MS has not been determined to date. Herein, Pt@MXene was synthesized by a simple and green approach (Scheme 1A), followed performed high-throughput detection and quantification of small-molecule natural products in two plant samples (Scheme 1B). Notably, the reaction is generated by self-reduction without the addition of a catalyst, enabling significant preservation of the original characteristics of MXene nanosheets. The efficacy of Pt@MXene has been demonstrated for the analysis of various small-molecule natural products. Pt@MXene has been found to have stronger UV‒Vis absorption than MXene and can effectively absorb laser energy. Compared with the commonly used substrates (GO), MXene, and the organic matrix (CHCA), Pt@MXene exhibits superior performance for the detection of small-molecule natural products (amino acids, phytohormones, organic acids, alkaloids, and carbohydrates) in terms of a lower background interference, high sensitivity, good salt and protein tolerance, and reliable stability. Pt@MXene has also been used to successfully quantify small-molecule natural products of medicinal plants, including gentiopicroside (GP) in Gentiana scabra Bge. (G. scabra) and tanshinone IIA (TS) in Salvia miltiorrhiza Bge. (S. miltiorrhiza). A multifunctional detection platform for the high-throughput qualitative and quantitative analysis of small-molecule natural products in complex plant samples has been developed in this study.

The synthesis approach of Pt@MXene as depicted in Scheme 1A. Single/few-layer MXene nanosheets were prepared by etching Ti3AlC2 based on LiF/HCl method, followed by ultrasonication. The Pt@MXene nanocomposite was constructed by a top-down synthesis method (a self-reduction reaction) using single/few-layer MXene nanosheets and H2PtCl6·6H2O as raw materials. Compared to other top-down synthetic methods (such as electrochemical exfoliation) and bottom-up methods (such as atomic layer deposition and electrodeposition), the top-down synthesis method (a self-reduction reaction) applied in this study has the advantages of simpler, greener, and lower cost. More importantly, the interference of mass spectrometry analysis produced by reducing agent residue could also be avoided by using this top-down synthesis method (a self-reduction reaction) because no reducing agent was applied to the entire operation. The transmission electron microscopy (TEM) image in Figure 1A shows the micromorphology of the MXene consisting of a monolayer or few layers of MXene nanosheets. The reaction produced Pt NPs dispersed on the surface of MXene nanosheets with an average particle size of approximately 2.56 nm (Figure 1B). The high-resolution TEM (HRTEM) image of Pt@MXene exhibited a 0.23-nm lattice fringe of Pt, assigned to the (111) plane (Figure 1C), which proved the successful doping of Pt NPs into MXene. The types and distribution of elements in Pt@MXene are shown in the energy-dispersive X-ray spectroscopy (EDS) mapping spectrum. Figure 1D shows that Pt NPs were dispersed homogeneously on the surface of MXene, which was consistent with the HRTEM detection result. The results presented above prove that the Pt NPs-doped MXene composite material was successfully synthesized. The optical characteristics of Pt@MXene were characterized by UV–Vis absorption. The typical broad peak of MXene at approximately 270 nm was observed in the UV–Vis spectrum (Figure 1E). Meanwhile, there has no obvious absorption peak in the UV–Vis region for Pt NPs, which is also compatible with the results of previous studies. Doping MXene with Pt NPs resulted in a significantly higher absorbance at 355 nm (the working wavelength of the LDI MS laser) than that of MXene, which indicated that Pt@MXene can be applied for LDI MS spectrometer and have the potentials to effectively absorb laser energy and improve the desorption/ionization efficiency of samples. The absorption peak of Pt@MXene appeared at a slightly higher wavelength (290 nm) than that of MXene, may be corresponding to the coupling between Pt NPs and MXene according to previous reports. These results prove that Pt@MXene has superior light-harvesting ability to MXene and can effectively absorb laser energy to promote the desorption/ionization process. The X-ray diffraction (XRD) patterns of Pt@MXene and MXene are shown in Figure 1F. From XRD analysis results, the (002) diffraction peak of Ti3AlC2, MXene, and Pt@MXene exists obvious difference. The (002) diffraction peak of MXene broadened and shifted toward the lower 2 theta angle from 9.5° to 6.3° compared to the Ti3AlC2, which because of the removal of the Al layer from Ti3AlC2 resulting in a widened d-spacing of MXene sheets. After the deposition of Pt NPs on MXene, the (002) peak appeared at a lower angle than in the original MXene pattern, which implies a further structural expansion of the MXene sheets. The peaks at 2θ = 39.7°, 46.2°, 67.5°, and 81.4° correspond to the (111), (200), (220), and (311) crystal planes of Pt, respectively, and are the same as those previously reported, confirming that Pt@MXene was successfully prepared. The functional groups of the Pt@MXene composite were characterized by Fourier transform infrared spectra (FT-IR). Figure 1G shows the FT-IR spectra, in which the peaks at 3369, 1620, 801, and 639 cm−1 were attributed to O-H, C=O, C-F, and Ti-O stretching vibrations, respectively.,,, Pt@MXene has basically the same functional groups as MXene, possibly because doping with Pt NPs forms a new nano hybrid without changing the surface structure of MXene. The design of the metal/MXene structure is essential for LDI MS. MXene nanosheets provide loading sites for Pt NPs because of the abundant functional groups (-O,-OH, and-F) and defects/vacancies on the surface. After a self-reduction reaction, [PtCl6]2- can be reduced to Pt NPs on MXene and be firmly adsorbed to the interlayer and surface of MXene sheets by electrostatic interactions, in which MXene functioned as the reducing agent. X-ray photoelectron spectroscopy (XPS) was performed to further confirm the elemental composition and valence state of the Pt@MXene. The survey spectra presented in Figure 1H shows the presence of F, O, Ti, and C elements in Pt@MXene. This result confirms the presence of MXene in the nanocomposite. A characteristic elemental Pt peak was observed in the Pt@MXene spectrum (Figure 1H). In the high-resolution XPS spectrum of Pt 4f (Figure 1I), the binding energies of 70.8 and 74.0 eV correspond to Pt0 4f7/2 and 4f5/2 orbitals, respectively, which demonstrate the successfully reduction of [PtCl6]2- to Pt NPs. Moreover, the binding energies of 71.9 and 74.8 eV correspond to oxidized PtOx,, which accounted for approximately half of the Pt0 in the material. The appearance of this peak was attributed to the formation of Ti-Pt and C-Pt coordination bonds and showed that Pt NPs were effectively doped in situ on the MXene nanosheets, which was consistent with the TEM results. In addition to the peaks of the above-mentioned elements, TiO2 2p1/2, Ti3+ 2p1/2, TiO2 2p3/2, Ti3+ 2p3/2, and TiO2 satellite peaks appeared (Figure 1J) in the Pt@MXene spectrum. The binding-energy peak of Ti3+ appeared at a slightly higher position in the Pt@MXene spectrum than in the MXene spectrum, consistent with the previous ref. 32. It has been reported that the shifting of the binding energy of elements may be because of interactions or electron exchange between different elements. Therefore, it is presumed that the binding energy shift of Ti3+ in Pt@MXene may be caused by the interaction or partial electron transfer between Pt and Ti. Thus, Pt@MXene is a favorable laser energy receptor for the desorption/ionization process. Compared with some popular high-performance inorganic materials, such as titanium nanosheets, non-metallic semiconductors (black phosphorus,, selenium), and composite hydrogel materials,, Pt@MXene has a maximum UV absorption peak position that better matches the operational laser wavelength, richer surface functional groups, and better stability.

The matrix/substrate concentration is an important factor affecting the efficacy of analyte desorption/ionization in LDI MS. We first evaluated the optimal concentration of Pt@MXene using natural small molecules (phenylalanine (Phe), abscisic acid (ABA), GP, TS). Pt@MXene aqueous dispersions of seven concentrations (0.01 mg·mL−1, 0.1 mg·mL−1, 0.5 mg·mL−1, 1 mg·mL−1, 1.5 mg·mL−1, 2 mg·mL−1 and 5 mg·mL−1) were used for SALDI MS to detect the target ions. The highest MS signal intensity was obtained at concentrations of 1.0 mg·mL−1 and 1.5 mg·mL−1 Pt@MXene aqueous dispersion (Figure S1), and there was no significant difference (p> 0.05) in the signal response obtained at these two concentrations. To obtain the best detection results and save costs, 1 mg·mL−1 was chosen as the Pt@MXene concentration for subsequent experiments. Next, the performance of Pt@MXene as a substrate was evaluated using natural small-molecule model analytes (amino acids and phytohormones). We performed a comparative study of the desorption/ionization efficiency of Pt@MXene against different substrates (MXene and GO) and an organic matrix (CHCA). GO is a carbon material that is considered an effective substrate because of its good thermoelectric properties and clean background in LDI MS detection of natural small molecules (such as amino acids, saccharides, lipids, and biogenic amines). Thus, the effects of using GO and Pt@MXene as substrates in SALDI MS were compared. Clearly lower background noise interference was obtained by using Pt@MXene than by using MXene, GO, and CHCA matrix (Figure S2). Figures 2A and 2B show that Pt@MXene exhibited superior performance to MXene, GO, and CHCA for the detection of small molecules. All 6 amino acids were detected with high intensity by using Pt@MXene, whereas MXene and GO could only detect all amino acids with relatively low intensity (Figure 2A). All 7 phytohormones were detected by using Pt@MXene (Figure 2B). However, only 6 phytohormones were detected using MXene, and only 4 phytohormones were detected using GO. The complex background interference of the organic matrices significantly reduced the detection efficiency. Only 4 amino acids and 5 phytohormones were detected using CHCA. More analyte signal peaks could be identified by using Pt@MXene than by using the other substrates and matrix, which may facilitate preliminary identification of a target (Figure 2C). The superior SALDI MS efficiency of Pt@MXene is mainly ascribed to its synergetic effect, exhibit excellent electron conductivity and efficient catalytic properties., Strong electronic coupling between Pt NPs and MXene enhances the efficiency of separation of electrons and holes., Under laser irradiation, the metal NPs exhibit unique localized surface plasma resonance properties. Pt NPs act as an antenna to directly convert absorbed laser energy into hot carriers and promote rapid charge transfer., Thus, the Pt@MXene offered a stronger desorption/ionization efficiency of analytes. The applicability of Pt@MXene to the detection of different small-molecule natural products, including carbohydrates (glucose, sucrose, and maltose), organic acids (succinic acid, malic acid, and ascorbic acid), alkaloids (strychnine, matrine, and berberine), and other products (isoquercitrin, loganin, and stearic acid), by SALDI MS in positive ion mode was tested (Figures 3 and S3). As expected, Pt@MXene exhibited a higher LDI efficiency than MXene, GO, and CHCA. The highest signal intensity for all the analytes was obtained by using Pt@MXene compared to only a few low-abundance signals using MXene and GO and no alkaloid and stearic acid signals using CHCA. Overall, Pt@MXene is an effective substrate for SALDI MS analysis of small-molecule natural products because it provides relatively low background noise, high detection sensitivity, wide molecular coverage, and enhanced the desorption/ionization efficiency of analytes.

Complex biological samples often contain salt ions or protein molecules that may interfere with the desorption/ionization of the analytes. Therefore, it is essential for a substrate to have good salt and protein tolerance. The anti-interference ability of Pt@MXene to salt ions and proteins was investigated by simulating an environment with a high concentration of salt and protein. A representative analyte (ABA) was used as the analysis model for protein tolerance and repeatability tests. Figure 4A shows that for the sample with an added salt solution (0.5 M NaCl and 0.5 M KCl), high-intensity target signal peaks were still obtained by using Pt@MXene. The [M + K]+ peak was significantly suppressed in the high-concentration protein solution, but a high-intensity [M + Na]+ peak was clearly observed. Hence, the Pt@MXene substrate has good salt and protein tolerance that can be used to enhance LDI MS analysis of complex biological samples. The repeatability of Pt@MXene was investigated by using standard samples of two concentrations (5 ng·μL−1 and 15 ng·μL−1) at 8 different positions on the same substrate (intraspot points) and 8 different substrates (interspot points) (Figures 4B and 4C). The results showed no significant differences in the signal-to-noise ratios (S/N) of the analytes at the intraspot and interspot points, and all the corresponding coefficients of variation (CVs) were less than 8%. This result showed that using the Pt@MXene substrate enhanced the signal reproducibility. Next, the response of the Pt@MXene substrate to low concentration samples was verified. GP and TS were used as analytical models to investigate the limit of detection (LOD) (S/N = 3) and the limit of quantitation (LOQ) (S/N = 10). Analytes were deposited on the substrate and serially diluted until S/N = 3 and S/N = 10. The LOD and LOQ of GP and TS were as follows: GP LOD = 0.5 ng·μL−1, LOQ = 1 ng·μL−1; TS LOD = 0.1 ng·μL−1, and LOQ = 0.2 ng·μL−1. The proposed approach exhibits higher sensitivity than some conventional liquid chromatography., Note that existing analytical methods with high stability and precision are usually time-consuming and require the use of large quantities of solvents and complex operating procedures.,, By contrast, Pt@MXene-assisted LDI MS has the advantages of facile operation, the use of a low solvent volume (only 1 μL), rapid analysis (only seconds per sample), and no derivatization required.

Many studies have shown that the secondary metabolites of medicinal plants are extremely valuable. GP is a major iridoid compound in Gentianaceae herbs. It has been proven that GP has anti-inflammatory and gastric acid secretion-promoting biological activities. In addition, Labiatae plants are widely planted worldwide. S. miltiorrhiza is a representative species with high medicinal value. S. miltiorrhiza is rich in tanshinone compounds and has been used for a long period to treat blood diseases. Therefore, qualitative and quantitative analyses of GP and TS in medicinal plants are necessary. Currently, the organic matrix and sample mixture often form non-uniform crystals because of the solvent effect. This phenomenon results in uneven laser energy during analysis or ionization and desorption processes. Therefore, quantification by MALDI MS is unsatisfactory. To solve these problems, an internal-standard quantitative method based on a Pt@MXene substrate was developed in this study to quantify GP in G. scabra and TS in S. miltiorrhiza. Here, vitamin A acid (VAA) was chosen as the internal standard (IS) because it has stable chemical properties and high signal peak intensity with no interferences from other analytes. Samples at different concentrations were mixed with equal volumes of VAA solution for detection. Figure S4 shows the characteristic signal peaks for GP ([M + Na]+, m/z 379.1; [M + K]+, m/z 395.2) and TS ([M + Na]+, m/z 317.1; [M + K]+, m/z 333.2) in the mass spectra. The sum of the peak areas of [M + Na]+ and [M + K]+ was selected for the test sample to objectively include all ionic behaviors and simulate the detection scenario of real samples. The calibration curves were linearly regressed using the ratio of the peak areas of the sample and the IS as the Y axis and the sample concentration as the X axis. In Figures 5A and 5B, the detected characteristic signal peaks of [M + Na]+ and [M + K]+ of selected analytes are marked with asterisks, and detailed information for the MS peaks is shown in Table S1. The following standard curve was obtained using Pt@MXene: GP (R2 = 0.9954, 25–400 ng·μL−1, Y = 0.0164X+0.0852) for an IS concentration of 20 ng·μL−1; TS (R2 = 0.9956, 15–100 ng·μL−1, Y = 0.257X+0.333) for an IS concentration of 30 ng·μL−1 (Figures 5A and 5B). The good linear relationship between the peak area ratio (of GP and TS) and the sample concentration (R2> 0.99) proved the effectiveness of this method for quantification. Next, the practicability of the method was further assayed by analysis of the content of small-molecule natural products in two medicinal plants (G. scabra and S. miltiorrhiza). The original mass spectra of the two medicinal plants were shown in Figures 5C and 5D. The results were shown in Table 1, the content of the target compounds determined by SALDI MS was almost consistent with the HPLC detection results (Figures S7–S9). This result demonstrated that Pt@MXene platform can be used to obtain accurate quantitative (relative error <3%) information on natural products in medicinal plants. LDI-TOF/TOF MS/MS was used to confirm the structure of the target analytes (Figures S5 and S6), and information on all the MS peaks is presented in Tables S2 and S3.

In this study, a Pt@MXene nanocomposite was successfully prepared by a one-step reduction method. This material is an ideal SALDI MS substrate for the analysis of small-molecule natural products in positive mode. The structural properties, elemental composition, and valence state of Pt@MXene were characterized by UV‒Vis, TEM, XRD, XPS, and FT-IR. Compared with a traditional organic matrix (CHCA) and two nanosubstrates (MXene and GO), Pt@MXene exhibited the lowest background, the strongest signal peak intensity, and the widest molecular detection coverage. Moreover, Pt@MXene showed excellent salt and protein tolerance, good repeatability, and high detection sensitivity. This substrate was used in conjunction with the internal standard method to yield an accurate content of target molecules in medicinal plants, including GP in G. scabra and TS in S. miltiorrhiza. Hence, this method has high application potential for the rapid and accurate detection of small-molecule natural products and serves as a valuable reference for the design of other high-performance substrates for SALDI MS.

An efficient Pt@MXene was synthesized as a substrate for high-throughput detection and quantification of natural small molecules by SALDI MS in this study. However, more biosamples need to be evaluated to determine the applicability of this substrate to the detection of natural small molecules in different types of complex samples (such as body fluids, organs, or microbial fermentation products).



Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Chunxia Ma (machunxia8927@163.com).

This study did not generate new unique reagents.

This study does not involve animal and cellular experiments.

H2PtCl6·6H2O and CHCA (≥99%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Pt nanoparticle was obtained from Hangzhou Nano-Mall Technology. Serine (Ser), glutamic acid (Glu), histidine (His), phenylalanine (Phe), arginine (Arg), and tryptophan (Trp) were purchased from the National Institutes for Food and Drug Control (Beijing, China). 3-Indoleacetic acid (IAA), trans-zeatin (t-ZT), jasmonic acid (JA), methyl jasmonate (MeJA), abscisic acid (ABA), strigolactone (SL), trans-zeatin-riboside (t-ZR) and vitamin A acid (VAA) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). All other standard samples were HPLC grade and purchased from Chengdu DeSiTe Biological Technology Co. Ltd. (Chengdu, China). Single/few-layer MXene powders and graphene oxide (GO) were obtained from Beike 2D Materials Co., Ltd. (Jiangsu, China). Chromatographic grade methanol (MeOH), acetonitrile (ACN), ethanol absolute (EtOH), and trifluoroacetic acid (TFA) were purchased from Merck (Darmstadt, Germany). Deionized water was obtained from a Milli-Q ultrapure water system in the laboratory (Millipore Corporation, Bedford, MA, USA). G. scabra was obtained from a pharmacy in China (Batch No: JN-SYPM-20220427001, Jinan, Shandong). S. miltiorrhiz was obtained from the planting base of medicinal plants in China (Laiwu, Shandong).

Single/few-layer MXene was prepared according to our previously reported method. Firstly, 0.67 g of LiF powder was mixed with HCl (6 moL·mL−1, 10 mL), and 0.1 g of Ti3AlC2 power was slowly added to the above solution. The obtained mixture was stirred continuously at room temperature for 24 h. The reaction produce was washed by multiple centrifugation (3500 rpm, 5 min) until the solution pH > 6. To obtain single/few-layer MXene, the precipitate was dispersed in 100 mL of deionized water and then ultrasonicated (500 W) for 3 h in argon atmosphere. Finally, the supernatant was collected by centrifugation (3500 rpm) for 1 h, followed by freeze-drying. Pt@MXene was synthesized by a one-step reduction method according to previous reports., In this study, MXene promoted the spontaneous reduction of H2PtCl6·6H2O to Pt NPs. Finally, Pt nanoparticles were anchored on the surface of MXene nanosheets. Specifically, 10 mg of single/few-layer MXene powder were added to 20 mL of deionized water, and the sample solution was then ultrasonicated at 400 W for 20 min. Subsequently, a H2PtCl6·6H2O (1 mg·mL-1, 1.33 mL) solution was slowly added to the MXene solution under continuous stirring. The entire reaction was carried out for 6 hours in the dark. After the reduction reaction was completed, the resulting reaction mixtures were collected by centrifugation (9000 rpm, 5 min) and purified by washing with water three times. Finally, the composite Pt@MXene powder was obtained by the freeze-drying method. The proportion of platinum in the Pt@MXene composite was determined to be 5% by a weight calculation.

UV–Vis absorption spectra of Pt@MXene and MXene at the same concentration were recorded by a UV-2700 (Shimadzu, Japan) absorption spectrophotometer. Data were collected in the visible region between 200∼800 nm. Sample solutions (5 mg·mL-1, 1 mL) were prepared for X-ray diffraction (XRD) analysis by deposition on a 25 mm×25 mm transparent glass plate followed by drying. The crystal structures of the samples were determined using a D8 Advance (Bruker Daltonik GmbH, Germany) in the 2θ range of 5°∼90° (scanning speed 4°·min-1). Fourier transform infrared spectra (FT-IR) were obtained using a Vertex 70v (Bruker Daltonik GmbH, Germany), and the sample was prepared according to a reported KBr method. Briefly, Pt@MXene (2 mg) and KBr (300 mg) powders were homogeneously mixed, ground well and placed in a tablet press to prepare samples for analysis. X-ray photoelectron spectroscopy (XPS) was performed by using a ESCALAB Xi+ (Thermo Fisher Scientific, USA) with a mono Alka X-ray source at a voltage of 1486.6 eV. The sample solution (5 mg·mL-1) was dropped on the surface of a silicon wafer for subsequent analysis. 10 μL of the Pt@MXene or MXene solution were dropped onto a square silicon chip with dimensions of 5 mm × 5 mm and dried in a vacuum at room temperature. The aforementioned operation was repeated in triplicate. The obtained data were analyzed using Thermo Avantage v5.976 software (Thermo Fisher Scientific, USA), and the binding energies were calibrated using the C 1s peak at 284.8 eV corresponding to graphitic carbon. Transmission electron microscopy (TEM), high-angle annular dark field-scanning transmission electron microscopy (HAADF-STEM), and energy-dispersive X-ray spectroscopy (EDS) mapping were performed using a Talos™ F200s instrument (Thermo Fisher Scientific, USA). The Pt@MXene powder was dispersed in EtOH and dropped onto a carbon-coated Cu grid, followed by drying at room temperature for 12 h.

Solutions of six amino acids (Ser, Glu, His, Phe, Arg, and Trp) were prepared in deionized water at a concentration of 100 ng·μL-1. Solutions of seven phytohormones (IAA, t-ZT, JA, MeJA, ABA, SL, and t-ZR) were prepared in MeOH at a concentration of 100 ng·μL-1. The following analytes were dissolved in appropriate solvents to produce 1 mg·mL-1 solutions: organic acids (succinic acid and malic acid, dissolved in MeOH and ascorbic acid, dissolved in pure water), saccharides (glucose, maltose, and sucrose, dissolved in pure water), alkaloids (matrine, berberine, and strychnine, dissolved in MeOH), and other analytes (loganin, isoquercitrin, and stearic acid, dissolved in MeOH). GP (1 mg·mL-1), TS (1 mg·mL-1), and VAA (40 and 60 ng·μL-1) standard solutions were prepared in MeOH. A 1 mg·mL-1ABA standard was prepared in MeOH/water (1:1, v/v) and mixed with an equal volume of a salt solution (NaCl and KCl, 1 M) or protein (BSA, 10 mg·mL-1) to investigate the detection efficiency for complex samples. Pt@MXene powder was dispersed in deionized water at a concentration of 5 mg·mL-1. The 1 mg·mL-1 graphene oxide (GO) and MXene dispersion were prepared in deionized water, respectively. CHCA was prepared at a concentration of 1 mg·mL-1 in 0.1% TFA buffer (ACN/water, 70:30, v/v). The ionization efficiencies of different nanomaterials and organic matrices were compared based on the signal intensity of the target peaks.

In this experiment, natural products from two medicinal plants, G. scabra and S. miltiorrhiza, were extracted using ultrasonic-assisted extraction. In brief, 1 g of the crude powder of each medicinal material and 10 mL of EtOH were mixed in a conical flask and left at room temperature for 1 hour. Next, the mixture was ultrasonicated at 15 °C for 1 h. The output power of the ultrasonic machine was set at 300 W. The extracts were filtered through a 0.22-μm membrane before analysis. The internal standard (IS) method was used for the quantitative analysis of the metabolites. The internal standard solution (40 ng·μL-1 and 60 ng·μL-1 VAA (IS)) was added to the extracts of G. scabra and S. miltiorrhiza in a 1:1 volume ratio, respectively. Finally, a standard curve was generated by plotting the ratio of the sum of the peak areas of the target to that of the IS as the ordinate and the target concentration as the abscissa. The final experimental result was taken as the average measurement of 10 repeated tests.

The external standard method was used to quantify single compounds (GP and TS) in G. scabra and S. miltiorrhiza. A Waters e2695 HPLC instrument with a diode array detector (DAD) and reverse-phase Symmetry C-18 (Waters) column (250 mm × 4.6 mm i.d., 5 μm) was used at 25 °C. Gradient elution was employed using solution A (0.1% formic acid in water) and solution B (ACN) as mobile phases. The LC gradient elution conditions were referred to and improved from previously reported., Among them, the HPLC analysis conditions of GP were as follows: 0∼1 min, 90%∼85% A; 1∼10 min, 85%∼70% A; 10∼15 min, 70%∼90% A; and 15.5∼20 min, 90% A, at a flow rate of 1 mL·min-1. The UV‒Vis wavelength of the detector was set at 270 nm, and the sample injection volume was 3 μL; TS was analyzed according to the following procedure: 0∼6 min, 39% A; 6∼20 min, 39%∼10% A; 20∼25 min, 10% A; 25∼25.5 min, 10%∼39% A; and 25.5∼30 min, 39% A, at a flow rate of 1 mL·min-1. Detection was performed at a maximum absorption wavelength of 270 nm, and the sample injection volume was 3 μL. Two analyte standard solutions were determined under the above mentioned chromatographic conditions. A quantitative linear relationship was established between the standard concentration and peak area for GP and TS. The extracts of the medicinal materials were determined under the same chromatographic conditions. The actual content of the metabolites in the samples was calculated from the average peak area and regression equation.

Mass spectrometry was performed on a rapifleX MALDI TissuetyperTM TOF/TOF MS (Bruker Daltonics, Billerica, MA) equipped with a 355-nm SmartBeam II laser. The original spectrum was obtained in positive reflection mode, the molecular weight detection range was 100∼1000 Da, and the global attenuator shift was adjusted to 15%. The mass spectra were superimposed by 200 laser shots at a frequency of 5000 Hz. The laser energy was adjusted to 45%∼60%. MALDI LIFT-TOF/TOF MS/MS operated at a pulsed voltage of 2.65 kV was used for in situ tandem MS/MS analysis. For LDI MS detection, 1 μL of the nanomaterial was deposited onto a stainless-steel sample target plate, and the solvent was dried in a vacuum at 25 °C. Next, 1 μL of the sample solution was placed on the dried substrate on the target plate. Samples in the CHCA matrix were prepared using the reverse order presented above, that is, the sample was first dropped on the substrate and then covered. The original mass spectrogram was imported into Flexanalysis version 4.0 (Build 9) software (Bruker Daltonics) for peak screening and baseline calibration.

Substrates are synthesized in a well-controlled environment and no harmful chemicals are used throughout the process. All synthesis processes were carried out in controlled acid-resistant fume hoods. Vacuum drying was performed for 5∼20 minutes after the substrate was deposited on the steel target to prevent impurities from entering the mass spectrometer. The proper use of all instrumentation ensures that experimenter is not at risk.

The data were statistically analyzed, including determining the standard deviation (S.D.), one-way analysis of variance (ANOVA), and performing the t test, Duncan’s test, and Tukey’s test using SPSS (Version 21.0 Armonk, NY, USA). p< 0.05 was considered statistically significant.