Application of Cu2O and Au heterogeneous composite nanomaterial as SALDI matrix in small molecule substance detection

Abstract

The invention discloses an application of a Cu2O and Au heterogeneous composite nanomaterial as an SALDI (selective absorption laser desorption ionization) matrix in detection of small molecular substances. The preparation method comprises the following steps: by taking CuCl2. 2H2O as a raw material, regulating and controlling through PVP (Polyvinyl Pyrrolidone), and synthesizing Cu2O with a truncated regular octahedral structure; based on the reducibility of Cu2O, regulation and control are carried out through PVP, in-situ reduction of gold nanoparticles is achieved, and the Cu2O and Au heterogeneous composite nanomaterial is obtained. When the Cu2O and Au heterogeneous composite nanomaterial is used as a matrix for analyzing an analyte, the composite nanomaterial has multiple advantages of high desorption ionization efficiency, excellent salt tolerance and good universal detection capability for metabolic molecules such as amino acid and saccharides. Compared with a traditional organic matrix, the matrix has a clean and simple background peak, greatly reduces interference, especially has excellent performance in saccharide molecule detection, and is a potential SALDI nano material matrix.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry detection, and in particular to an application of a Cu2O@Au heterogeneous composite nanomaterial as a SALDI matrix in the detection of small molecule substances. Background Art

[0002] Matrix-assisted laser desorption ionization (MALDI) is a well-known soft ionization technique that is very popular for analyzing proteins, peptides, and other large biomolecules in biological samples. MALDI's advantages, such as high ionization efficiency, high sensitivity, simple sample preparation, and high tolerance to salts and common chemical buffer components, have led to its wide application in biology, genetics, food chemistry, medical research, and clinical diagnostics.

[0003] While MALDI-TOF MS offers numerous advantages, organic matrices can strongly interfere with detection results in the molecular weight region (m / z <700 Da). Furthermore, the matrix and analyte crystallization are heterogeneous, impacting both reproducibility and quantitation. Consequently, a new technique, surface-assisted laser desorption ionization (SALDI), has been developed. Metal oxides, due to their unique electronic structure and chemical stability, have shown great potential as SALDI matrices. They not only provide a stable chemical environment but also enhance the ionization of small molecules through their surface properties. Precious metal nanomaterials, such as gold (Au) and silver (Ag) NPs, are used as matrices due to their unique optical and electronic properties. The surface plasmon resonance (SPR) effect of these nanomaterials can significantly enhance the interaction between the laser and the sample, thereby improving ionization efficiency. Furthermore, the high chemical stability and biocompatibility of precious metal nanomaterials make them ideal candidates for SALDI matrices.

[0004] Therefore, the green in situ method is used to synthesize Cu2O@Au heterogeneous composite nanomaterials, which does not require a reducing agent, has a fast reaction time, and improves the desorption ionization efficiency and heat transfer efficiency, excellent salt tolerance, and extremely high reproducibility, broadening its detection types and fully combining the advantages of Cu2O and Au NPs on the matrix. Summary of the Invention

[0005] To address the technical challenges faced by metal oxide nanomaterials in laser desorption ionization mass spectrometry (SALDI-MS) applications, this paper proposes an innovative heterogeneous composite nanomaterial solution: the Cu2O@Au heterogeneous composite nanomaterial. This novel material aims to address the limitations of Cu2O in detecting a limited number of analytes, as well as low heat transfer and desorption ionization efficiencies. This paper comprehensively describes the preparation method of the Cu2O@Au heterogeneous composite nanomaterial and explores its potential application as a SALDI matrix for small molecule detection. The introduction of this novel material is expected to significantly improve the accuracy, reproducibility, and detection efficiency of SALDI-MS analysis, paving a new path for the development of mass spectrometry technology.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides the following technical solutions: The present invention provides an application of a Cu2O@Au heterogeneous composite nanomaterial as a SALDI matrix.

[0007] Furthermore, the Cu2O@Au heterogeneous composite nanomaterial is Au nanoparticles uniformly loaded on the surface of Cu2O to form a core-shell structure; the particle size of the Au nanoparticles is about 1.5-2.5 nm, the Cu2O is a truncated octahedral structure, and the mass percentage of the Au nanoparticles to Cu2O is 0.017%-0.09%.

[0008] Furthermore, the preparation method of the above-mentioned Cu2O@Au heterogeneous composite nanomaterial comprises the following steps: Cu2O was uniformly dispersed in a PVP aqueous solution, and a chloroauric acid aqueous solution was added. After stirring evenly, the mixture was allowed to stand for reaction, and then washed and dried to obtain a Cu2O@Au heterogeneous composite nanomaterial.

[0009] Cu2O has reducing properties, and the reducing properties of Cu2O itself are used to prepare gold nanoparticles. Through the regulation of PVP, Au NPs are evenly attached to the surface of Cu2O.

[0010] Wherein, the Cu2O is prepared by the following method: (1) Add polyvinyl pyrrolidone (PVP-K30) to the CuCl2·2H2O aqueous solution and stir evenly at room temperature to obtain a mixed solution.

[0011] The mass ratio of PVP-K30 to CuCl2·2H2O is (10-30):1, preferably 20:1; The concentration of PVP-K30 in the mixed solution is 30-40 mg / mL.

[0012] (2) Add NaOH aqueous solution dropwise to the above solution and stir at 600 rpm for 30 min.

[0013] The concentration of the NaOH aqueous solution is 2 M, and the volume ratio of the NaOH aqueous solution to the mixed solution is 1:10.

[0014] (3) Add ascorbic acid solution dropwise to the solution of step (2), age for 3 h, wash with distilled water 3 times, wash with anhydrous ethanol 3 times, and finally dry in a vacuum at 60°C for 6 h to obtain Cu2O with a truncated octahedral structure.

[0015] The molar ratio of CuCl2·2H2O, NaOH and ascorbic acid is 1:20:6, and the volume ratio of the ascorbic acid solution to NaOH is 1:1.

[0016] In the technical solution of the above-mentioned method for preparing Cu2O@Au heterogeneous composite nanomaterials, the average molecular weight of PVP is between 30,000 and 50,000; preferably, the average molecular weight of PVP is 40,000.

[0017] The technical solution of the preparation method of the above-mentioned Cu2O@Au heterogeneous composite nanomaterial is preferably: the mass ratio of Cu2O to PVP is 6.5:(50-200); more preferably, the mass ratio of Cu2O to PVP is 6.5:100; and the mass volume concentration of PVP is 1% w / v.

[0018] In the technical solution of the preparation method of the above-mentioned Cu2O@Au heterogeneous composite nanomaterial, the mass ratio of Cu2O to HAuCl4 is 6.5:(0.002-0.01); more preferably, the mass ratio of Cu2O to HAuCl4 is 6.5:0.01; the mass volume concentration of chloroauric acid is preferably 0.02% w / v.

[0019] Furthermore, the present invention provides the use of Cu2O@Au heterogeneous composite nanomaterials as SALDI-MS matrix in the detection of small molecule substances.

[0020] The molecular weight of the small molecule substance is m / z < 700 Da, preferably m / z < 500 Da.

[0021] The small molecule substances include sugars and amino acids. The small molecule sugar compounds include but are not limited to lactose, galactose, raffinose, fructose, glucose, maltose, sucrose, etc., and the small molecule amino acid compounds include but are not limited to lysine, aspartic acid, histidine, threonine, alanine, valine, arginine, serine, asparagine, glutamic acid, glutamine, tryptophan, cysteine, etc.

[0022] The present invention also provides the application of Cu2O@Au heterogeneous composite nanomaterials as a matrix in the fields of mass spectrometry imaging, metabolomics, etc.

[0023] As described above, the Cu2O@Au heterogeneous composite nanomaterial, its preparation method, and its use as a SALDI matrix in the detection of metabolic small molecules of the present invention have the following beneficial effects: The present invention adds PVP during the synthesis of Cu2O, and uses PVP to regulate the shape and size of its nanoparticles, ultimately obtaining truncated octahedral Cu2O nanoparticles. Utilizing the reducing property of Cu2O, without the need to add a separate reducing agent, HAuCl4 is greenly reduced in situ on the Cu2O nanoparticles to form gold nanoparticles, and the particle size of the gold nanoparticles is regulated by PVP, ultimately forming a Cu2O@Au heterogeneous composite nanomaterial uniformly loaded with gold nanoparticles. By regulating the loading amount of gold nanoparticles, the optical absorption of the Cu2O@Au heterogeneous composite nanomaterial is significantly increased, and it possesses excellent photothermal and photoelectric conversion capabilities, greatly improving its desorption and ionization efficiency. When the present invention uses the Cu2O@Au heterogeneous composite nanomaterial with truncated octahedrons as a matrix to analyze analytes, it exhibits multiple advantages: high desorption and ionization efficiency, excellent salt tolerance, and good universal detection capabilities for metabolic molecules such as amino acids and sugars. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 SEM images of Cu2O and Cu2O@Au prepared in Examples 1-6 of the present invention.

[0025] Figure 2 This is the SEM image of Cu2O@Au prepared in Example 7 of the present invention.

[0026] Figure 3 The ultraviolet absorption spectra of Cu2O and Cu2O@Au prepared in Examples 1-6 of the present invention.

[0027] Figure 4 Laser threshold spectra of Cu2O and Cu2O@Au prepared in Examples 1-6 of the present invention for detecting 4-C-BP.

[0028] Figure 5 SY value diagram of Cu2O and Cu2O@Au prepared in Examples 1-6 of the present invention for detecting 4-C-BP.

[0029] Figure 6 The mass spectra of Cu2O and Cu2O@Au prepared in Examples 1-6 of the present invention detecting sodium tetraphenylborate.

[0030] Figure 7The mass spectra of 13 amino acids detected in the Cu2O@Au heterogeneous composite nanomaterial matrix prepared in Example 6 of the present invention are lysine, aspartic acid, histidine, threonine, alanine, valine, arginine, serine, asparagine, glutamic acid, glutamine, tryptophan, and cysteine, and the amino acid concentration is 0.01 mol / L.

[0031] Figure 8 The mass spectra of seven sugars detected by the Cu2O@Au heterogeneous composite nanomaterial matrix prepared in Example 6 of the present invention are respectively analyzed for lactose, galactose, raffinose, fructose, glucose, maltose, and sucrose; wherein M is the corresponding analyte, and the analyte concentration is 0.01 mol / L.

[0032] Figure 9 The mass spectra of the Cu2O@Au heterogeneous composite nanomaterial matrix prepared in Example 6 of the present invention were detected under high salt concentrations; the salt concentrations were 10 mM, 100 mM, and 1000 mM; and the concentrations of histidine and glucose were 0.001 mol / L.

[0033] Figure 10 This is the relative standard deviation (RSD) graph of glucose and amino acids detected by the Cu2O@Au heterogeneous composite nanomaterial matrix prepared in Example 6 of the present invention. DETAILED DESCRIPTION

[0034] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Example 1

[0035] A Cu2O material is prepared by the following method: (1) Add 3.4 g of polyvinylpyrrolidone (PVP-K30) to 100 mL of CuCl2·2H2O aqueous solution (10 mM) and stir at room temperature to obtain a mixed solution; (2) Add 10.0 mL of 2 M NaOH aqueous solution dropwise to the above solution and stir at 600 rpm for 30 min. (3) Add 10.0 mL of ascorbic acid solution (0.6 M) dropwise to the dark brown solution and age for 3 h; (4) Wash with distilled water three times, then with anhydrous ethanol three times, and finally dry in a vacuum at 60°C for 6 h to obtain a brick-red powder. Example 2

[0036] A Cu2O@Au heterogeneous composite nanomaterial is prepared by the following method: (1) The preparation method of Cu2O is the same as that of Example 1; (2) 6.5 mg of Cu2O was added to 10 mL of PVP (1% w / v) aqueous solution; (3) Add 1 mL of HAuCl4 (0.02% w / v) aqueous solution, stir for 2 min, and then let it stand for 10 min; (4) Centrifuge in a high-speed centrifuge (9000 rpm), and then wash with water and ethanol three times to obtain Cu2O@Au-1. Example 3

[0037] A Cu2O@Au heterogeneous composite nanomaterial is prepared by the following method: (1) The preparation method of Cu2O is the same as that of Example 1; (2) 6.5 mg of Cu2O was added to 10 mL of PVP (1% w / v) aqueous solution; (3) Add 2 mL of HAuCl4 (0.02% w / v) aqueous solution, stir for 2 min, and then let it stand for 10 min; (4) Centrifuge in a high-speed centrifuge (9000 rpm), and then wash with water and ethanol three times to obtain Cu2O@Au-2. Example 4

[0038] A Cu2O@Au heterogeneous composite nanomaterial is prepared by the following method: (1) The preparation method of Cu2O is the same as that of Example 1; (2) 6.5 mg of Cu2O was added to 10 mL of PVP (1% w / v) aqueous solution; (3) Add 3 mL of HAuCl4 (0.02% w / v) aqueous solution, stir for 2 min, and then let it stand for 10 min; (4) Centrifuge at 9000 rpm and then wash with water and ethanol three times to obtain Cu2O@Au-3. Example 5

[0039] A Cu2O@Au heterogeneous composite nanomaterial is prepared by the following method: (1) The preparation method of Cu2O is the same as that of Example 1; (2) 6.5 mg of Cu2O was added to 10 mL of PVP (1% w / v) aqueous solution; (3) Add 4 mL of HAuCl4 (0.02% w / v) aqueous solution, stir for 2 min, and then let it stand for 10 min; (4) Centrifuge at 9000 rpm and then wash with water and ethanol three times to obtain Cu2O@Au-4. Example 6

[0040] A Cu2O@Au heterogeneous composite nanomaterial is prepared by the following method: (1) The preparation method of Cu2O is the same as that of Example 1; (2) 6.5 mg of Cu2O was added to 10 mL of PVP (1% w / v) aqueous solution; (3) Add 5 mL of HAuCl4 (0.02% w / v) aqueous solution, stir for 2 min, and then let it stand for 10 min; (4) Centrifuge at 9000 rpm and then wash with water and ethanol three times to obtain Cu2O@Au-5. Example 7

[0041] A Cu2O@Au heterogeneous composite nanomaterial is prepared by the following method: (1) The preparation method of Cu2O is the same as that of Example 1; (2) 6.5 mg of Cu2O was added to 10 mL of aqueous solution; (3) Add 5 mL of HAuCl4 (0.02% w / v) aqueous solution, stir for 2 min, and then let it stand for 10 min; (4) Centrifuge in a high-speed centrifuge (9000 rpm), and then wash with water and ethanol three times to obtain Cu2O@Au-6.

[0042] Performance characterization:

[0043] 1. Take SEM photos of the Cu2O and Cu2O@Au heterogeneous composite nanomaterials prepared in Examples 1-6, respectively. Figure 1 As shown in the figure, pure Cu2O presents a regular polyhedral structure with a smooth surface and clear edges, indicating its good crystal structure and high crystallinity. As the amount of HAuCl4 added increases, the number of Au NPs on the Cu2O surface gradually increases and the distribution becomes more dense. In Cu2O@Au-1 to Cu2O@Au-3, Au NPs begin to form on the Cu2O surface but have not yet completely covered it. In Cu2O@Au-4 and Cu2O@Au-5, Au NPs almost completely cover the Cu2O surface, forming a continuous Au layer, which significantly changes the original morphology of Cu2O and forms some defects. Figure 2 The scanning electron microscopy image of Cu2O@Au NPs without PVP is shown in the figure. It can be seen that the AuNPs are unevenly distributed and have a large particle size. However, after adding PVP, the Figure 1, with small particle size and uniformly distributed on the surface of Cu2O.

[0044] 2. The Cu2O and Cu2O@Au heterogeneous composite nanomaterials in the process of Examples 1-6 were subjected to ultraviolet absorption spectrum analysis. Figure 3 As shown in the figure, Cu2O has a clear absorption peak at 747 nm, which is consistent with the intrinsic absorption characteristics of Cu2O. With the loading of AuNPs, the absorption spectrum of Cu2O@Au heterogeneous composite nanomaterials changes significantly, and Cu2O has a new absorption peak near 360 nm and 545 nm, which is caused by the surface plasmon resonance (SPR) effect of Au NPs.

[0045] 3. 4-(4-Chlorobenzyl)pyridine (4-C-BP), which can undergo specific fragmentation in positive ion mode to produce a stable ion signal, is used to detect the laser threshold of Cu2O and Cu2O@Au heterogeneous composite nanomaterials. Figure 4 As shown in the figure, with the addition of Au NPs, the laser threshold is significantly reduced. In Cu2O@Au-3-5, at the same laser energy of 2%, the analyte signal gradually increases, and the best matrix is ​​Cu2O@Au-5.

[0046] 4. 4-Chlorobenzyl-pyridine produces a characteristic ion fragment peak F⁺ (m / z 125) and a parent ion peak M⁺ (m / z 204) under the same laser energy conditions. The parent ion fragmentation degree SY value (defined as the ratio of the fragment ion intensity to the parent ion intensity, i.e., SY = I((fragment) / I(parent ion)) is related to the heat transfer performance of the matrix. The Cu2O@Au and Cu2O@Au heterogeneous composite nanomaterials prepared in the process of Examples 1-6 were detected by MALDI-MS instrument for 4-C-BP, and the SY value was calculated as follows: Figure 5 As shown in the figure, the addition of Au NPs significantly improved the heat transfer efficiency of the matrix, among which Cu2O@Au-3 had the best heat transfer efficiency.

[0047] 5. Sodium tetraphenylborate is an organic salt with negative charge, which is often used to evaluate the desorption efficiency of the substrate. The Cu2O@Au and Cu2O@Au heterogeneous composite nanomaterials prepared in the process of Examples 1-6 were tested with sodium tetraphenylborate in negative ion mode to evaluate the desorption efficiency. Figure 6 As shown, under standardized experimental conditions (laser energy 40%, n=5), BPh4 - The signal intensity analysis of the characteristic peak (m / z 319.1) showed that Cu2O@Au-5 had the best desorption performance.

[0048] 6. Figure 7Cu2O@Au NPs were used as the matrix to analyze 13 amino acids, including lysine, aspartic acid, histidine, threonine, alanine, valine, arginine, serine, asparagine, glutamic acid, glutamine, tryptophan, and cysteine. The concentration of the amino acids was 0.01 mol / L.

[0049] 8. Figure 8 Using Cu2O@Au as the matrix, the mass spectra of lactose, galactose, raffinose, fructose, glucose, maltose, and sucrose were analyzed respectively; where M is the corresponding analyte, and the concentration of the analyte is 0.01 mol / L.

[0050] 9. Figure 9 Using Cu2O@Au NPs as the matrix, histidine and glucose were detected at high salt concentrations of 10 mM, 100 mM, and 1000 mM, respectively. The concentrations of histidine and glucose were 0.001 mol / L.

[0051] 10. Figure 10 This is the relative standard deviation diagram of Cu2O@Au NPs as a matrix for the detection of glucose and histidine. The RSD of Cu2O@Au NPs for sugar detection is <10%, and the RSD for histidine detection is 10.28%, which shows excellent reproducibility for both.

[0052] In summary, the present invention first adjusts the particle size and shape of nanoparticles through PVP, and finally synthesizes Cu2O with uniform particle size and truncated octahedral structure, and then obtains Cu2O@Au heterogeneous composite nanomaterials through in-situ green reduction, and AuNPs are uniformly attached to the Cu2O surface. Through a series of tests, the Cu2O@Au has better heat transfer performance and laser desorption ionization efficiency. It also shows extremely high salt tolerance and extremely high reproducibility, and the detection of analyte types is richer. Compared with traditional organic matrices, its background peak is clean and concise, greatly reducing interference, especially in the detection of sugar molecules. Therefore, the present invention effectively overcomes the various shortcomings of the existing technology and has high industrial utilization value.

[0053] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. Application of a Cu2O@Au heterogeneous composite nanomaterial as a SALDI matrix, characterized in that: The Cu2O@Au heterogeneous composite nanomaterial is Au nanoparticles uniformly loaded on the surface of Cu2O to form a core-shell structure; the particle size of the Au nanoparticles is 1.5-2.5 nm, the Cu2O is a truncated octahedral structure, and the mass percentage of the Au nanoparticles to Cu2O is 0.017%-0.09%.

2. The use according to claim 1, characterized in that The Cu2O is prepared by the following method: (1) Add polyvinyl pyrrolidone to a CuCl2·2H2O aqueous solution and stir uniformly at room temperature to obtain a mixed solution; wherein the mass ratio of polyvinyl pyrrolidone to CuCl2·2H2O is (10-30):1; (2) Add the NaOH aqueous solution dropwise to the mixed solution and stir at 600 rpm for 30 min; (3) Adding the ascorbic acid solution dropwise to the solution of step (2), aging for 3 h, washing, and drying to obtain Cu2O having a truncated octahedral structure; wherein the molar ratio of CuCl2·2H2O, NaOH, and ascorbic acid is 1:20:

6.

3. The use according to claim 1, characterized in that The preparation method of the Cu2O@Au heterogeneous composite nanomaterial comprises the following steps: uniformly dispersing Cu2O in a PVP aqueous solution, adding a chloroauric acid aqueous solution, stirring evenly, standing for reaction, washing and drying to obtain the Cu2O@Au heterogeneous composite nanomaterial.

4. The use according to claim 3, characterized in that The mass ratio of Cu2O to PVP is 6.5:(50-200).

5. The use according to claim 3, characterized in that The mass ratio of Cu2O to HAuCl4 is 6.5:(0.002-0.01).

6. The use according to claim 3, characterized in that The mass volume concentration of the PVP aqueous solution is 1% w / v; the mass volume concentration of the chloroauric acid aqueous solution is 0.02% w / v.

7. The use according to claim 1, characterized in that Application of Cu2O@Au heterogeneous composite nanomaterials as SALDI matrix in the detection of small molecule compounds.

8. The use according to claim 7, characterized in that The molecular weight m / z of the small molecule compound is less than 700 Da.

9. The use according to claim 7, characterized in that The small molecule compounds include sugars and amino acids; the sugars include at least one of lactose, galactose, raffinose, fructose, glucose, maltose, and sucrose; the amino acids include at least one of lysine, aspartic acid, histidine, threonine, alanine, valine, arginine, serine, asparagine, glutamic acid, glutamine, tryptophan, and cysteine.

10. The use according to claim 1, characterized in that Application of Cu2O@Au heterogeneous composite nanomaterials as SALDI matrix in mass spectrometry imaging and metabolomics.

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