Gold nanoparticle array with strong stability and reusability and preparation and application thereof
By functionalizing the surface of gold nanoparticles and forming covalent bonds with a glass support, the stability and salt resistance issues of gold nanoparticle arrays in SALDI-MS were solved, enabling high-sensitivity and reusable small molecule detection suitable for complex sample analysis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANTOU UNIV
- Filing Date
- 2022-11-09
- Publication Date
- 2026-06-26
AI Technical Summary
In existing SALDI-MS technology, the stability and salt resistance of gold nanoparticle arrays are insufficient, resulting in unstable analytical results and severe signal fluctuations. Furthermore, background interference caused by traditional organic matrices affects the mass spectrometry detection effect.
By functionalizing the surface of gold nanoparticles with hexadecyltrimethylammonium chloride and homocysteine solution, the interparticle spacing was adjusted by Coulomb repulsion, and the nanoparticles were fixed on a glass support by forming covalent bonds with amino groups, thus forming a stable array of gold nanoparticles.
It achieves high stability and reusability of gold nanoparticle arrays, reduces background interference, and improves detection sensitivity and salt resistance, making it suitable for the detection of complex samples, especially small molecule analysis in high-salt samples.
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Figure CN116148340B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a gold nanoparticle array with strong stability and reusability, and its preparation and application. Background Technology
[0002] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) utilizes the co-crystallization of analytes with a photosensitive organic matrix, inducing desorption and ionization of the analytes under laser irradiation. MALDI offers numerous advantages and is an indispensable tool for polymer analysis. However, the presence of the organic matrix leads to high background interference in the mass spectrometry region below m / z 800, posing a significant challenge for the analysis of small molecules. Surface-assisted laser desorption / ionization mass spectrometry (SALDI-MS) using inorganic nanomaterials as the matrix can effectively address this issue. Inorganic nanomaterials have relatively simple compositions and, compared to traditional organic matrices, do not cause severe background interference during detection. Furthermore, their large specific surface area and strong adsorption properties result in high adsorption efficiency for analyte molecules. They also exhibit excellent light energy absorption and utilization in the ultraviolet region, effectively transferring energy to the analyte molecules to assist in their desorption and ionization. In recent years, SALDI has become a powerful tool for small molecule metabolism analysis due to its advantages such as high throughput, high sensitivity, and immunity to matrix interference in the low mass region of mass spectrometry. It has been widely used in fields such as rapid clinical sample screening, biological tissue imaging analysis, and metabolomics research.
[0003] Gold nanoparticles are simple to prepare, highly stable, and exhibit strong absorption in the ultraviolet region, allowing for effective use of laser energy to assist in the desorption ionization of analytes. They are a commonly used matrix in SALDI-MS. The shape, particle size, and arrangement of gold nanomaterials significantly influence SALDI performance. For example, specific arrangements or structures of gold nanomaterials can generate special effects such as local electric field coupling and plasmonic coupling. These coupling effects can increase the number and energy of excited electrons generated by gold nanomaterials, enhancing their desorption ionization performance. However, currently, weak interactions such as van der Waals forces, electrostatic interactions, and hydrogen bonds are commonly used to prepare nanomaterials with specific arrangements or structures. These weak interactions are easily affected by the complex environment of the sample (high salt content, interfering substances, etc.), leading to the disruption of the original distribution of gold nanomaterials and thus affecting the analytical results. Furthermore, gold nanoparticles dried directly on glass surfaces are prone to aggregation, and uneven distribution of gold nanoparticles can result in poor analytical stability and severe signal fluctuations.
[0004] Therefore, developing a gold nanoparticle array matrix with high stability, regular arrangement, and high salt resistance is of great significance for SALDI-MS analysis. Summary of the Invention
[0005] The purpose of this invention is to provide a gold nanoparticle array with strong stability and reusability, as well as its preparation and application, to overcome the shortcomings of existing SALDI technology.
[0006] A method for preparing a gold nanoparticle array with strong stability and reusability includes the following steps:
[0007] (1) Preparation of gold nanoparticles;
[0008] (2) Add hexadecyltrimethylammonium chloride solution to gold nanoparticles, stir vigorously to react, and centrifuge to wash; then add a solution with amino groups, stir vigorously to react, and wash away excess ligands to obtain functionalized gold nanoparticles; preferably use 3000g, centrifuge for 15min to wash, and then redissolve in water to achieve the purpose of cleaning gold nanoparticles.
[0009] (3) Add KCl solution to the functionalized gold nanoparticle solution in step (2) to change the distance between gold nanoparticles by regulating the Coulomb repulsion on the surface of gold nanoparticles.
[0010] (4) Functionalize the glass substrate so that the surface of the glass substrate has groups that can react with amino groups to form covalent bonds;
[0011] (5) The gold nanoparticle solution obtained in step (3) is dropped onto a functionalized glass support. The reaction is preferably carried out at room temperature and 100% humidity. Stable covalent bonds are formed by the reaction of the amino groups of the ligands on the surface of the gold nanoparticles with groups on the surface of the glass support that can react with the amino groups to form covalent bonds. This allows the gold nanoparticles to be stably fixed onto the glass. The mixture is then rinsed to remove unreacted gold nanoparticles, and dried to obtain a uniformly arranged and dense array of gold nanoparticles. Preferably, 4 μL of the gold nanoparticle solution obtained in step (3) with a concentration of 2E+12NPs / mL is dropped into each 3 mm diameter well.
[0012] Furthermore, the solution containing amino groups is a homocysteine solution; the group that can react with the amino groups to form covalent bonds is an ethylene oxide group. Since directly replacing the ligands on the surface of gold nanoparticles with homocysteine is not stable, it is necessary to first replace them with hexadecyltrimethylammonium chloride as a transition to prevent the gold nanoparticles from precipitating during the reaction.
[0013] Further, the preparation method of gold nanoparticles in step (1) includes: adding potassium chloroaurate and sodium citrate solutions to a reactor, mixing thoroughly with magnetic stirring in an ice bath to form a precursor solution; then adding sodium borohydride solution to the vigorously stirred precursor solution, followed by the simultaneous addition of potassium chloroaurate and ascorbic acid solutions in multiple portions. Preferably, the concentration of potassium chloroaurate solution added to the reactor is 0.75 mmol / L, and the volume of potassium chloroaurate solution is 5 mL; the concentration of sodium citrate solution is 3.75 mmol / L, and the volume of sodium citrate solution is 5 mL; the concentration of sodium borohydride solution is 3 mmol / L, and the volume of sodium borohydride solution is 5 mL. Potassium chloroaurate and ascorbic acid solutions were added twice. In the first addition, the concentration of potassium chloroaurate solution was 1.42 mmol / L and the volume was 1 mL; the concentration of ascorbic acid solution was 4.24 mmol / L and the volume was 1 mL. In the second addition, the concentration of potassium chloroaurate solution was 5.68 mmol / L and the volume was 1 mL; the concentration of ascorbic acid solution was 16.95 mmol / L and the volume was 1 mL.
[0014] Furthermore, the gold nanoparticles have a particle size of 10-60 nm, preferably 26 nm.
[0015] Furthermore, in step (2), the molar ratio of hexadecyltrimethylammonium chloride solution, homocysteine solution, and gold nanoparticles is 200-260:40-80:4.15E-9, and the concentration of the ligand increases with the increase of the total surface area of the gold nanoparticles. Preferably, when the particle size of the gold nanoparticles is 26 nm, the concentration of the hexadecyltrimethylammonium chloride solution is 44 mmol / L, and the volume of the hexadecyltrimethylammonium chloride solution is 5 mL; the concentration of the homocysteine solution is 10 mmol / L, and the volume of the homocysteine solution is 5 mL.
[0016] Furthermore, in step (3), the concentration of KCl is 2-5 mmol / L, the ratio of gold nanoparticle solution to KCl solution is 1:0.8-1.5 (V / V), and the concentration of gold nanoparticle solution after treatment is 2E+12NPs / mL.
[0017] Further, the glass substrate functionalization treatment in step (4) includes: first, cleaning the glass with a piranha solution of sulfuric acid:hydrogen peroxide = 3:1 to expose the silanol groups; then immersing the glass slide in the piranha solution; after complete treatment, washing the glass with tap water and distilled water; after drying the glass, immersing it in a toluene solution of γ-glycidoxypropyltrimethoxysilane with ethylene oxide at the end, so that the surface of the glass substrate has ethylene oxide groups; after immersion, rinsing the glass sequentially with toluene, ethanol, and acetone; and finally drying it with argon gas to obtain the functionalized glass. Preferably, the volume ratio of γ-glycidoxypropyltrimethoxysilane to toluene is 1:50.
[0018] The above preparation method yields a gold nanoparticle array with strong stability and reusability.
[0019] The aforementioned gold nanoparticle arrays, characterized by strong stability and reusability, can be used to detect and analyze samples with high salt content in surface-assisted laser desorption / ionization mass spectrometry and to perform rapid analysis of small molecule compounds in both positive and negative ion modes.
[0020] Furthermore, the small molecule compounds are compounds with a molecular weight of less than 1200 Daltons, including peptides, carbohydrates, bases, drugs, and serum metabolites. Standards such as peptides, carbohydrates, drugs, and bases are used at a concentration of 2E-5 mol / L, and human serum is used after protein precipitation. Samples with high salt content include serum, urine, and sweat.
[0021] This invention first prepares high-cysteine (terminal amino group) stable gold nanoparticles, and uses the coulombic repulsion between gold nanoparticles to change the spacing between them. Then, the gold nanoparticle solution is dropped onto a glass surface modified with γ-glycidyl etheroxypropyltrimethoxysilane (terminal ethylene oxide group) to react and form stable covalent bonds. After the reaction is complete, a gold nanoparticle array with uniform distribution on the glass surface can be obtained.
[0022] Compared with existing technologies, this invention provides a gold nanoparticle array matrix material with strong stability and reusability, and its preparation method. The preparation process does not require high temperature, and the experimental operation is simple and convenient. The gold nanoparticle array matrix of this invention enables the gold nanoparticles to be arranged in an orderly and compact manner, resulting in high detection sensitivity. It also has strong ultraviolet absorption capacity and good stability, reproducibility, reusability, and salt resistance, with low background interference; it can also be reused. The gold nanoparticle array matrix of this invention eliminates the influence of high salinity samples on matrix stability, and can detect complex samples (such as small molecule metabolites in serum with high salt content). It can realize the detection and analysis of samples with high salt content in surface-assisted laser desorption / ionization mass spectrometry. It also eliminates the background interference problem that has always existed in MALDI-MS, enabling rapid detection of low molecular weight substances. It can be applied to the rapid analysis of small molecule compounds such as carbohydrates, peptides, bases, and drugs in positive and negative ion dual modes. Attached Figure Description
[0023] Figure 1 Scanning electron microscope images of gold nanoparticle array (a) without KCl in Comparative Example 1 and gold nanoparticle array (b) with 5 mmol / L KCl in Example 1.
[0024] Figure 2 Mass spectra of adenine phosphate (ai) and leucine-enkephalin (a-ii) detected in positive ion mode and adenine phosphate (bi) and leucine-enkephalin (b-ii) detected in negative ion mode using gold nanoparticle array as matrix.
[0025] Figure 3 Scanning electron microscope images of blank gold nanoparticle arrays (a) and gold nanoparticle arrays containing 0.5 mM (b), 10 mM (c), 50 mM (d), 100 mM (e), and 200 mM (f) PBS, used to test the salt resistance of gold nanoparticle arrays.
[0026] Figure 4 Mass spectra of L-alanine detected on a conventional DHB matrix (a) and a gold nanoparticle array (b); where * represents background interference peaks;
[0027] Figure 5 The signal intensity of berberine hydrochloride was analyzed multiple times for reuse of the gold nanoparticle array (a) and the signal intensity of the residue after each cleaning (b);
[0028] Figure 6 This is a mass spectrum of blood samples from a patient with early-stage lung cancer (a) and a healthy volunteer (b) analyzed by mass spectrometry using a gold nanoparticle array for clinical blood sample analysis. Detailed Implementation
[0029] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.
[0030] Example 1:
[0031] A gold nanoparticle array with strong stability and reusability is prepared through the following steps:
[0032] Step S1, Preparation of precursor solution: Add 5 mL of 0.75 mmol / L potassium chloroaurate solution and 5 mL of 3.75 mmol / L sodium citrate solution to the reactor, place in an ice bath, and stir magnetically for 30 s to mix thoroughly to form the precursor solution;
[0033] Step S2, synthesis of 26 nm gold nanoparticle solution: 5 mL of sodium borohydride solution with a concentration of 3 mmol / L was added dropwise to the precursor solution. After reacting for 30 min, 1 mL of potassium chloroaurate solution with a concentration of 1.42 mmol / L and ascorbic acid solution with a concentration of 4.24 mmol / L were added dropwise. After reacting for 45 min, 1 mL of potassium chloroaurate solution with a concentration of 5.68 mmol / L and ascorbic acid solution with a concentration of 16.95 mmol / L were added dropwise. After reacting for 45 min, a 26 nm gold nanoparticle solution with uniform particle size was obtained.
[0034] Step S3, preparation of functionalized 26nm gold nanoparticles: Taking 1mL of gold nanoparticle solution as an example, add 5mL of 44mmol / L hexadecyltrimethylammonium chloride solution to the vigorously stirred 26nm gold nanoparticle solution, react for 2 hours, wash to remove excess ligands, then add 5mL of 10mmol / L homocysteine solution, react for 2 hours, wash to remove excess ligands, and finally reconstitute to 1mL to obtain functionalized 26nm gold nanoparticles;
[0035] In step S4, the functionalized gold nanoparticle solution is mixed with 5 mol / L KCl solution at a ratio of 1:1 (V / V) to reduce the Coulomb repulsion on the surface of the gold nanoparticles, thereby reducing the distance between the gold nanoparticles. Finally, the concentration of the gold nanoparticle solution is 2E+12NPs / mL.
[0036] Step S5, Preparation of gold nanoparticle array: A functionalized 26nm gold nanoparticle solution is dropped onto a functionalized glass support, 4μL per well. The reaction is carried out overnight at room temperature and 100% humidity, allowing the gold nanoparticles with amino groups on their surface to react with the glass substrate with epoxy groups on its surface to form covalent bonds, firmly attaching the gold nanoparticles to the glass surface. The substrate is then rinsed with distilled water to remove unreacted gold nanoparticles and allowed to air dry to obtain the gold nanoparticle array (e.g., ...). Figure 1 (The right image in the middle), from Figure 1It can be seen that the gold nanoparticles are arranged closely and uniformly on the glass surface without aggregation. Furthermore, due to the reduction in Coulomb repulsion between the gold nanoparticles, the distance between the gold nanoparticles in the array is reduced from 35 nm to 16 nm.
[0037] The excess ligands of the gold nanoparticles were cleaned by centrifugation and resolution. The centrifugation conditions were 3000g for 15min. The nanoparticles were then resolvated with water.
[0038] The glass substrate is functionalized before use. The functionalization process is as follows: First, a piranha solution (sulfuric acid: hydrogen peroxide = 3:1) is prepared. Then, the glass slide is immersed in the piranha solution for 30 minutes to expose the hydroxyl groups on the glass surface. The glass is then washed with tap water and distilled water. Next, the glass is immersed in a toluene solution (V / V) containing 2% γ-glycidoxypropyltrimethoxysilane at room temperature. After immersion for 19 hours, the glass is rinsed with toluene, ethanol, and acetone in sequence. Finally, it is dried with argon gas to obtain the functionalized glass.
[0039] Example 2:
[0040] The surface-assisted laser desorption / ionization mass spectrometry (SALDS) of the gold nanoparticle array prepared in Example 1 was used to analyze small molecules in both positive and negative ion modes. The specific procedures are as follows:
[0041] Leucine-enkephalin and adenine hydrochloride were prepared into solutions of various concentrations. 2 μL of each solution was dropped onto the surface of a gold nanoparticle array. After drying, surface-assisted laser desorption / ionization mass spectrometry (SALMS) analysis was performed in both positive and negative ion modes (mass spectra are shown below). Figure 2 (As shown). From Figure 2 It can be observed that both analytes can be detected in both positive and negative ion modes with minimal background interference and a high signal-to-noise ratio (S / N). These results indicate that the gold nanoparticle array prepared in Example 1 can be used as a SALDI matrix for the analysis of small molecule analytes.
[0042] Example 3:
[0043] The reproducibility of the gold nanoparticle array matrix prepared in Example 1 was investigated as follows: Leucine-enkephalin and berberine hydrochloride solutions with concentrations of 2E-5 mol / L were prepared, and 2 μL of each standard solution was sequentially added to the surface of the gold nanoparticle array for surface-assisted laser desorption / ionization mass spectrometry (LASIK). Signal intensity analysis was performed on different regions of the matrix (point-to-point within a single matrix pore). Data were collected from five locations around and in the center of each sample's matrix pore, and the relative standard deviation (RSD) of the detection signal intensity for each substance was calculated (as shown in Table 1). 2 μL of each of the five standard solutions was sequentially added to the surface of the gold nanoparticle array for LSIK analysis. Signal intensity analysis was performed on different regions of the matrix (between matrix pores). Four data points were collected for each sample, and the relative standard deviation of the detection signal intensity for each substance was calculated (as shown in Table 2). The tables show that the gold nanoparticle array exhibits good reproducibility.
[0044] Table 1. Reproducibility of the gold nanoparticle array matrix (point-to-point within a single matrix pore)
[0045]
[0046] Table 2. Examination of the reproducibility of the gold nanoparticle array matrix (between matrix pores)
[0047]
[0048] Example 4:
[0049] The salt tolerance of the gold nanoparticle array prepared in Example 1 was analyzed, and the specific procedures are as follows:
[0050] Prepare PBS solutions with concentrations of 5, 10, 50, 100, and 200 mM (Note: 10 mM = 1x PBS contains 137 mM NaCl, 2.7 mM KCl, 110 mM Na2HPO4, and 2 mM KH2PO4), and drop them onto the surface of a gold nanoparticle array. Then, observe the microstructure of the gold nanoparticle array using a field emission scanning electron microscope (PES) (e.g., ...). Figure 3 As shown in the figure, compared with the blank matrix, more and more salt crystals are generated on the matrix surface with increasing PBS concentration. However, such a high concentration of PBS does not disrupt the uniform distribution of gold nanoparticles on the glass surface. Mass spectrometry signal analysis was also performed on different regions of the PBS-containing gold nanoparticle array (point-to-point within a single matrix pore and between matrix pores). Even with high salt content, the analytes could still be detected. As shown in Tables 3 and 4, the detection signal fluctuation remained at around 10%, indicating that the gold nanoparticle array has good salt resistance and stability.
[0051] Table 3. Reproducibility of the gold nanoparticle array matrix containing 10 mM PBS (point-to-point within a single matrix pore)
[0052]
[0053] Table 4. Reproducibility of the gold nanoparticle array matrix containing 10 mM PBS (between matrix pores)
[0054]
[0055] Example 5:
[0056] Example 1 describes the analysis of peptides, carbohydrates, drugs, and base compounds using the gold nanoparticle array matrix and the traditional DHB matrix in positive ion mode. The specific procedures are as follows: 20 mg of DHB was weighed and dissolved in 1 mL of TA 30 (acetonitrile / 0.2% TFA, V / V, 3:7). Compounds (adenine hydrochloride, L-alanine-glutamic acid, maltose, berberine hydrochloride, and leucine enkephalin) were mixed to prepare a 2E-5M solution. 2 μL of this mixed solution was dropped onto the surface of the gold nanoparticle array. Then, 2 μL of the mixed solution was mixed with 2 μL of the DHB matrix and dropped onto the target plate. After drying, laser desorption / ionization mass spectrometry (LASIK) analysis was performed in positive ion mode. The results for berberine hydrochloride are shown in the figure below. Figure 4 As shown in the figure, compared with the traditional DHB matrix, the mass spectrum obtained by the gold nanoparticle array matrix has a higher signal-to-noise ratio, fewer matrix background peaks, and higher sensitivity. The ratio of the signal intensity of each analyte in the gold nanoparticle array and the traditional DHB matrix was calculated (as shown in Table 5). It can be seen from the table that the detection sensitivity of the gold nanoparticle array is higher than that of DHB, indicating that the gold nanoparticle array is more suitable for the detection of small molecule analytes than the traditional DHB matrix.
[0057] Table 5 Comparison of signal intensity between gold nanoparticle arrays and traditional DHB matrix
[0058]
[0059] Example 6:
[0060] The reusability of the gold nanoparticle array matrix prepared in Example 1 was investigated as follows: A 5E-6 mol / L berberine hydrochloride solution was added dropwise to the surface of the gold nanoparticle array. After mass spectrometry analysis, the gold nanoparticle array was washed, and the washed matrix was also analyzed by mass spectrometry. This process was repeated 11 times (e.g., ...). Figure 5 After (as shown), the signal intensity of the analyte remained essentially unchanged during 10 repeated cleaning and detection cycles, with a relative standard deviation of 9.0%, and no carryover effect was observed, indicating that covalent bond fixation allows the gold nanoparticle array to be used multiple times.
[0061] Example 7:
[0062] The content of metabolites in human blood was measured using the gold nanoparticle array matrix prepared in Example 1. The specific procedure was as follows: Blood samples were collected from 21 patients with early-stage lung cancer and 30 healthy volunteers. Proteins in the serum were removed using a protein precipitant (methanol / acetonitrile, V / V, 1:1). After centrifugation, 0.6 μL of the supernatant was dropped onto the surface of the gold nanoparticle array for mass spectrometry analysis (e.g., ...). Figure 6 As shown), from Figure 6 The results show that the gold nanoparticle array matrix performs well in detecting complex serum samples. Further analysis of the data successfully distinguished early-stage lung cancer patients from healthy individuals, demonstrating the significant application potential of the gold nanoparticle array matrix in detecting relatively complex samples.
[0063] Comparative Example 1
[0064] Step S4 was not included, meaning that no 5 mmol / L KCl solution was added to the functionalized gold nanoparticle solution for mixing at a 1:1 (v / v) ratio; otherwise, it was the same as in Example 1. The final product was obtained by natural drying, as shown in Example 1. Figure 1 As shown in the left figure, the distance between the gold nanoparticles in the array is 35 nm.
[0065] Comparative Example 2
[0066] In step S3, hexadecyltrimethylammonium chloride solution was not added; homocysteine solution was added directly, and the rest was the same as in Example 1. Step S3 was as follows: Preparation of functionalized 26nm gold nanoparticles: Taking 1mL of gold nanoparticle solution as an example, 5mL of 10mmol / L homocysteine solution was added to the vigorously stirred 26nm gold nanoparticle solution, reacted for 2 hours, excess ligands were washed away, and finally the solution was reconstituted to 1mL. It was found that directly replacing the ligands on the surface of the gold nanoparticles with homocysteine was not stable, and the gold nanoparticles precipitated during the reaction.
[0067] In summary, this invention provides a gold nanoparticle array matrix material with strong stability and reusability, as well as its preparation method. The preparation process does not require high temperatures, and the experimental operation is simple and convenient. The gold nanoparticle array matrix of this invention allows for the orderly and compact arrangement of gold nanoparticles, resulting in high detection sensitivity. This matrix also exhibits good stability and salt resistance, enabling the detection of complex samples, such as small molecule metabolites in serum with high salt content. The gold nanoparticle array matrix of this invention is reusable.
[0068] The gold nanoparticle array matrix of the present invention can eliminate the background interference problem that has always existed in MALDI-MS, and realize the rapid detection of low molecular weight substances.
[0069] The gold nanoparticle array matrix material of the present invention can be used to detect low molecular weight compounds in both positive and negative ion modes.
[0070] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.
Claims
1. A method for preparing a gold nanoparticle array with strong stability and reusability, characterized in that, Includes the following steps: (1) Preparation of gold nanoparticles; (2) Add hexadecyltrimethylammonium chloride solution to gold nanoparticles, stir to react, and centrifuge to wash; then add a solution with amino groups, stir to react, and wash away excess ligands to obtain functionalized gold nanoparticles. (3) Add KCl solution to the functionalized gold nanoparticle solution in step (2) to change the distance between the gold nanoparticles; (4) Functionalize the glass carrier so that the surface of the glass carrier has groups that can react with amino groups to form covalent bonds; (5) The gold nanoparticle solution obtained in step (3) is dropped onto the functionalized glass carrier to fix the gold nanoparticles stably onto the glass. Then, it is rinsed to remove the unreacted gold nanoparticles and dried to obtain a gold nanoparticle array. The glass substrate functionalization process in step (4) includes: first, cleaning the glass with a piranha solution of sulfuric acid: hydrogen peroxide = 3:1 to expose the silanol groups, then immersing the glass in the piranha solution. After the treatment is complete, the glass is washed and dried, and then immersed in a toluene solution of γ-glycidyl etheroxypropyltrimethoxysilane to make the surface of the glass substrate have epoxy ethylene groups. After rinsing and drying, functionalized glass is obtained. The solution containing amino groups is a homocysteine solution; the group that can react with amino groups to form covalent bonds is an ethylene oxide group.
2. The preparation method according to claim 1, characterized in that, The preparation method of gold nanoparticles in step (1) includes: adding potassium chloroaurate and sodium citrate solution to the reactor, using an ice bath, and stirring magnetically to mix thoroughly to form a precursor solution; then adding sodium borohydride solution to the precursor solution, and then adding potassium chloroaurate and ascorbic acid solution simultaneously in multiple batches.
3. The preparation method according to claim 1, characterized in that, The gold nanoparticles have a particle size of 10-60 nm.
4. The preparation method according to claim 2, characterized in that, In step (2), the molar ratio of hexadecyltrimethylammonium chloride solution, homocysteine solution, and gold nanoparticles is 200-260 : 40-80 : 4.15E. -9 .
5. The preparation method according to claim 1, characterized in that, In step (3), the concentration of KCl is 2-5 mmol / L, and the ratio of functionalized gold nanoparticles to KCl solution is 1:0.8-1.5 (V / V).
6. A gold nanoparticle array with strong stability and reusability obtained by the preparation method according to any one of claims 1-5.
7. The application of the gold nanoparticle array with strong stability and reusability according to claim 6, characterized in that, It can be used to detect and analyze samples with high salt content in surface-assisted laser desorption / ionization mass spectrometry and to perform rapid analysis of small molecule compounds in both positive and negative ion modes.
8. The application according to claim 7, characterized in that, The small molecule compounds are compounds with a molecular weight of less than 1200 Daltons, including polypeptides, sugars, bases, drugs, and serum metabolites; the samples with high salt content include serum, urine, and sweat.