CN116148340A

CN116148340A - Gold nano-particle array with strong stability and reusability and preparation and application thereof

Info

Publication number: CN116148340A
Application number: CN202211396430.0A
Authority: CN
Inventors: 苏洋; 梁凯晴; 吴坤明
Original assignee: Shantou University
Current assignee: Shantou University
Priority date: 2022-11-09
Filing date: 2022-11-09
Publication date: 2023-05-23

Abstract

The invention relates to a gold nanoparticle array preparation method with strong stability and reusability, which comprises the steps of firstly preparing gold nanoparticles with stable homocysteine, changing the interval between the gold nanoparticles by utilizing coulomb repulsion between the gold nanoparticles, then dripping gold nanoparticle solution onto the glass surface modified by gamma-glycidol ether oxypropyl trimethoxy silane for reaction to form stable covalent bonds, and obtaining the gold nanoparticle array with uniform distribution on the glass surface after the reaction. The preparation of the invention does not need high temperature, the experimental operation is simple and convenient, the gold nano particles are orderly and tightly arranged, and the detection sensitivity is high; the ultraviolet light absorption capacity is strong, the stability, the reproducibility, the reusability and the salt tolerance are good, and the background interference is low; and can be reused. The method can be used for detecting and analyzing the sample with high salt content in the surface-assisted laser desorption ionization mass spectrum and rapidly analyzing the small molecular compound in the positive and negative ion double mode.

Description

Gold nano-particle array with strong stability and reusability and preparation and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a gold nano-particle array with strong stability and reusability, and preparation and application thereof.

Background

Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) performs co-crystallization with an analyte through a photosensitive organic matrix, induces desorption and ionization of the analyte under the action of laser, and MALDI has many advantages and is an indispensable tool for polymer analysis. However, the presence of an organic matrix can lead to high background interference of the mass spectrum in the region below m/z 800, which presents a great challenge in analyzing small molecule analytes. The surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) using inorganic nano-materials as the matrix can well solve the problem. The inorganic nanometer matrix has relatively simple components, and compared with the traditional organic matrix, the inorganic nanometer matrix does not cause serious background interference during detection; meanwhile, the adsorption efficiency of the molecules to be detected is high due to the extremely large specific surface area and extremely strong adsorption performance; the ultraviolet light energy absorption and utilization ratio in the ultraviolet light region is excellent, and energy can be effectively transferred to the molecules of the object to be detected to assist desorption and ionization of the object to be detected. In recent years, SALDI has become a powerful tool for small molecule metabolic analysis due to the advantages of high flux, high sensitivity, no matrix interference in a mass spectrum low-mass region and the like, and is widely applied to the fields of rapid clinical sample screening, biological tissue imaging analysis, metabonomics research and the like.

The gold nanoparticles are simple to prepare, high in stability, strong in absorption in ultraviolet region, capable of effectively utilizing laser energy to assist analyte in desorption ionization, and one of the common matrixes of SADLI-MS. The shape, particle size and arrangement of the gold nano-materials have important influence on the performance of SALDI. For example, the specific arrangement or structure of the gold nano material can generate special effects such as local electric field coupling, plasma coupling and the like, and the coupling effects can improve the quantity and energy of excitation electrons generated by the gold nano material and enhance the desorption ionization performance of the gold nano material. However, at present, weak forces such as van der waals force, electrostatic interaction, hydrogen bond and the like are commonly used to prepare nano materials with specific arrangement or structure, and the weak forces are extremely susceptible to complex environments (high salt content, interferents and the like) in a sample, so that the original distribution of the gold nano materials is destroyed, thereby influencing analysis results. Meanwhile, gold nanoparticles directly dried on the surface of glass are easy to aggregate, and uneven distribution of the gold nanoparticles can cause poor stability of analysis results and serious signal fluctuation.

Therefore, developing gold nanoparticle array substrates with high stability, regular arrangement and high salt tolerance is of great significance to research of SALDI-MS analysis.

Disclosure of Invention

The invention aims to provide a gold nano-particle array with strong stability and reusability, and preparation and application thereof, so as to overcome the defects of the existing SALDI technology.

A preparation method of a gold nanoparticle array with strong stability and reusability comprises the following steps:

(1) Preparing gold nanoparticles;

(2) Adding cetyl trimethyl ammonium chloride solution into the gold nanoparticles, carrying out intense stirring reaction, and carrying out centrifugal cleaning; adding a solution with amino groups, and carrying out vigorous stirring reaction to clean redundant ligands to obtain functionalized gold nanoparticles; preferably using 3000g for 15min centrifugation to clean, and re-dissolving in water to clean gold nano particles;

(3) Adding KCl solution into the functionalized gold nanoparticle solution in the step (2), and changing the distance between gold nanoparticles by regulating and controlling the coulomb repulsion of the surfaces of the gold nanoparticles;

(4) Performing functionalization treatment on the glass carrier to enable the surface of the glass carrier to be provided with groups which can react with amino groups to form covalent bonds;

(5) And (3) dropwise adding the gold nanoparticle solution obtained in the step (3) on a functionalized glass carrier, preferably reacting at normal temperature and in an environment with 100% humidity, forming a stable covalent bond through the reaction of an amino group of a ligand on the surface of the gold nanoparticle and a group capable of forming a covalent bond through reaction with the amino group on the surface of the glass carrier, so that the gold nanoparticle is stably fixed on the glass, then washing, removing unreacted gold nanoparticle, and drying to obtain a gold nanoparticle array which is uniformly and tightly distributed. Preferably, 4. Mu.L of gold nanoparticle obtained in the step (3) is dropwise added into each hole with a diameter of 3mm, and the concentration of the gold nanoparticle solution is 2E+12NPs/mL.

Further, the solution with amino groups is homocysteine solution; the group that can react with an amino group to form a covalent bond is an ethylene oxide group. Because the ligand on the surface of the gold nanoparticle is directly changed into homocysteine and is unstable, cetyl trimethyl ammonium chloride is needed to be firstly changed into hexadecyl trimethyl ammonium chloride to be used as transition, and the gold nanoparticle is prevented from precipitating in the reaction process.

Further, the preparation method of the gold nanoparticles in the step (1) comprises the following steps: adding potassium chloroaurate and sodium citrate solution into the reactor, ice-bathing, and magnetically stirring to fully mix to form a precursor solution; then, a sodium borohydride solution was added to the vigorously stirred precursor solution, and then potassium chloroaurate and ascorbic acid solution were added simultaneously multiple times. Preferably, the concentration of the potassium chloroaurate solution is 0.75mmol/L, and the volume of the potassium chloroaurate solution is 5mL; the concentration of the sodium citrate solution was 3.75mmol/L and the volume of the sodium citrate solution was 5mL. The concentration of the sodium borohydride solution was 3mmol/L, and the volume of the sodium borohydride solution was 5mL. Adding twice potassium chloroaurate and ascorbic acid solution, wherein the concentration of the potassium chloroaurate solution is 1.42mmol/L for the first time, and the volume of the potassium chloroaurate solution is 1mL; the concentration of the ascorbic acid solution is 4.24mmol/L, and the volume of the ascorbic acid solution is 1mL; the second time, the concentration of the potassium chloroaurate solution is 5.68mmol/L, and the volume of the potassium chloroaurate solution is 1mL; the concentration of the ascorbic acid solution was 16.95mmol/L, and the volume of the ascorbic acid solution was 1mL.

Further, the particle diameter of the gold nanoparticles is 10-60nm, preferably, the particle diameter of the gold nanoparticles is 26nm.

Further, in the step (2), the molar ratio of the cetyltrimethylammonium chloride solution, the homocysteine solution and the 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 nano-particles is 26nm, the concentration of the cetyltrimethylammonium chloride solution is 44mmol/L, and the volume of the cetyltrimethylammonium chloride solution is 5mL; the concentration of the homocysteine solution was 10mmol/L, and the volume of the homocysteine solution was 5mL.

Further, in the step (3), the concentration of KCl is 2-5mmol/L, the dosage ratio of the gold nanoparticle solution to the KCl solution is 1:0.8-1.5 (V/V), and the concentration of the gold nanoparticle solution after treatment is 2E+12NPs/mL.

Further, the glass carrier functionalization treatment in the step (4) includes: firstly, cleaning glass with sulfuric acid and hydrogen peroxide=3:1 piranha solution, exposing silicon hydroxyl groups, then soaking glass sheets in the piranha solution, washing the glass with tap water and distilled water after the glass is completely treated, drying the glass, soaking the glass in a toluene solution of gamma-glycidoxypropyl trimethoxysilane with ethylene oxide groups at the tail end, enabling the surface of a glass carrier to be provided with the ethylene oxide groups, washing the glass with toluene, ethanol and acetone in sequence after the soaking is finished, and finally drying the glass with argon gas to obtain the functionalized glass. Preferably, the volume ratio of gamma-glycidoxypropyl trimethoxysilane to toluene is 1:50.

The gold nano-particle array with strong stability and reusability is obtained by the preparation method.

The application of the gold nano-particle array with strong stability and reusability can realize the detection analysis of samples with high salt content in the surface-assisted laser desorption ionization mass spectrum and the rapid analysis of small molecular compounds in positive and negative ion double modes.

Further, the small molecule compounds are compounds having a molecular weight of less than 1200 daltons, including polypeptides, sugars, bases, drugs, and serum metabolites. The standard substances such as polypeptide, sugar, medicine, base and the like are used at the concentration of 2E-5mol/L, and the protein precipitation is carried out on human serum for reuse. The sample with high salt content comprises serum, urine and sweat.

The gold nanoparticle array with uniform distribution on the glass surface can be obtained after the reaction.

Compared with the prior art, the gold nano-particle array matrix material with strong stability and reusability and the preparation method thereof provided by the invention have the advantages that high temperature is not needed in the preparation process, and the experimental operation is simple and convenient. The gold nano-particle array substrate ensures that gold nano-particles are orderly and tightly arranged, and has higher detection sensitivity; the ultraviolet light absorption capacity is strong, the stability, the reproducibility, the reusability and the salt tolerance are good, and the background interference is low; and can be reused. The gold nano-particle array substrate eliminates the influence of a high-salinity sample on the stability of the substrate, can detect complex samples (such as small molecular metabolites in serum with high salt content), and can realize detection and analysis of the samples with high salt content in surface-assisted laser desorption ionization mass spectrometry; the method also eliminates the background interference problem existing in MALDI-MS, can realize the rapid detection of low molecular weight substances, and can be applied to the rapid analysis of small molecular compounds such as saccharides, polypeptides, bases, medicaments and the like under positive and negative ion double modes.

Drawings

FIG. 1 is a scanning electron microscope image of a gold nanoparticle array (a) of comparative example 1 without KCl mixed and a gold nanoparticle array (b) of example 1 with 5mmol/L KCl mixed;

FIG. 2 is a mass spectrum of gold nanoparticle arrays as a substrate for detection of adenine phosphate (a-i), leucine-enkephalin (a-ii) and adenine phosphate (b-i), leucine-enkephalin (b-ii) in positive ion mode;

FIG. 3 is a scanning electron microscope image of an array of blank gold nanoparticles (a) and gold nanoparticle arrays containing 0.5mM (b), 10mM (c), 50mM (d), 100mM (e), 200mM (f) PBS for testing the salt tolerance of the gold nanoparticle arrays;

FIG. 4 is a mass spectrum of a conventional DHB matrix (a) and gold nanoparticle array (b) for detecting L-propyl-glutamic acid; wherein, is the background interference peak;

FIG. 5 shows the re-use of gold nanoparticles for multiple analyses of berberine hydrochloride (a) signal intensity and residue after each wash (b);

FIG. 6 is a mass spectrum of gold nanoparticle arrays for analysis of clinical blood samples for mass spectrometric detection analysis of blood samples from early stage lung cancer patients (a) and healthy volunteers (b).

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.

Example 1:

a gold nanoparticle array with strong stability and reusability is prepared by the following steps:

step S1, preparing a precursor solution: adding 5mL of potassium chloroaurate solution with the concentration of 0.75mmol/L and 5mL of sodium citrate solution with the concentration of 3.75mmol/L into a reactor, carrying out ice bath, and magnetically stirring for 30s to fully mix to form a precursor solution;

step S2, synthesizing a 26nm gold nanoparticle solution: dropwise adding 5mL of sodium borohydride solution with the concentration of 3mmol/L into the precursor solution, reacting for 30min, simultaneously dropwise adding 1mL of potassium chloroaurate solution with the concentration of 1.42mmol/L and 4.24mmol/L of ascorbic acid solution, reacting for 45min, simultaneously dropwise adding 1mL of potassium chloroaurate solution with the concentration of 5.68mmol/L and 16.95mmol/L of ascorbic acid solution, and reacting for 45min to obtain 26nm gold nanoparticle solution with uniform particle size;

step S3, preparing functionalized 26nm gold nanoparticles: taking 1mL of gold nanoparticle solution as an example, adding 5mL of 44mmol/L cetyltrimethylammonium chloride solution into the vigorously stirred 26nm gold nanoparticle solution, reacting for 2 hours, cleaning, removing redundant ligand, then adding 5mL of 10mmol/L homocysteine solution, reacting for 2 hours, cleaning, removing redundant ligand, and finally redissolving to 1mL to obtain functionalized 26nm gold nanoparticle;

and S4, mixing the functionalized gold nanoparticle solution with a KCl solution of 5mol/L (V/V) at a ratio of 1:1, and reducing coulomb repulsion on the surfaces of the gold nanoparticles so as to reduce the distance between the gold nanoparticles, thereby finally obtaining the gold nanoparticle solution with the concentration of 2E+12NPs/mL.

Step S5, preparing a gold nanoparticle array: the functionalized 26nm gold nanoparticle solution was added dropwise to the functionalized glass support, with 4 μl drop-wise per well. The gold nanoparticles with amino groups on the surface and the glass with ethylene oxide groups on the surface react overnight under the environment of normal temperature and 100% humidity to form covalent bonds, the gold nanoparticles are firmly connected to the surface of the glass, then distilled water is used for washing, unreacted gold nanoparticles are removed, and the gold nanoparticles are naturally dried to obtain a gold nanoparticle array (as shown in the right diagram in fig. 1), as can be seen from fig. 1, the gold nanoparticles are closely and uniformly distributed on the surface of the glass without aggregation, and the distance between the gold nanoparticles in the array is reduced from 35nm to 16nm due to the reduction of coulomb repulsion between the gold nanoparticles.

Wherein, the redundant ligand of the gold nano particles is cleaned by using a means of centrifugal re-dissolution, and the centrifugal conditions are 3000g and 15min; it is reconstituted with water.

Wherein the glass carrier is used after functionalization, and the functionalization process is as follows: firstly preparing a piranha solution (sulfuric acid: hydrogen peroxide=3:1), then soaking a glass sheet in the piranha solution for 30min to expose hydroxyl groups on the surface of the glass, then washing the glass with tap water and distilled water, then soaking the glass in a toluene solution (V/V) containing 2% of gamma-glycidoxypropyl trimethoxysilane, reacting at normal temperature, washing the glass with toluene, ethanol and acetone in sequence after soaking for 19h, and finally drying with argon to obtain the functionalized glass.

Example 2:

the gold nanoparticle surface prepared in example 1 was analyzed for small molecular substances by laser-assisted desorption ionization mass spectrometry in positive and negative ion modes, and the specific operation was as follows:

leucine-enkephalin and adenine hydrochloride are prepared into a series of solutions with a concentration, 2 mu L of the solutions are dripped on the surfaces of gold nanoparticle arrays, and after the solutions are dried, surface-assisted laser desorption ionization mass spectrometry (the mass spectrogram is shown in figure 2) is carried out under positive ion and negative ion modes respectively. As can be seen from fig. 2, both analytes can be detected in both positive and negative ion modes, the background interference is small, and a higher signal-to-noise ratio (S/N) is exhibited. The results indicate that the gold nanoparticle arrays prepared in example 1 can be used as SALDI matrices for analysis of small molecule analytes.

Example 3:

the gold nanoparticle array matrix prepared in example 1 was examined for reproducibility, and the specific procedure was as follows: and (3) preparing leucine-enkephalin and berberine hydrochloride solution with the concentration of 2E-5mol/L respectively, taking 2 mu L of standard substance solution, and sequentially dripping the standard substance solution onto the surface of the gold nanoparticle array for surface-assisted laser desorption ionization mass spectrometry analysis. Analysis of signal intensities from different areas of the matrix (point-to-point within a single matrix well), collecting data at the periphery and middle 5 of each sample within the matrix well, and calculating the Relative Standard Deviation (RSD) of the detected signal intensities for each substance (as shown in table 1); and sequentially dripping 2 mu L of five standard substance solutions on the surface of the gold nanoparticle array to perform surface-assisted laser desorption ionization mass spectrometry. The signal intensities of different areas of the matrix (between matrix wells) were analyzed, 4 data were collected for each sample, and the relative standard deviation of the detected signal intensities for each substance was calculated (as shown in table 2). It can be seen from the table that the gold nanoparticle array has good reproducibility.

TABLE 1 examination of matrix reproducibility of gold nanoparticle arrays (Point-to-Point within a single matrix pore)

Table 2 examination of matrix reproducibility of gold nanoparticle arrays (between matrix pores)

Example 4:

the analysis of salt tolerance of the gold nano-example array prepared in example 1 is carried out as follows:

PBS solutions with concentrations of 5, 10, 50, 100, 200mM (note: 10 mM=1xPBS contains 137mM NaCl, 2.7mM KCl, l10mM Na) ₂ HPO ₄ 、2mM KH ₂ PO ₄ ) Respectively dripping the gold nano-particles on the surface of the gold nano-particle surface,then observing the microscopic morphology of the gold nanoparticle array by a field emission scanning electron microscope (as shown in fig. 3), it can be seen from the figure that more and more salt crystals are generated on the surface of the matrix along with the increase of the concentration of PBS compared with the blank matrix. However, such high concentrations of PBS do not disrupt the uniform distribution of gold nanoparticles on the glass surface. At the same time, mass spectrum signal analysis is also carried out on different areas (between a single substrate Kong Nadian and a point and a substrate hole) of the gold nanoparticle array containing PBS, and under the condition of high salt content, the analyte can still be detected, and as can be seen from tables 3 and 4, the fluctuation of the detection signal is still kept at about 10%, which indicates that the gold nanoparticle array has good salt tolerance and stability.

TABLE 3 examination of matrix reproducibility of gold nanoparticle arrays with 10mM PBS (Point-to-Point within a single matrix well)

TABLE 4 examination of matrix reproducibility of gold nanoparticle arrays with 10mM PBS (between matrix wells)

Example 5:

the gold nanoparticle array substrate prepared in example 1 and the conventional substrate DHB analyze polypeptides, saccharides, drugs, and base compounds in a positive ion mode, and the specific operations are as follows: weighing 20mg of DHB, dissolving in 1mL of TA 30 (acetonitrile/0.2% TFA, V/V, 3:7), mixing the compounds (adenine hydrochloride, L-propyl-glutamic acid, maltose, berberine hydrochloride and leucine enkephalin) to prepare a 2E-5M solution, dripping 2 mu L of the mixed solution onto the surface of a gold nanoparticle array, dripping 2 mu L of the mixed solution and 2 mu L of DHB matrix onto a target plate, drying, and performing laser desorption ionization mass spectrometry in a positive ion mode, wherein the result diagram of berberine hydrochloride is shown in fig. 4, and compared with the traditional DHB matrix, the gold nanoparticle array matrix detection has higher signal-to-noise ratio, less matrix background peak and higher sensitivity. The Ratio (Ratio) of the signal intensities of each analyte in the gold nanoparticle array and the conventional DHB matrix was calculated (as shown in table 5), from which it can be seen that the gold nanoparticle array has higher detection sensitivity than DHB, indicating that the gold nanoparticle array is more suitable for detection of small molecule analytes than the conventional DHB matrix.

TABLE 5 comparison of gold nanoparticle arrays and conventional DHB matrix Signal intensities

Example 6:

the reusability of the gold nanoparticle array matrix prepared in example 1 is examined, and the specific operation is as follows: and (3) dropwise adding 5E-6mol/L berberine hydrochloride to the surface of the gold nanoparticle array, washing the gold nanoparticle array after mass spectrometry, and carrying out mass spectrometry on the washed matrix. After 11 repetitions of this procedure (as shown in fig. 5), the signal intensity of the analyte remained essentially unchanged over 10 repeated wash and detection cycles with a relative standard deviation of 9.0% in intensity and no carry-over effect was observed, indicating that covalent immobilization enabled multiple use of the gold nanoparticle array.

Example 7:

the content of metabolites in human blood was measured using the gold nanoparticle array matrix prepared in example 1, and the specific procedure was as follows: blood samples of 21 lung cancer early stage patients and 30 healthy volunteers were collected, protein in serum was removed using protein precipitants (methanol/acetonitrile, V/V, 1:1), 0.6 μl of supernatant was collected after centrifugation and was dropped on the surface of gold nano-particle array for mass spectrometry (as shown in fig. 6), and it can be seen from fig. 6 that the gold nano-particle array matrix performed well in detecting complex serum samples. By further analyzing the data, the early lung cancer patients and healthy people can be successfully distinguished, and the great application potential of the gold nanoparticle array matrix in more complex sample detection is illustrated.

Comparative example 1

The procedure of example 1 was repeated except that step S4 was not included, i.e., no 5mmol/L KCl solution 1:1 (V/V) was added to the functionalized gold nanoparticle solution. And finally, naturally drying to obtain a gold nanoparticle array, wherein the distance between gold nanoparticles in the array is 35nm as shown in the left graph in FIG. 1.

Comparative example 2

In step S3, the hexadecyl trimethyl ammonium chloride solution was not added, and the homocysteine solution was directly added, and the procedure was the same as in example 1. The step S3 is as follows: preparing functionalized 26nm gold nanoparticles: taking 1mL of gold nanoparticle solution as an example, 5mL of 10mmol/L homocysteine solution is added into the vigorously stirred 26nm gold nanoparticle solution to react for 2 hours, excessive ligand is removed by washing, and finally, the solution is redissolved to 1mL. It was found that the direct exchange of ligands on the surface of gold nanoparticles into homocysteine was not stable and gold nanoparticles precipitated during the reaction.

In conclusion, the gold nano-particle array matrix material with strong stability and reusability and the preparation method thereof provided by the invention have the advantages that high temperature is not needed in the preparation process, and the experimental operation is simple and convenient. The gold nano-particle array substrate ensures that gold nano-particles are orderly and tightly arranged, and has higher detection sensitivity. The matrix also has good stability and salt tolerance, and can be used for detecting complex samples, such as small molecule metabolites in serum with high salt content. The gold nanoparticle array substrate of the invention can be reused.

The gold nano-particle array matrix can eliminate the background interference problem existing in MALDI-MS all the time, and realize the rapid detection of low molecular weight substances.

The gold nano-particle array matrix material can be applied to detection of low molecular weight compounds in positive and negative ion double modes.

The above-described embodiments serve to describe the substance of the present invention in detail, but those skilled in the art should understand that the scope of the present invention should not be limited to this specific embodiment.

Claims

1. The preparation method of the gold nanoparticle array with strong stability and reusability is characterized by comprising the following steps of:

(1) Preparing gold nanoparticles;

(2) Adding cetyl trimethyl ammonium chloride solution into the gold nanoparticles, stirring for reaction, and centrifugally cleaning; adding a solution with amino groups, stirring for reaction, and cleaning redundant ligands to obtain functionalized gold nanoparticles;

(3) Adding KCl solution into the functionalized gold nanoparticle solution in the step (2) to change the distance between the gold nanoparticles;

(5) And (3) dripping the gold nanoparticle solution obtained in the step (3) on a functionalized glass carrier, stably fixing the gold nanoparticles on the glass, washing, removing unreacted gold nanoparticles, and drying to obtain the gold nanoparticle array.

2. The method of claim 1, wherein the solution having amino groups is a homocysteine solution; the group that can react with an amino group to form a covalent bond is an ethylene oxide group.

3. The method of claim 1, wherein the method of preparing gold nanoparticles in step (1) comprises: adding potassium chloroaurate and sodium citrate solution into the reactor, carrying out ice bath, and fully mixing by magnetic stirring to form a precursor solution; then adding sodium borohydride solution into the precursor solution, and then adding potassium chloroaurate and ascorbic acid solution simultaneously for a plurality of times.

4. The method of claim 1, wherein the gold nanoparticles have a particle size of 10-60nm.

5. The method according to claim 2, wherein the molar ratio of cetyltrimethylammonium chloride solution, homocysteine solution, gold nanoparticles in step (2) is 200-260:40-80:4.15e-9.

6. The method according to claim 1, wherein the concentration of KCl in the step (3) is 2-5mmol/L, and the ratio of the functionalized gold nanoparticles to KCl solution is 1:0.8-1.5 (V/V).

7. The method of claim 2, wherein the glass carrier functionalization process of step (4) comprises: firstly, cleaning glass with sulfuric acid and a piranha solution with hydrogen peroxide=3:1, exposing silicon hydroxyl groups, then soaking glass sheets in the piranha solution, after the glass sheets are completely treated, cleaning and drying the glass, and then soaking the glass sheets in a toluene solution of gamma-glycidoxypropyl trimethoxysilane to enable the surface of a glass carrier to be provided with ethylene oxide groups, and washing and drying the glass sheets to obtain the functionalized glass.

8. Gold nanoparticle arrays with strong stability and reusability obtained by the preparation method according to any one of claims 1-7.

9. The application of the gold nano-particle array with strong stability and reusability according to claim 8, wherein the detection analysis of samples with high salt content and the rapid analysis of small molecular compounds under positive and negative ion double modes can be realized in surface-assisted laser desorption ionization mass spectrometry.

10. The use according to claim 9, wherein the small molecule compound is a compound having a molecular weight of less than 1200Dalton, including polypeptides, sugars, bases, drugs and serum metabolites; the sample with high salt content comprises serum, urine and sweat.