A nanoESI-MS real-time analysis method for studying the molecular mechanism of gold nanoparticles against bacteria

Abstract

This invention belongs to the field of chemical analysis and detection, specifically relating to a method for real-time analysis of the molecular mechanism of gold nanoparticles against bacteria using nanoESI-MS. The method includes the following steps: co-culturing gold nanoparticles with bacteria, collecting the supernatant by centrifugation, using the supernatant as the sample solution, and analyzing it using nanoESI-MS to obtain spectral data; performing orthogonal partial least squares discriminant analysis on the spectral data to establish a predictive model of metabolites and sample categories; screening differentially expressed metabolites based on the predictive model and the importance of projected variables; and constructing a correlation network diagram based on the differentially expressed metabolites to analyze the antibacterial mechanism of gold nanoparticles. This method allows analysis without sacrificing sample integrity, avoiding the drawbacks of traditional mass spectrometry techniques using extraction reagents, and improving resolution efficiency and coverage.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis and detection, specifically involving a method for detecting the interaction between small molecule modified antibacterial gold nanoparticles and bacteria, as well as the resulting changes in bacterial primary and secondary metabolites using nanoESI-MS, in order to explore the antibacterial mechanism of nanomaterials at the molecular level. Background Technology

[0002] Antibiotics are widely used in clinical treatment, but due to persistent misuse and abuse, bacterial resistance is becoming increasingly serious, leading to a gradual decline in the efficacy of antibiotics against many infectious diseases. Therefore, the development of novel antibacterial drugs is urgently needed. Gold nanomaterials, due to their excellent physical properties, simple surface chemical modification, and high biocompatibility, show great promise in antibacterial applications. Previous work has shown that gold nanomaterials can disrupt bacterial cell wall structure, trigger oxidative stress to produce reactive oxygen species, reduce bacterial adenosine triphosphate (ATP) levels, and damage DNA, thereby achieving antibacterial effects. Although gold nanomaterials can exert antibacterial effects through multiple pathways, limitations in the methods for observing the interaction between gold nanomaterials and bacteria prevent a deep correlation between changes in bacterial state and material transformation, making it difficult to obtain accurate antibacterial mechanisms. Therefore, it is urgent to develop a new method to comprehensively explore the effects of gold nanomaterials on bacterial metabolism, in order to elucidate their antibacterial mechanisms and provide strategies for the development of novel antibacterial drugs.

[0003] Domestic and international scholars have employed various methods to analyze the metabolism of biological samples and explored methodological approaches for detection. In bacterial metabolism detection, Chinese patent (Patent Publication No.: CN109576172A) discloses a pretreatment method for GC-MS detection of bacterial metabolomics. This method involves sample preparation through bacterial culture, bacterial quenching, and extraction of intracellular metabolites. However, this process is complex, relies on ultrasonic disruption instruments, and is costly, failing to meet the requirement of real-time observation of bacterial metabolism. Chinese patent (CN107941938B) discloses a method for extracting and determining the degradation pathways of bacterial polycyclic aromatic hydrocarbon (PAH) metabolites, using HPLC-MS for separation and identification. This method is time-consuming, and cross-contamination may occur between samples during continuous injection. Furthermore, it only focuses on PAHs in terms of methodological exploration. Therefore, developing a simple detection method that requires no complex sample pretreatment is crucial for detecting bacterial metabolites, analyzing metabolite changes resulting from the interaction between gold nanomaterials and bacteria, revealing the antibacterial mechanisms of gold nanomaterials, and guiding the development of new drugs. Summary of the Invention

[0004] To address the aforementioned technical challenges, this invention provides a method for real-time analysis of the molecular mechanisms by which gold nanoparticles combat bacteria using nanoESI-MS. NanoESI, with its unique miniaturized nozzle and low flow rate characteristics, enables detailed analysis without sacrificing sample integrity. This not only avoids the drawbacks of traditional mass spectrometry techniques using extraction solvents but also significantly improves resolution efficiency and coverage. This allows for the uncovering of the complex biological reactions behind the antibacterial activity of p-aminophenylborate-modified gold nanoparticles (ABA-AuNPs), establishing a direct link between ABA-AuNPs and bacterial metabolism, and laying the foundation for further elucidating its molecular-level antibacterial mechanism.

[0005] The specific technical solution provided by this invention is as follows:

[0006] This invention provides a method for real-time analysis of the molecular mechanism of gold nanoparticles against bacteria using nanoESI-MS, comprising the following steps:

[0007] Gold nanoparticles were mixed with bacteria and cultured for different time periods. The supernatant was collected by centrifugation and used as the sample solution. The sample was analyzed by nanoESI-MS in positive and negative ion modes to obtain the spectral data of the sample at each time period.

[0008] Orthogonal partial least squares discriminant analysis was performed on the spectral data to establish a predictive model for metabolites and sample categories;

[0009] Based on the prediction model, differential metabolites are screened by calculating the numerical importance of the projected variables.

[0010] Based on the differentially metabolized substances, an association network diagram was constructed to analyze the antibacterial mechanism of gold nanoparticles.

[0011] As a preferred embodiment of the present invention, the detection conditions for analysis using nanoESI-MS are as follows:

[0012] The ionization voltage is 0.5kV~3.5kV, the ion transmission tube temperature is 200℃, the capillary voltage is set to 35 V, the lens voltage is set to 55 V, and the sample introduction distance is 5mm.

[0013] As a preferred embodiment of the present invention, the gold nanoparticles and bacteria are cultured together for different times by culturing the bacteria to the logarithmic growth phase, diluting the bacterial solution and mixing it with the gold nanoparticles, and culturing at 37°C for 0.5h, 2h, 4h and 6h.

[0014] In a preferred embodiment of the present invention, the gold nanoparticles are prepared according to the following steps:

[0015] After mixing p-aminophenylborate acid salt with chloroauric acid trihydrate, a reduction reaction and electrostatic adsorption were carried out under the action of a reducing agent to obtain gold nanoparticles modified with p-aminophenylborate acid salt.

[0016] In a preferred embodiment of the present invention, the molar ratio of p-aminophenylborate acid salt to chloroauric acid trihydrate is 1~5:1~5.

[0017] In a preferred embodiment of the present invention, the reaction of p-aminophenylborate acid salt with chloroauric acid trihydrate is carried out at 0°C for 2-3 hours and the reaction speed is 800-1200 rpm.

[0018] The reducing agent is any one of sodium borohydride, sodium citrate, ascorbic acid, hydroxylamine hydrochloride, H2, and CO.

[0019] As a preferred embodiment of the present invention, the antibacterial effect of gold nanoparticles is the inhibitory effect of gold nanoparticles on Gram-negative bacteria.

[0020] In a preferred embodiment of the present invention, the Gram-negative bacteria is Klebsiella pneumoniae or drug-resistant Klebsiella pneumoniae.

[0021] In a preferred embodiment of the present invention, after gold nanoparticles act on Klebsiella pneumoniae, nine differentially metabolites are upregulated: acetic acid, acetyl-CoA, glutamic acid, glutamine, cysteine, ornithine, putrescine, p-coumaric acid, and phenazine-1-carboxamide. These nine differentially metabolites serve as markers for judging the inhibitory effect of gold nanoparticles on Klebsiella pneumoniae.

[0022] In previous studies, our team used ND-EESI-MS online analysis to explore the effects of ABA-AuNPs on bacterial metabolism, identifying 10 significantly altered metabolites. However, the use of methanol (100%) as the extraction solvent directly damaged the biological sample, limiting its ability to screen for differentially expressed substances and hindering in-depth data mining. Compared to traditional ESI, nano-electrospray ionization (nanoESI), with its ultra-micro nozzle and low flow rate, eliminates the need for an extraction solvent, avoiding damage to the biological sample structure and enabling precise analysis of biological samples and their metabolites, significantly improving analytical sensitivity and range. Simultaneously, nanoESI-MS utilizes an ultra-small diameter nozzle to ensure more precise flow rate control, thereby promoting ionization efficiency. After the analyte solution is ejected from the nano-scale nozzle, it is instantly dispersed into micron- or even nano-sized droplets under electrostatic repulsion. These droplets then form charged droplets under the balance of surface tension and electric field. As the flow rate decreases to the sub-nanoscale level (typically <1000 nL / min), the droplets undergo continuous evaporation and contraction phases, prompting an adjustment in the internal distribution of dissolved protons, ultimately condensing into highly discrete charged particles. Ions are arranged according to their mass-to-charge ratio (…). m / zThe components are separated, captured, and recorded by a mass spectrometer, enabling precise identification and quantification of sample components. This method not only avoids the drawbacks of traditional mass spectrometry techniques using extraction reagents, such as sample damage and information distortion, but also significantly improves resolution efficiency and coverage. This allows for the exploration of the complex biological responses behind ABA-AuNPs' antibacterial activity, establishing a direct link between ABA-AuNPs and bacterial metabolism, and laying the foundation for further elucidating its molecular-level antibacterial mechanism.

[0023] This invention utilizes a reducing agent, particularly sodium borohydride as a strong reducing agent (providing hydride anions H⁻), to reduce chloroauric acid (Au) 3+ Gold ions in the sample are reduced to zero-valent gold (Au). 0 Au reduction in NaBH4 3+ While generating gold nanoparticles, small amino molecules (such as -NH3) are also produced. + The direct coordination or electrostatic adsorption between the molecule and the surface of the newly formed gold nanoparticles enables simultaneous reduction and functionalization modification. Furthermore, the choice of p-aminophenylborate salt in this invention improves the solubility of the small molecule and stabilizes the synthetic system. Compared to ligand exchange methods, the preparation method provided by this invention is a one-step synthesis that is faster and more convenient, making it suitable for large-scale industrial production.

[0024] The small molecule modified gold nanoparticles prepared by this invention have inhibitory effects on both Klebsiella pneumoniae and drug-resistant Klebsiella pneumoniae, and can be used as candidate drugs for the preparation of antibacterial products. Attached Figure Description

[0025] Figure 1 This is an antibacterial characterization of ABA-AuNPs and AuNPs;

[0026] Figure 2 It is a capillary tube used to load samples;

[0027] Figure 3 This is a schematic diagram of the nanoESI-MS experimental setup;

[0028] Figure 4 This is an optimized diagram of the nanoESI-MS test voltage;

[0029] Figure 5 After removing the culture medium background Kp The first-order spectra, (A) positive ion mode, (B) negative ion mode;

[0030] Figure 6 MDR after removing the culture medium background Kp The first-order spectra, (A) positive ion mode, (B) negative ion mode;

[0031] Figure 7It is before and after ABA-AuNPs treatment at different times. Kp PCA analysis chromatograms of differential ions, (A) positive ion mode, (B) negative ion mode;

[0032] Figure 8 It is before and after ABA-AuNPs treatment at different times. Kp OPLS-DA differential ion analysis diagrams, (A) positive ion mode, (B) negative ion mode;

[0033] Figure 9 After 2 hours of ABA-AuNPs treatment Kp Differential ion VIP value, (A) positive ion mode, (B) negative ion mode;

[0034] Figure 10 It is before and after ABA-AuNPs treatment at different times. Kp OPLS-DA efficiency analysis of differential ions, (A) positive ion mode, (B) negative ion mode;

[0035] Figure 11 These are secondary mass spectrometry data of 17 differentially metabolites screened in positive ion mode;

[0036] Figure 12 These are secondary mass spectrometry data of 14 differentially metabolites screened in negative ion mode;

[0037] Figure 13 This study analyzes the content of differentially identified compounds in bacteria after ABA-AuNPs treatment for different durations, detected by 31 positive and negative ion modes. Serial numbers 1-17 were identified under positive ion mode, and 18-31 were identified under negative ion mode.

[0038] Figure 14 This is a heatmap showing the changes in 61 differentially expressed metabolites identified in bacteria after ABA-AuNPs treatment for different durations. Plus signs indicate upregulation, minus signs indicate downregulation, and the intensity of the color indicates the degree.

[0039] Figure 15 This is a network diagram of metabolic substances. The dashed boxes represent substances to be identified, and the solid boxes represent metabolic substances that are upregulated overall. Detailed Implementation

[0040] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0041] The Klebsiella pneumoniae and Enterococcus faecalis involved in this invention were purchased from the China General Microbiological Culture Collection Center, while the drug-resistant Klebsiella pneumoniae and vancomycin-resistant Enterococcus faecalis were purchased from the Henan Provincial Industrial Microbial Culture Collection Center.

[0042] Example 1

[0043] Preparation of gold nanoparticles modified with p-aminophenylborate salt

[0044] (1) In a round-bottom flask, p-aminophenylborate salt (molecular weight 173.4, Aldrich, 5M, 10 µL) and chloroauric acid trihydrate (molecular weight 393.83, Sinopharm Chemical Reagent Co., Ltd., 10M, 5 µL) were dissolved in 10 mL of deionized water. The round-bottom flask was placed on a magnetic stirrer with the parameters set to 0℃ and 1000 rpm. 3 mg of sodium borohydride was dissolved in 1 mL of deionized water and added dropwise to 1000 µL of the solution in the rapidly stirred round-bottom flask. The solution in the flask immediately turned dark brown. The entire reaction lasted for 3 h.

[0045] (2) The obtained p-aminophenylborate modified gold nanoparticles (ABA-AuNPs) were dialyzed for 24 h using a dialysis bag (10 kDa MW cut-off, Solarbio) to remove unreacted chemicals and stored at 4°C for later use.

[0046] Example 2

[0047] Antibacterial properties of ABA-AuNPs

[0048] Antimicrobial properties are determined by the minimum inhibitory concentration (MIC) of ABA-AuNPs.

[0049] Klebsiella pneumoniae ( Kp Drug-resistant Klebsiella pneumoniae (MDR) Kp ), Enterococcus faecalis ( Ef ( ) and vancomycin-resistant Enterococcus faecalis (VRE) were inoculated into liquid LB medium and cultured in a shaker at 260 rpm, 30°C, for 4 h to obtain bacteria in the logarithmic growth phase. The bacteria were then diluted to 1×10⁻⁶ with LB medium. 4 CFU / mL was inoculated into 96-well plates. ABA-AuNPs were diluted 2, 4, 8, 16, 32, 64, and 128 times and added to the culture medium containing bacteria. After 24 h, the turbidity of the bacterial suspension at 600 nm was measured using a microplate reader. 600nm Record the MIC, and the result is as follows: Figure 1 As shown.

[0050] The results showed that ABA-AuNPs had antibacterial activity only against Gram-negative bacteria, and their MIC values ​​were... Kp and MDR Kp It is 6 μg / mL, while for Gram-positive bacteria EfIf VRE is present, the MIC value is greater than 64 μg / mL.

[0051] Example 3

[0052] Preparation and antibacterial properties of unmodified gold nanoparticles

[0053] (1) In a round-bottom flask, chloroauric acid trihydrate (molecular weight 393.83, Sinopharm Chemical Reagent Co., Ltd., 10M, 5 µL) was dissolved in 10 mL of deionized water, and 3 mg of sodium borohydride was dissolved in 1 mL of deionized water. 1000 µL of the solution was added dropwise to the rapidly stirred round-bottom flask. The round-bottom flask was placed on a magnetic stirrer with the parameters set to 0℃ and 1000 rpm. The solution in the flask immediately turned dark brown, and the entire reaction lasted for 3 h.

[0054] (2) The purification steps for unmodified gold nanoparticles (AuNPs) are the same as in Example 1.

[0055] (3) The characterization of the antibacterial properties of AuNPs was the same as in Example 2, and the results are as follows: Figure 1 As shown in the figure. The results indicate that AuNPs have no antibacterial activity against either Gram-negative or Gram-positive bacteria.

[0056] Example 4

[0057] Klebsiella pneumoniae metabolomics detection

[0058] 1. Preparation of capillary glass sample inlet tube

[0059] Capillary tubes for mass spectrometry sample loading were prepared using a horizontally programmed microelectrode puller (P-1000). The puller parameters were set as follows: heating temperature 473℃, pulling force 105 N, speed 70 m / s, delay time 50 ms, and pressure 200 Pa. The instrument was preheated to a steady state for 15 minutes before use. Glass capillary tubes with an inner diameter of 0.78 nm and an outer diameter of 1.00 nm were used. Both ends of the glass tube were fixed to glass tube clamps, and the pulling program was started. The puller uses an advanced microcontroller to precisely control the entire pulling process, achieving sample breakage through the combined action of heating and electric or elastic force. After pulling, the desired capillary glass tube was obtained, as shown below. Figure 2 As shown.

[0060] 2. nano-ESI device

[0061] The experiment used a nano-ESI device for detection, and a schematic diagram of the device is shown below. Figure 3As shown in the diagram, in this apparatus, one end of the platinum electrode is inserted into a capillary containing the sample solution, and the other end is connected to a resistor connected to a high voltage source, driven by an externally applied voltage. The tip of the capillary is aligned with the mass spectrometer port, with a distance of 5 mm between them. Experiments were conducted in both positive and negative ion modes, with the temperature, capillary voltage, and lens voltage all using optimized system settings: the ion transmission tube temperature was set to 200℃, the capillary voltage to 35 V, and the lens voltage to 55 V. The ionization voltage was selected within a range of 0.5–3.5 kV to explore the optimal mass spectral peak response intensity. The ionization voltage condition was optimized by selecting the mass-to-charge ratio (MTBR) in the first-order spectrum. m / z Using a compound ion with a value of 175 as a standard, the effect under different ionization voltages was evaluated by observing changes in its signal intensity. Figure 4 It can be concluded that when the ionization voltage is set to 3 kV, the intensity of the mass spectrum peak reaches its highest value.

[0062] 3. Mass spectrometry analysis

[0063] Will Kp The culture was inoculated into LB medium and cultured on a shaker at 37°C and 260 rpm for 4 hours until the logarithmic growth phase. The OD value of the bacterial culture was measured using a microplate reader. 600nm The bacterial concentration was determined by measuring the optical density. Subsequently, the bacterial concentration was diluted to 10⁻⁶ using LB medium. 5 CFU / mL. The bacterial culture sample was then centrifuged at 8000 rpm for 3 min, and the supernatant was used as the sample solution and loaded into a capillary for mass spectrometry analysis.

[0064] 4. Fingerprint pattern

[0065] Using the optimized conditions for the mass spectrometry experiment described above, the blank group (LB medium) and the experimental group (inoculated with LB medium) were tested in both positive and negative ion modes. Kp Primary fingerprint data were collected using LB medium (the medium containing the blank LB medium). Using Xcalibur software (ThermoScientific, USA), background data from the blank LB medium were removed by subtraction to obtain the primary fingerprint data. Kp The primary fingerprint data was obtained. Origin (version 2022, OriginLab, USA) software was used to process the data, and the corresponding fingerprint was plotted. The results are as follows: Figure 5 As shown.

[0066] from Figure 5From A, it can be concluded that under positive ion conditions, after removing the background from the blank LB medium, the primary fingerprint peak signals of 1,5-pentanediamine, γ-aminobutyric acid, cysteine, leucine, histidine, phenylalanine, arginine, tyrosine, citric acid, and myricetin can be observed. Figure 5 From the results in B, it can be concluded that under negative ion conditions, after removing the background of the blank LB medium, the primary fingerprint peak signals of succinic acid, N-methyl-L-proline, isoleucine, asparagine, malic acid, tyrosine, arginine, tryptophan, linolenic acid, and naringenin can be observed additionally.

[0067] Example 5

[0068] Metabolic profiling of drug-resistant Klebsiella pneumoniae

[0069] (1) The capillary drawing process is the same as in Example 4.

[0070] (2) The nano-ESI experimental parameters are the same as those in Example 4.

[0071] (3) MDR Kp Inoculated into LB medium and cultured at 260 rpm and 37°C. o Under C conditions, the culture was carried out on a shaker for 4 hours until the logarithmic growth phase. The OD value of the bacterial culture was measured using a microplate reader. 600nm The bacterial concentration was determined by measuring the optical density. Subsequently, the bacterial concentration was diluted to 10⁻⁶ using LB medium. 5 CFU / mL. The bacterial culture sample was then centrifuged at 8000 rpm for 3 min, and the supernatant was used as the sample solution and loaded into a capillary for mass spectrometry analysis.

[0072] (4) Using the same mass spectrometry testing conditions as in Example 3, the blank group (LB medium) and the experimental group (inoculated with MDR) were tested in positive and negative ion modes, respectively. Kp Primary fingerprint data were collected from LB medium (blank LB medium). Using Xcalibur software, background data from blank LB medium were removed by subtraction to obtain the MDR. Kp The primary fingerprint data was obtained. The data was processed using Origin software, and the corresponding fingerprint was plotted.

[0073] from Figure 6 From A, it can be concluded that under positive ion conditions, after removing the background from the blank LB medium, the primary fingerprint peak signals of 1,5-pentanediamine, γ-aminobutyric acid, valine, leucine, histidine, phenylalanine, arginine, and myricetin can be observed. Figure 6From the results in B, it can be concluded that under negative ion conditions, after removing the background of blank LB medium, the primary fingerprint peak signals of N-methyl-L-proline, isoleucine, malic acid, phenylalanine, tyrosine, arginine, and spermine can be observed.

[0074] Example 6

[0075] Effects of p-aminophenylborate modified gold nanoparticles on bacterial metabolism

[0076] (1) The capillary drawing process is the same as in Example 4.

[0077] (2) The nano-ESI experimental parameters are the same as those in Example 4.

[0078] (3) Kp The bacterial culture was inoculated into LB medium and cultured on a shaker at 260 rpm and 37 °C for 4 h to reach the logarithmic growth phase. The OD value of the bacterial culture was measured using a microplate reader. 600nm The bacterial concentration was determined by measuring the optical density. Subsequently, the bacterial concentration was diluted to 10⁻⁶ using LB medium. 5 CFU / mL. An experimental group and a control group were set up. In the experimental group, 180 μL of diluted bacterial culture was placed in a centrifuge tube, and 20 μL of 100 μg / mL ABA-AuNPs solution (A-) was added. Kp The control group received an equal volume of LB medium (group ); Kp The bacterial cultures were cultured at 37℃ for different times (0.5, 2, 4, 6 h). The treated bacterial cultures were centrifuged at 8000 rpm for 3 min, and the supernatant was used as the sample solution and loaded into a capillary for mass spectrometry analysis.

[0079] (4) Xcalibur software was used to analyze the sample spectral data collected in positive and negative ion modes at various time intervals. Subsequently, the data were processed in Microsoft Excel software (Office 2016 version, Microsoft Corporation, USA) to map the relationship between mass-to-charge ratio and intensity. Multivariate analysis was performed based on the obtained primary mass spectrometry data. Matlab R2016a software (version 9.0, Mathworks Corporation, USA) was used for normalization, and principal component analysis (PCA) was performed.

[0080] from Figure 7 It can be concluded that, under positive and negative ion modes Kp With A- Kp Significant differences exist between the components, and these differences become more pronounced over time, indicating that the two sets of data exhibit significant differences in several key components.

[0081] Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed using Simca software (version 14.1, Umetrics, Sweden) to establish a predictive framework between metabolic characteristics and sample attributes for predicting sample categories. Figure 8 The results show that the OPLS-DA model reveals the positive and negative ion modes. Kp With A- Kp The data show significant differences in composition within the coordinate graph space, indicating that under supervised mode, the two sets of data exhibit essential differences in several key components.

[0082] Taking 2 hours of mass spectrometry data as an example, by calculating the importance of projected variables (VIP), metabolites with high contributions to classification are screened. The screening results are as follows: Figure 9 As shown, with a threshold of 1, 29 compounds were screened in positive ion mode and 29 compounds were screened in negative ion mode. Among them, 18 compounds overlapped, indicating that after ABA-AuNPs treatment, the above substances have an important influence on the interpretation of the variability in the model and may be key biomarkers affecting bacterial metabolism.

[0083] Using Simca software, 200 displacement tests were performed in both positive and negative ion modes, demonstrating that the OPLS-DA model did not exhibit overfitting in either mode, thus validating the model's effectiveness. The results are as follows: Figure 10 As shown. Using the MetaboAnalyst website (version 6.0, McGill University, Canada), 1000 displacements were performed separately in positive and negative ion modes to verify orthogonal OPLS-DA analysis. The results are as follows. Figure 10 As shown, R 2 Greater than Q 2 The value of R. In statistics, R... 2 Q represents the degree to which the model fits the data, while Q... 2 This represents the predictive power of the model. When R... 2 Greater than Q 2 When the value is , the model is generally considered to be stable and reliable, and has good explanatory and predictive capabilities.

[0084] Example 7

[0085] ABA-AuNPs Kp Effects of metabolites

[0086] (1) The capillary drawing process is the same as in Example 4.

[0087] (2) The nano-ESI experimental parameters are the same as those in Example 4.

[0088] (3) KpThe bacterial culture was inoculated into LB medium and cultured on a shaker at 260 rpm and 37 °C for 4 h to reach the logarithmic growth phase. The OD value of the bacterial culture was measured using a microplate reader. 600nm The bacterial concentration was determined by measuring the optical density. Subsequently, the bacterial concentration was diluted to 10⁻⁶ using LB medium. 5 CFU / mL. An experimental group and a control group were set up. In the experimental group, 180 μL of diluted bacterial culture was placed in a centrifuge tube, and 20 μL of 100 μg / mL ABA-AuNPs solution (A-) was added. Kp The control group received an equal volume of LB medium (group ); Kp The bacterial cultures were cultured at 37℃ for 0.5, 2, 4, and 6 h, respectively. The bacterial culture samples treated for different times were centrifuged at 8000 rpm for 3 min, and the supernatant was used as the sample solution and loaded into capillary tubes for mass spectrometry analysis.

[0089] Of the 61 differential metabolites identified throughout the entire action phase, 31 had overlapping positive and negative ion patterns. These 31 metabolites were then subjected to further quantitative analysis.

[0090] (4) The 31 predicted standards purchased were prepared into 10 μg / mL standard solutions. Secondary collision-induced dissociation experiments were performed under the same mass spectrometry conditions. The compounds were confirmed by comparing the mass spectrometric fingerprints of the standards and bacterial samples. Among them, 17 standards showed more stable CID fingerprints in positive ion mode. Figure 11 The remaining 14 standards were tested using negative ion mode to obtain stable signals. Figure 12 The results showed that the CID mass spectrometry fingerprints of the bacterial samples and the standards were consistent, confirming the compounds corresponding to the 31 differentially expressed ions.

[0091] (4) By preparing standard solutions of different concentrations, ranging from 0.01 to 10 μg / mL, a standard curve was established between the concentration of the standard solution and the relative intensity of the mass spectrometry peak. The results are as follows: Figure 13 As shown in the figure. The results indicate a clear linear relationship between the concentration of the standard and the relative intensity of the mass spectrometry peaks. Measurements were taken after ABA-AuNPs treatment for different times. Kp The relative intensities of the corresponding mass spectrometry peaks of various compounds were used to calculate the content of the corresponding compounds through linear relationships, and the results are as follows: Figure 13 As shown.

[0092] Example 8

[0093] Before and after ABA-AuNPs treatment Kp Differences in metabolites

[0094] (1) The capillary drawing process is the same as in Example 4.

[0095] (2) The nano-ESI experimental parameters are the same as those in Example 4.

[0096] (3) Kp The bacterial culture was inoculated into LB medium and cultured on a shaker at 37°C and 260 rpm for 4 hours until it reached the logarithmic growth phase. The OD value of the bacterial culture was measured using a microplate reader. 600nm The bacterial concentration was determined by measuring the optical density. Subsequently, the bacterial concentration was diluted to 10⁻⁶ using LB medium. 5 CFU / mL. 180 μL of diluted bacterial culture was placed in a centrifuge tube, and 20 μL of 100 μg / mL ABA-AuNPs solution was added. The culture was incubated at 37℃ for 0.5, 2, 4, and 6 h, respectively. The bacterial culture samples treated at different times were centrifuged at 8000 rpm for 3 min, and the supernatant was collected as the sample solution and loaded into a capillary tube for mass spectrometry analysis.

[0097] (4) Continue to monitor the 61 differential metabolites identified throughout the treatment phase and use the MetaboAnalyst website to create heatmaps in positive and negative ion modes to visually observe the up- or down-regulation of bacterial metabolism of the compounds after different treatment times with ABA-AuNPs.

[0098] from Figure 14 The study found that the metabolism of most substances was downregulated, becoming more pronounced over time, peaking at 6 hours. This indicates a decrease in the concentration or activity of the corresponding metabolites after treatment, suppression of downstream reaction chains, or increased activity consuming these metabolites, leading to a reduction in their reserves. However, nine compounds showed upregulated metabolism after treatment, indicating an increase in their concentration or activity, enhanced upstream reactions synthesizing these metabolites, and subsequent accumulation.

[0099] Based on the identified biological functions of 61 substances, this invention constructed a metabolic correlation network diagram for 56 closely related substances. The nine substances showing significant increases are acetic acid, acetyl-CoA, glutamate, glutamine, cysteine, ornithine, putrescine, p-coumaric acid, and phenazine-1-carboxamide, corresponding to four pathways: energy metabolism, oxidative defense and redox homeostasis, membrane integrity and osmotic regulation, and quorum effects. Figure 15 Furthermore, it is expected to become a marker for judging antibacterial efficacy.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for studying the molecular mechanism of the antibacterial effect of gold nanoparticles in real time by nanoESI-MS, characterized in that, Includes the following steps: Gold nanoparticles were mixed with bacteria and cultured for different time periods. The supernatant was collected by centrifugation and used as the sample solution. The sample was analyzed by nanoESI-MS in positive and negative ion modes to obtain the spectral data of the sample at each time period. Orthogonal partial least squares discriminant analysis was performed on the spectral data to establish a predictive model for metabolites and sample categories; Based on the predicted model of metabolites and sample categories, and by calculating the numerical value of the importance of the projected variables, differential metabolites are screened. A correlation network diagram was constructed based on the differential metabolites to analyze the antibacterial mechanism of gold nanoparticles; The detection conditions for analysis using nanoESI-MS are as follows: The ionization voltage is 0.5kV~3.5kV, the ion transmission tube temperature is 200℃, the capillary voltage is set to 35 V, the lens voltage is set to 55 V, and the sample introduction distance is 5mm. The bacteria in question is Klebsiella pneumoniae; After gold nanoparticles acted on Klebsiella pneumoniae, 61 differential metabolites were identified. Nine differential metabolites that were upregulated were acetic acid, acetyl-CoA, glutamate, glutamine, cysteine, ornithine, putrescine, p-coumaric acid, and phenazine-1-carboxamide.

2. The method for real-time analysis of the molecular mechanism of gold nanoparticles against bacteria using nanoESI-MS according to claim 1, characterized in that, The gold nanoparticles were cultured with bacteria for different durations. After the bacteria reached the logarithmic growth phase, the bacterial solution was diluted and mixed with the gold nanoparticles, and then cultured at 37°C for 0.5h, 2h, 4h, and 6h.

3. The method for real-time analysis of the molecular mechanism of gold nanoparticles against bacteria using nanoESI-MS according to claim 1, characterized in that, The gold nanoparticles were prepared according to the following steps: After mixing p-aminophenylborate acid salt with chloroauric acid trihydrate, a reduction reaction and electrostatic adsorption were carried out under the action of a reducing agent to obtain gold nanoparticles modified with p-aminophenylborate acid salt.

4. The method for real-time analysis of the molecular mechanism of gold nanoparticles against bacteria using nanoESI-MS according to claim 3, characterized in that, The molar ratio of p-aminophenylborate salt to chloroauric acid trihydrate is 1~5:1~5.

5. The method for real-time analysis of the molecular mechanism of gold nanoparticles against bacteria using nanoESI-MS according to claim 4, characterized in that, The reaction of p-aminophenylborate acid salt with chloroauric acid trihydrate was carried out at 0℃ for 2-3 hours, and the reaction speed was 800-1200 rpm. The reducing agent is any one of sodium borohydride, sodium citrate, ascorbic acid, hydroxylamine hydrochloride, H2, and CO.

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