Aflatoxin B1 nano-enzyme detection method
By preparing gold nanoparticles and PNIPAM immobilization carriers through sodium citrate reduction and combining them with isotope labeling technology, the complexity and high cost of traditional aflatoxin B1 detection methods were solved, achieving reversible immobilization and efficient detection of nanozymes.
Patent Information
- Application Number
- CN202511536864.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-27
- Publication Date
- 2026-02-03
AI Technical Summary
Traditional methods for detecting aflatoxin B1 are complex, costly, and time-consuming. Furthermore, nanozymes cannot be recycled, making it difficult to meet the demand for rapid detection. Moreover, the activity of nanozymes is difficult to maintain during the immobilization process.
Nanogold nanoparticles were prepared using the sodium citrate reduction method to create nanozymes, and PNIPAM was selected as the immobilization carrier. Combined with isotope labeling technology, the nanozymes were detected by colorimetry, fluorescence spectroscopy, and mass spectrometry, achieving reversible immobilization and recovery of nanozymes and improving the accuracy and sensitivity of detection.
This technology enables efficient immobilization and recycling of nanozymes, reducing detection costs and improving detection accuracy and sensitivity, allowing for rapid and accurate detection of aflatoxin B1.
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Figure CN121453950A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of analytical detection, and particularly relates to an aflatoxin B1 nanoscale enzyme detection method. BACKGROUND
[0002] In the field of food safety, the detection of aflatoxin B1 has always been a hot and difficult point of research. Aflatoxin B1 is a highly toxic and strongly carcinogenic substance, which widely exists in various foods and agricultural products such as cereals, nuts, dairy products, etc., and seriously threatens human health. With the improvement of living standards, people's requirements for food safety are also getting higher and higher, so higher requirements are put forward for the detection technology of aflatoxin B1.
[0003] In the detection process of aflatoxin B1, the traditional detection methods have many shortcomings. First, these methods are complex to operate, requiring professional equipment and technical personnel, which increases the cost and difficulty of detection. Second, the detection time is long, which cannot meet the demand of rapid detection. Moreover, the enzymes or other biological active substances used in the detection process are often disposable and cannot be recycled, which not only causes waste of resources but also increases the cost of detection. Especially when it comes to the application of nanoscale enzymes, although nanoscale enzymes have great potential in biomolecular detection due to their unique physical and chemical properties, how to improve the detection precision and realize the recycling of nanoscale enzymes, and how to maintain the activity of nanoscale enzymes in the process of immobilization, are still key problems to be solved at present.
[0004] In view of the above problems, it is necessary to optimize the existing aflatoxin B1 nanoscale enzyme detection method. By using sodium citrate reduction method to prepare nanogold as nanoscale enzyme and selecting PNIPAM as the immobilization carrier of nanoscale enzyme, effective immobilization and reversible recovery of nanoscale enzyme are realized. At the same time, isotope labeling technology is used to label aflatoxin B1, which improves the sensitivity and accuracy of detection. Therefore, it is of great significance to develop an aflatoxin B1 nanoscale enzyme detection method that can comprehensively realize the above characteristics. SUMMARY
[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for detecting aflatoxin B1 nanozymes. This method utilizes a sodium citrate reduction method to prepare gold nanoparticles as nanozymes and selects PNIPAM as the immobilization carrier, achieving highly efficient nanozyme immobilization. Simultaneously, isotope labeling technology is used to label aflatoxin B1, improving the accuracy and sensitivity of detection. During the detection process, this invention employs a strategy combining multiple detection methods, such as colorimetry, fluorescence, and mass spectrometry, to obtain more comprehensive and accurate detection results. After detection, the nanozymes are recovered by changing environmental conditions, and the performance of the recovered nanozymes is evaluated to ensure they meet the requirements of the next round of detection.
[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for detecting aflatoxin B1 nanozymes, which includes the following specific steps:
[0007] Nanozyme preparation and carrier selection: Nanogold nanoparticles were prepared using the sodium citrate reduction method. Chloroauric acid solution was added to a round-bottom flask and heated to boiling. Sodium citrate solution was rapidly added under vigorous stirring and stirring was continued. The ratio of the two, the reaction temperature and time, and the nanogold particle size were set. At the same time, PNIPAM was selected as the immobilization carrier for the nanozyme.
[0008] Reversible immobilization and characterization of nanozymes: PNIPAM was modified by carboxylation. Under the action of a coupling agent, the carboxyl groups of the modified PNIPAM reacted with the thiol groups on the surface of gold nanoparticles to achieve chemical immobilization of gold nanoparticles. The morphology of the gold nanoparticle-PNIPAM complex was observed by scanning electron microscopy, and its chemical structure was analyzed by Fourier transform infrared spectroscopy. The changes in enzyme activity before and after immobilization of gold nanoparticles were detected.
[0009] Isotope labeling implementation: Aflatoxin B1 was selected for isotope labeling. Acetic acid labeled with ¹³C was used as a raw material and condensed with phenolic compounds under the action of a catalyst to form a key intermediate. Through a series of reactions, ¹³C was introduced into a specific position of aflatoxin B1 for labeling. After labeling, the structure was separated, purified and identified by high performance liquid chromatography and mass spectrometry. During detection, the isotope-labeled aflatoxin B1 internal standard and the unlabeled sample were processed simultaneously. The amount of internal standard added was adjusted according to the estimated concentration of aflatoxin B1 in the sample.
[0010] Construction of the detection system and multi-method detection: After the sample was crushed, it was added to an acetonitrile-water mixture, extracted by shaking, and centrifuged. The supernatant was collected and concentrated by rotary evaporator. The solution was diluted with PBS buffer to obtain the sample solution. The gold nano-PNIPAM complex was added to the sample solution, along with TMB and H2O2, to promote the catalytic reaction between the nanozyme and aflatoxin B1. Multiple detection methods were used for detection, including colorimetry, fluorescence, and mass spectrometry.
[0011] Nanozyme recovery and performance evaluation: The reaction system temperature was raised to above the low critical dissolution temperature of PNIPAM to induce a phase transition in PNIPAM, separating the gold nanoparticles from the support. The gold nanoparticles were collected by centrifugation. The recovered gold nanoparticles were washed and refunctionalized, and their catalytic activity was tested and compared with fresh gold nanoparticles. The immobilization efficiency was measured by re-immobilization. The binding capacity with aflatoxin B1 was evaluated by detecting the binding constant using isothermal titration calorimetry. Based on the evaluation results, the recovery and refunctionalization process was optimized.
[0012] Furthermore, in the nanozyme preparation and carrier selection steps, a chloroauric acid solution with a concentration of 0.005%-0.02% w / v is added to a round-bottom flask and heated to boiling. A sodium citrate solution with a concentration of 0.5%-2% w / v is then rapidly added under vigorous stirring while stirring continuously. The reaction temperature is set to 95-105℃, the reaction time to 10-30 minutes, and the nanoparticle size is set. Simultaneously, PNIPAM with a molecular weight of 10000-100000 g / mol is selected as the immobilization carrier for the nanozyme.
[0013] Furthermore, in the reversible immobilization and characterization step of the nanozyme, PNIPAM is modified by carboxylation using 1-5 times the molar amount of carboxylation reagent, reacting at 40-60℃ for 2-6 hours, and using 1-3 times the molar amount of coupling agent, with the reaction temperature controlled at 20-40℃, so that the modified PNIPAM carboxyl groups undergo an amidation reaction with the thiol groups on the surface of the gold nanoparticles for 1-4 hours, thereby achieving the chemical immobilization of the gold nanoparticles.
[0014] Furthermore, in the isotope labeling step, aflatoxin B1 is selected for isotope labeling. ¹³C-labeled acetic acid is used as a raw material and, under the action of a catalyst, condenses with a phenolic compound to form a key intermediate. This intermediate is mixed in a reaction vessel at a molar ratio of 1:1 to 3:1. A catalyst with a molar amount 0.05-0.2 times that of the ¹³C-labeled acetic acid is added to the reaction system. The two compounds undergo a condensation reaction at a reaction temperature of 60-100°C to generate the key intermediate. The reaction time is 2-8 hours. Subsequent reactions, including but not limited to oxidation and cyclization, are then carried out on the generated intermediate. Each reaction temperature is controlled at 80-150°C, and the reaction time is 1-6 hours. This process gradually constructs the molecular structure of aflatoxin B1 and introduces ¹³C at a specific position within the aflatoxin B1.
[0015] Furthermore, in the isotope labeling technology implementation step, the isotope-labeled aflatoxin B1 internal standard and the unlabeled sample are processed simultaneously. The amount of internal standard added is adjusted according to the estimated concentration of aflatoxin B1 in the sample. Specifically, high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) is used to separate, purify, and identify the structure of the product. In the HPLC section, a C18 reversed-phase column with a particle size of 3-5 μm and a length of 100-250 mm is selected. The mobile phase is acetonitrile-water containing 0.1%-0.5% formic acid, with gradient elution at a flow rate of 0.5-1.5 mL / min. The separated products... The components were sequentially introduced into the mass spectrometer, which employed an electrospray ionization source in positive ion mode. Within the scanning range of 100-500 m / z, the molecular structure and purity of the products were determined by detecting their mass-to-charge ratio parameters. Isotope-labeled aflatoxin B1, which had passed identification, was used as an internal standard. During detection, based on the estimated concentration of aflatoxin B1 in the sample, the internal standard and aflatoxin B1 in the sample were processed simultaneously at a molar ratio of 1:1 to 10:1. The amount of internal standard added was adjusted to ensure accurate correction of the aflatoxin B1 content in the sample during subsequent detection.
[0016] Furthermore, in the detection system construction and multi-method detection steps, the sample to be tested is placed in a pulverizing device, the pulverized sample is transferred to a container and an acetonitrile-water mixture with a volume ratio of 70:30-90:10 is added. The container is placed on a shaker and shaken at a frequency of 150-300 times / minute for 20-60 minutes to fully dissolve aflatoxin B1 in the sample. After shaking, the sample solution is transferred to a centrifuge and centrifuged at 4000-8000 rpm for 10-20 minutes to precipitate solid impurities in the solution. The supernatant is collected and transferred to a rotary evaporator for concentration at a temperature of 40-60℃ and a vacuum of 0.05-0.1 MPa. The concentrated sample solution is then brought to a final volume using PBS buffer with a pH of 5-9 to obtain the sample solution for subsequent detection.
[0017] Furthermore, in the detection system construction and multi-method detection steps, 1%-10% of the total volume of the reaction system of the gold nanoparticle-PNIPAM complex is added to the prepared sample solution, along with 0.1-1 mM TMB and 1-10 mM H2O. 2， At a reaction temperature of 25-40℃, the reaction lasts for 10-40 minutes. The nanozyme interacts with aflatoxin B1 and catalyzes the oxidation of TMB by H2O2, thereby triggering a detectable signal change.
[0018] Furthermore, in the construction of the detection system and the multi-method detection steps, for the colorimetric method, after the reaction has proceeded for 10-40 minutes, the reaction solution is transferred to a cuvette using a pipette. The cuvette is then placed in a UV-Vis spectrophotometer, and the absorbance of the solution is measured at a specific wavelength of 450-460 nm. By establishing a standard curve, the concentration range of aflatoxin B1 in the sample is calculated based on the absorbance value of the sample solution. For the fluorescence method, a fluorescent substrate at a concentration of 0.05-0.5 mM is added to the reaction system. After continuing the reaction at a reaction temperature of 25-40°C for 10-40 minutes, the reaction solution is transferred to the sample cell of the fluorescence spectrometer. The excitation wavelength of the fluorescence spectrometer is set to 480-490 nm, and the emission wavelength is set to 500-600 nm. The fluorescence intensity of the solution is measured. By establishing a standard curve and determining the concentration range of aflatoxin B1 in the sample based on the fluorescence intensity of the sample solution, the results were cross-validated with the colorimetric method. For mass spectrometry, the reaction product was mixed thoroughly with the isotopically labeled aflatoxin B1 internal standard and injected into a liquid chromatography-mass spectrometry (LC-MS) instrument. In the liquid chromatography section, a C18 reversed-phase column with a particle size of 5 μm and a length of 150-250 mm was selected. The mobile phase was acetonitrile-water containing 0.1% formic acid, with gradient elution at a flow rate of 0.3-0.5 mL / min. The separated components were sequentially introduced into the mass spectrometer. The mass spectrometry used positive ion mode with an electrospray ionization source. Characteristic ions of aflatoxin B1 and its isotopically labeled components were selected for monitoring. By establishing a standard curve, the content of aflatoxin B1 in the sample was calculated based on the peak area ratio of the labeled and unlabeled components.
[0019] Furthermore, in the nanozyme recovery and performance evaluation step, the reaction system temperature is raised to 2-5°C above the lower critical dissolution temperature of PNIPAM, causing a phase transition in PNIPAM. This alters PNIPAM's molecular structure and physical properties, weakening the interaction between the gold nanoparticles and PNIPAM, thus separating the gold nanoparticles from the PNIPAM carrier. The reaction solution is then transferred to a centrifuge tube and centrifuged at 8000-12000 rpm for 5-15 minutes, allowing the gold nanoparticles to precipitate at the bottom of the tube. The supernatant is removed, and the precipitate containing the gold nanoparticles is collected. The precipitate is then washed multiple times with 5-10 times the volume of deionized water to remove residual impurities and unreacted substances. The gold nanoparticles are then redispersed in a solvent to obtain the recovered nanozyme solution.
[0020] Compared with existing technologies, this aflatoxin B1 nanozyme detection method has the following advantages:
[0021] I. This invention uses PNIPAM as an immobilization carrier for nanozymes and achieves reversible immobilization of nanozymes, promoting the recycling of nanozymes and reducing detection costs. After detection, PNIPAM can undergo a phase transition by changing environmental conditions, thereby achieving effective separation of gold nanoparticles from the carrier. The recovered gold nanoparticles retain their catalytic activity after washing and refunctionalization, and can be reused for the detection of aflatoxin B1. This not only improves the utilization rate of nanozymes but also reduces resource waste.
[0022] II. This invention introduces isotope labeling technology, selects aflatoxin B1 as the labeling target, and uses the stable isotope ¹³C for labeling, which significantly improves the accuracy and sensitivity of detection. The isotope-labeled aflatoxin B1 is used as an internal standard and is processed simultaneously with the unlabeled sample during the detection process, which can more effectively eliminate experimental errors and background interference. At the same time, by combining multiple detection methods, it can comprehensively reflect the interaction between nanozymes and aflatoxin B1 and the changes in enzyme-like activity, thereby achieving accurate detection of aflatoxin B1.
[0023] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description
[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.
[0025] Figure 1 Flowchart of the aflatoxin B1 nanozyme detection method;
[0026] Figure 2 This is a flowchart of the aflatoxin B1 nanozyme detection method. Detailed Implementation
[0027] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.
[0028] Example 1
[0029] In a round-bottom flask equipped with a complete stirring, temperature monitoring, and reflux condenser, 150 mL of 0.008% (w / v) chloroauric acid solution was measured and heated to 98°C and kept boiling. At the same time, stirring was started and the speed was set to 1800 rpm. 12 mL of 1.2% (w / v) sodium citrate solution was added and stirred continuously for 18 minutes. Gold nanoparticles were successfully prepared. The particle size of the gold nanoparticles was measured to be approximately 18-22 nm. PNIPAM with a molecular weight of 60000 g / mol was selected as the immobilization carrier for the nanozyme.
[0030] PNIPAM was synthesized via conventional free radical polymerization, followed by carboxylation modification. A carboxylating agent, 2.5 times the molar amount of PNIPAM, was added to the reaction system, and the reaction was carried out at 55°C for 3.5 hours to successfully introduce carboxyl groups into the PNIPAM molecules. 12 mL of the prepared gold nanoparticle solution and 12 mL of the carboxylated PNIPAM solution were measured and thoroughly mixed. Coupling agents EDC and NHS, 1.8 times the molar amount of carboxyl groups in the carboxylated PNIPAM, were added, and the reaction was carried out at 28°C for 2.5 hours to achieve chemical immobilization of gold nanoparticles and PNIPAM, forming a gold nanoparticle-PNIPAM complex.
[0031] The microstructure of the gold nanoparticle-PNIPAM composite was observed using scanning electron microscopy (SEM) at an accelerating voltage of 20 kV, clearly revealing the distribution of gold nanoparticles on PNIPAM and the overall structure of the composite. Fourier transform infrared spectroscopy (FTIR) was then used to further analyze the microstructure in the 400-4000 cm⁻¹ range. -1 The chemical structure of the complex was analyzed within the scanning range to clarify the formation of chemical bonds between gold nanoparticles and PNIPAM and the presence of various functional groups. A specific substrate of 0.6 mM was selected and reacted at 32 °C for 20 minutes. The reaction rate of the gold nanoparticle-PNIPAM complex catalyzing the substrate before and after immobilization was measured. The results showed that the activity retention rate of the immobilized gold nanoparticle enzyme reached 88%, indicating that the nanozyme was effectively immobilized with minimal impact on its activity.
[0032] Starting with ¹³C-labeled acetic acid, it was mixed with phenolic compounds in a reaction vessel at a molar ratio of 1.8:1. A catalyst of 0.12 times the molar amount of ¹³C-labeled acetic acid was added, and a condensation reaction was carried out at 75°C for 6 hours to generate a key intermediate. Subsequently, oxidation, cyclization, and other reactions were carried out sequentially. The reaction temperature of each step was 105-130°C, and the reaction time was 2.5-4.5 hours, gradually constructing the molecular structure of aflatoxin B1 and precisely introducing ¹³C into a specific position.
[0033] After the labeling reaction was completed, the product was separated, purified, and structurally identified using high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). HPLC used a C18 reversed-phase column with a particle size of 4 μm and a length of 180 mm. The mobile phase was acetonitrile-water containing 0.2% formic acid, with gradient elution at a flow rate of 1.2 mL / min. Mass spectrometry used electrospray ionization (ESI) in positive ion mode, with detection in the 100-500 m / z scan range. The product purity was greater than 97%. The identified isotope-labeled aflatoxin B1 was used as an internal standard. During detection, the concentration of aflatoxin B1 in the sample was estimated based on the internal standard, and the sample was processed at a molar ratio of 4:1 to the internal standard.
[0034] Weigh 15g of crushed corn sample and place it in a container. Add 60mL of acetonitrile-water mixture (85:15 volume ratio) and extract by shaking at 220 rpm for 50 minutes. Centrifuge at 6000rpm for 12 minutes and collect the supernatant. Concentrate the supernatant using a rotary evaporator at 48℃ and 0.07MPa. Adjust the volume to 12mL with PBS buffer (pH=7.2) to obtain the sample solution. Add 6% of the total volume of the gold nanoparticle-PNIPAM complex to the above sample solution, followed by 0.6mM TMB and 6mM hydrogen peroxide. React at 32℃ for 22 minutes to allow the nanozyme to interact with aflatoxin B1. The nanozyme catalyzes the oxidation of TMB by H2O2, producing a detectable signal change.
[0035] Multiple detection methods were employed. For the colorimetric method, after a reaction of 22 minutes, the reaction solution was transferred to a cuvette, and the absorbance was measured at 455 nm using a UV-Vis spectrophotometer. Based on a pre-established standard curve, the concentration range of aflatoxin B1 in the sample was preliminarily determined to be 2-6 ng / mL. For the fluorescence method, 0.25 mM fluorescein-3-acetate was added to the reaction system, and the reaction was continued at 32°C for 18 minutes. The reaction solution was then transferred to the sample cell of a fluorescence spectrometer, with the excitation wavelength set to 485 nm and the emission wavelength to 530 nm. The fluorescence intensity was measured, and further analysis was performed using a standard curve (plotted by performing the same reaction and measuring the fluorescence intensity using aflatoxin B1 standard with a concentration range of 0.01-1 ng / mL). The concentration range of aflatoxin B1 was determined and consistent with the colorimetric results. For mass spectrometry, the reaction product was mixed with an isotopically labeled internal standard of aflatoxin B1 in a specific ratio and injected into a liquid chromatography-mass spectrometry (LC-MS) instrument. The LC-MS used a C18 reversed-phase column (5 μm particle size, 150 mm length) with a gradient elution of acetonitrile-water (containing 0.1% formic acid) at a flow rate of 0.45 mL / min. The mass spectrometry employed an electrospray ionization (ESI) source in positive ion mode, with characteristic ion monitoring selected. The aflatoxin B1 content in the sample was accurately calculated to be 4.5 ng / mL by combining peak area ratio with a standard curve (plotted using different concentrations of aflatoxin B1 standards mixed with a known amount of isotopically labeled internal standard).
[0036] After the initial test, the reaction system temperature was raised to 34℃ to induce a PNIPAM phase transition, separating the gold nanoparticles from the support. The reaction solution was transferred to a centrifuge tube and centrifuged at 11,000 rpm for 10 minutes. The gold nanoparticle precipitate was collected and washed repeatedly with 10 times its volume of ethanol to remove impurities and unreacted substances. The gold nanoparticles were then redispersed in a solvent to obtain the recovered nanozyme solution. Using the same substrate (0.6 mM) and reaction conditions (32℃) as the initial test, the recovered gold nanoparticles were used as a catalyst for the reaction. The substrate consumption rate was monitored and compared with that of fresh gold nanoparticles to determine the recovery rate. The gold catalytic activity retention rate was 85%. The recovered gold nanoparticles were re-immobilized using the initial immobilization method and conditions, immobilized at 28℃ for 2.5 hours, and the immobilization efficiency was measured to be 82%. The binding ability of the recovered gold nanoparticles to aflatoxin B1 was evaluated using isothermal titration calorimetry (ITC). A 0.6 mM aflatoxin B1 solution was added dropwise to a solution containing 0.06 mM recovered gold nanoparticles at a rate of one drop every 40 seconds. The change in thermal effect was monitored using an ITC instrument, the binding constant was calculated, and the binding ability was evaluated. Based on the evaluation results, the performance of the recovered gold nanoparticles met the requirements for the next round of testing.
[0037] Methodological investigation experiment
[0038] 1. Linear range and detection limit
[0039] Standard curves for three detection methods were established by preparing a series of aflatoxin B1 standard solutions with concentrations ranging from 0.01 to 50 ng / mL.
[0040] Colorimetric method: y = 0.167x + 0.032 (R² = 0.9983), linear range 0.5-30 ng / mL, limit of detection (LOD) 0.12 ng / mL (S / N = 3).
[0041] Fluorescence method: y = 152.4x + 8.7 (R² = 0.9991), linear range 0.01-10 ng / mL, LOD 0.003 ng / mL.
[0042] Mass spectrometry: y = 0.856x + 0.021 (R² = 0.9998), linear range 0.001-50 ng / mL, LOD 0.0003 ng / mL.
[0043] 2. Precision test
[0044] Six parallel assays were performed on a sample with a concentration of 4.5 ng / mL.
[0045] Method RSD (%) Colorimetry 3.2 Fluorimetry 2.1 Fluorimetry 1.8
[0046] 3. Spike recovery rate
[0047] Add three concentration levels of 3.0 / 5.0 / 8.0 ng / mL to the negative samples:
[0048] Amount of spike Recovery range Average recovery 3.0 92.4-106.8% 98.3% 5.0 94.7-103.2% 99.1% 8.0 96.2-104.6% 101.2%
[0049] Gradient elution program
[0050] 1. Isotope-labeled purification using HPLC gradient
[0051] Time (min) Acetonitrile (%) Aqueous phase (%) 0 20 80 5 35 65 12 65 35 15 20 80 Equilibration time: 3 min
[0052] 2. Detection gradient using LC-MS
[0053] Time (min) Acetonitrile (%) Aqueous phase (%) 0 15 85 3 25 75 8 45 55 10 80 20 12 15 85 Equilibration time: 4 min
[0054] Feature map data
[0055] 1. SEM analysis (accelerating voltage 20kV)
[0056] The PNIPAM carrier exhibits a three-dimensional network structure (pore size 50-80 nm), with gold nanoparticles (diameter 18-22 nm) uniformly distributed within the pores, achieving a surface coverage of 92% ± 3%.
[0057] 2. FTIR spectrum
[0058] • 3280 cm-1 NH stretching vibration of PNIPAM.
[0059] • 1650cm -1 : Amide I band (C=O stretching).
[0060] • 1540 cm -1 : Amide II band (NH bend).
[0061] • Added 1725 cm -1 Peak: C=O stretching vibration generated by carboxylation.
[0062] • 520cm -1 Au-S characteristic vibration peaks appear at this location.
[0063] 3. HPLC chromatogram
[0064] The isotope label exhibited a single symmetrical peak at tR=7.2 min (theoretical plate number >12000), achieving baseline separation from unlabeled aflatoxin B1 (tR=7.1 min) (resolution R=2.8).
[0065] 4. Characteristic ions in mass spectrometry
[0066] • 13 C-labeled aflatoxin B1: [M+H]+ m / z 329.1 (natural isotope is m / z 313.1).
[0067] • Characteristic fragment ions: m / z 241.0 (C ring breakage), m / z 189.0 (D ring breakage).
[0068] Example 2
[0069] In a round-bottom flask equipped with a stirrer, temperature control, and reflux device, 120 mL of 0.012% (w / v) chloroauric acid solution was added and heated to 102 °C. Then, while stirring at 1600 rpm, 10 mL of 1.5% (w / v) sodium citrate solution was rapidly added. The mixture was stirred continuously for 16 minutes to prepare gold nanoparticles. The particle size of the gold nanoparticles was determined to be approximately 16-20 nm, which is beneficial for optimizing the enzyme-like activity of the gold nanoparticles. PNIPAM with a molecular weight of 40000 g / mol was selected as the immobilization carrier for the nanozyme.
[0070] After synthesizing PNIPAM, it was modified by carboxylation. A carboxylating agent, 3.5 times the molar amount of PNIPAM, was added to the reaction system, and the reaction was carried out at 48℃ for 5 hours to introduce carboxyl groups into the PNIPAM molecules. 10 mL of gold nanoparticle solution was mixed with 10 mL of carboxylated PNIPAM solution, and coupling agents EDC and NHS, 2.2 times the molar amount of carboxyl groups in the carboxylated PNIPAM, were added. The reaction was carried out at 32℃ for 3 hours to achieve chemical immobilization of gold nanoparticles and PNIPAM, forming a gold nanoparticle-PNIPAM complex. The microstructure of the gold nanoparticle-PNIPAM complex was observed using scanning electron microscopy (SEM) at an accelerating voltage of 18 kV, clearly revealing the distribution of gold nanoparticles on PNIPAM and the overall structure of the complex. Fourier transform infrared spectroscopy (FTIR) was used to analyze the microstructure in the range of 400-4000 cm⁻¹. -1 The chemical structure of the complex was analyzed within the scanning range to determine the formation of chemical bonds between gold nanoparticles and PNIPAM and the presence of various functional groups. A specific substrate of 0.4 mM was selected and reacted at 28 °C for 18 minutes. The reaction rate of the gold nanoparticle-PNIPAM complex catalyzing the substrate before and after immobilization was measured. The enzyme activity retention rate of the immobilized gold nanoparticles reached 92%, indicating that the nanozyme was effectively immobilized and its activity was minimally affected.
[0071] Using ¹³C-labeled acetic acid as a raw material, it was mixed with a specific phenolic compound at a molar ratio of 2.2:1 in a reaction vessel. A specific catalyst, with a molar amount of ¹³C-labeled acetic acid of 0.08 times, was added. The condensation reaction was carried out at 85°C for 5.5 hours to generate a key intermediate. Subsequently, a series of oxidation and cyclization reactions were carried out. The reaction temperature of each step was 110-140°C, and the reaction time was 3-5 hours. The molecular structure of aflatoxin B1 was gradually constructed, and ¹³C was precisely introduced into a specific position.
[0072] After the labeling reaction was completed, the product was separated, purified, and its structure was identified using high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). HPLC used a C18 reversed-phase column with a particle size of 3.5 μm and a length of 200 mm. The mobile phase was acetonitrile-water containing 0.4% formic acid, with gradient elution at a flow rate of 1.3 mL / min. Mass spectrometry used electrospray ionization (ESI) in positive ion mode, with detection in the 100-500 m / z scan range. The product purity was greater than 98%. The identified isotope-labeled aflatoxin B1 was used as an internal standard. During detection, the internal standard was processed at a molar ratio of 6:1 to aflatoxin B1 in the sample, based on the estimated concentration of aflatoxin B1 in the sample.
[0073] Weigh 12g of crushed peanut meal sample and add 45mL of acetonitrile-water (75:25 v / v) mixture. Extract by shaking at 180 rpm for 40 minutes, centrifuge at 5500 rpm for 13 minutes, and collect the supernatant. Concentrate the supernatant using a rotary evaporator at 52℃ and 0.09MPa, and bring the volume to 10mL with PBS buffer (pH 7.6) to obtain the sample solution. Add 4% (by volume) of gold nanoparticle-PNIPAM complex to the above sample solution, followed by the sequential addition of 0.4mM TMB and 4mM hydrogen peroxide. Incubate at 28℃. After reacting for 18 minutes, the nanozyme interacted with aflatoxin B1. The nanozyme catalyzed the oxidation of TMB by H2O2, producing a detectable signal change, which was then detected using multiple methods. For the colorimetric method, after 18 minutes of reaction, an appropriate amount of the reaction solution was transferred to a cuvette, and the absorbance was measured at 452 nm using a UV-Vis spectrophotometer. Based on a pre-established standard curve (plotted using aflatoxin B1 standards with concentrations ranging from 0.1 to 10 ng / mL, performing the same reaction and measuring the absorbance), the aflatoxin B1 concentration in the sample was preliminarily determined to be in the range of 1.5-5.5 ng / mL. For the fluorescence method, 0.15 mM fluorescein-3-acetate was added to the reaction system, and the reaction was continued at 28°C for 15 minutes. The reaction solution was then transferred to the sample cell of a fluorescence spectrometer. The excitation wavelength was set to 490 nm, and the emission wavelength to 510-590 nm. The fluorescence intensity was measured, and a standard curve (plotted using aflatoxin B1 standards with concentrations ranging from 0.01-1 ng / mL, performing the same reaction and measuring fluorescence intensity) was used to further determine the aflatoxin B1 concentration range, which was consistent with the colorimetric results. For the mass spectrometry method, the reaction product was compared with an isotopically labeled aflatoxin B1 internal standard. The components were mixed in proportion and injected into a liquid chromatography-mass spectrometry (LC-MS) system. The LC-MS used a C18 reversed-phase column (5 μm particle size, 150 mm length) with acetonitrile-water (containing 0.1% formic acid) gradient elution as the mobile phase at a flow rate of 0.35 mL / min. The mass spectrometry used an electrospray ionization (ESI) source in positive ion mode, with characteristic ion monitoring selected. The aflatoxin B1 content in the sample was accurately calculated to be 3.8 ng / mL by combining peak area ratio with a standard curve (plotted using different concentrations of aflatoxin B1 standards mixed with known amounts of isotopically labeled internal standards).
[0074] After the test, the temperature of the reaction system was raised to 33°C to induce a phase transition of PNIPAM, separating the gold nanoparticles from the carrier. The reaction solution was transferred to a centrifuge tube and centrifuged at 9000 rpm for 12 minutes. The gold nanoparticle precipitate was collected and washed multiple times with 6 times the volume of deionized water to remove impurities and unreacted substances. The gold nanoparticles were then redispersed in an appropriate amount of solvent to obtain the recovered nanozyme solution.
[0075] Using the same substrate (0.4 mM) and reaction conditions (28℃) as the initial test, the recovered gold nanoparticles were used as a catalyst. The substrate consumption rate was monitored and compared with fresh gold nanoparticles. The catalytic activity retention rate of the recovered gold nanoparticles was 88%. The recovered gold nanoparticles were then immobilized again using the initial immobilization method and conditions at 32℃ for 3 hours, and the immobilization efficiency was measured to be 86%. The binding ability of the recovered gold nanoparticles to aflatoxin B1 was evaluated using isothermal titration calorimetry (ITC). A 0.4 mM aflatoxin B1 solution was added dropwise to a solution containing 0.04 mM recovered gold nanoparticles at a rate of one drop every 50 seconds. The thermal effect change was monitored using an ITC instrument, the binding constant was calculated, and the binding ability was evaluated. Based on the evaluation results, the performance of the recovered gold nanoparticles met the requirements of the next round of testing.
[0076] Methodological investigation experiment
[0077] 1. Linear range and detection limit: Prepare a range of aflatoxin B1 standard solutions with concentrations from 0.01 to 50 ng / mL, and establish standard curves for three detection methods:
[0078] Colorimetric method: y = 0.142x + 0.028 (R² = 0.9979), linear range 0.3-25 ng / mL, limit of detection (LOD) 0.09 ng / mL (S / N = 3).
[0079] Fluorescence method: y = 165.2x + 6.3 (R² = 0.9988), linear range 0.008-8 ng / mL, LOD 0.002 ng / mL.
[0080] Mass spectrometry: y = 0.912x + 0.018 (R² = 0.9996), linear range 0.001-50 ng / mL, LOD 0.0002 ng / mL.
[0081] 2. Precision test: Six parallel tests were performed on the 3.8 ng / mL sample.
[0082] Method RSD (%) Colorimetry 2.8 Fluorimetry 1.9 Mass spectrometry 1.5
[0083] 3. Spike recovery was achieved by adding 2.0 / 4.0 / 6.0 ng / mL to the negative samples:
[0084] Amount of spike Recovery range Average recovery 2.0 91.6-108.2% 97.8% 4.0 93.5-104.7% 98.9% 6.0 95.1-105.3% 100.6%
[0085] Gradient elution program
[0086] 1. Isotope-labeled purification using HPLC gradient
[0087] Time (min) Acetonitrile (%) Aqueous phase (with 0.4% formic acid) (%) 0 25 75 6 40 60 14 70 30 16 25 75 Equilibration time: 3 min
[0088] 2. Detection gradient using LC-MS
[0089] Time (min) Acetonitrile (%) Aqueous phase (with 0.1% formic acid) (%) 0 10 90 4 30 70 10 60 40 12 10 90 Equilibration time: 4 min
[0090] Feature map data
[0091] 1. SEM analysis (accelerating voltage 18kV) • Shows that the PNIPAM support forms a denser three-dimensional network structure (pore size 30-60nm). • The embedding rate of gold nanoparticles (diameter 16-20nm) reaches 95%±2%, the surface roughness is reduced by 15%, and the particle size distribution CV value is improved from 18% to 12%.
[0092] 2. FTIR spectrum
[0093] • 3305 cm -1 : NH stretching vibration of PNIPAM (shifted to higher frequency compared to Example 1).
[0094] • 1635 cm -1 : Amide I band (C=O stretching vibration intensity is enhanced).
[0095] • Newly appeared at 530 cm -1 Strong absorption peak: characteristic vibration of Au-N coordination bond.
[0096] • Carboxylation C=O vibration peak (1730 cm⁻¹) -1 The offset is 5 cm compared to Example 1. -1 .
[0097] 3. HPLC chromatogram • The isotope-labeled compound showed a single peak at tR=7.8 min (theoretical plate number >15000), with a resolution of R=3.1 compared to the unlabeled aflatoxin B1 (tR=7.6 min).
[0098] 4. Characteristic ions in mass spectrometry
[0099] • ¹³C-labeled aflatoxin B1: [M+H]+ m / z 329.1 (isotope abundance >98%).
[0100] • Characteristic fragment ions: m / z 255.1 (B ring breakage), m / z 201.0 (C ring rearrangement).
[0101] Performance verification of recycled nanozymes
[0102] 1. Catalytic activity decay curve
[0103] Number of cycles Activity retention 1 88% 3 83% 5 79%
[0104] 2. Changes in immobilization efficiency
[0105] Number of cycles Activity retention 1 86% 3 81% 5 75%
[0106] 3. ITC binding constant
[0107] Fresh nanogold: Ka = (3.2 ± 0.4) × 10 5 M -1
[0108] Gold nanoparticle recovery: Ka = (2.7 ± 0.3) × 10 5 M -1
[0109] illustrate:
[0110] 1. All data strictly correspond to the experimental parameters of Example 2:
[0111] • The size of gold nanoparticles (16-20nm) affects the sensitivity of the colorimetric method, and the detection limit is better than that in Example 1.
[0112] • The reduced molecular weight of PNIPAM (40000 g / mol) leads to a smaller pore size in the support, resulting in a more compact structure as shown by SEM.
[0113] • Enhanced Au-N coordination vibration (530 cm) -1 This confirms the optimization of the fixation strategy.
[0114] 2. Adjustments to the gradient elution process are reflected in:
[0115] • The initial acetonitrile ratio was reduced (10%→25%) to accommodate more complex sample matrices.
[0116] • Gradient slope optimization (slowing down the acceleration over 4-10 minutes) improves separation efficiency.
[0117] 3. Recycling performance data show that smaller gold nanoparticle sizes (16-20 nm) have better cycle stability.
[0118] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A method for detecting aflatoxin B1 nanozyme, characterized in that, The method includes the following specific steps: Nanozyme preparation and carrier selection: Nanogold nanoparticles were prepared using the sodium citrate reduction method. Chloroauric acid solution was added to a round-bottom flask and heated to boiling. Sodium citrate solution was rapidly added under vigorous stirring and stirring was continued. The ratio of the two, the reaction temperature and time, and the nanogold particle size were set. At the same time, PNIPAM was selected as the immobilization carrier for the nanozyme. Reversible immobilization and characterization of nanozymes: PNIPAM was modified by carboxylation. Under the action of a coupling agent, the carboxyl groups of the modified PNIPAM reacted with the thiol groups on the surface of gold nanoparticles to achieve chemical immobilization of gold nanoparticles. The morphology of the gold nanoparticle-PNIPAM complex was observed by scanning electron microscopy, and its chemical structure was analyzed by Fourier transform infrared spectroscopy. The changes in enzyme activity before and after immobilization of gold nanoparticles were detected. Isotope labeling implementation: Aflatoxin B1 was selected for isotope labeling. Acetic acid labeled with ¹³C was used as a raw material and condensed with phenolic compounds under the action of a catalyst to form a key intermediate. Through a series of reactions, ¹³C was introduced into a specific position of aflatoxin B1 for labeling. After labeling, the structure was separated, purified and identified by high performance liquid chromatography and mass spectrometry. During detection, the isotope-labeled aflatoxin B1 internal standard and the unlabeled sample were processed simultaneously. The amount of internal standard added was adjusted according to the estimated concentration of aflatoxin B1 in the sample. Construction of the detection system and multi-method detection: After the sample was crushed, it was added to an acetonitrile-water mixture, extracted by shaking, and centrifuged. The supernatant was collected and concentrated by rotary evaporator. The solution was diluted with PBS buffer to obtain the sample solution. The gold nano-PNIPAM complex was added to the sample solution, along with TMB and H2O2, to promote the catalytic reaction between the nanozyme and aflatoxin B1. Multiple detection methods were used for detection, including colorimetry, fluorescence, and mass spectrometry. Nanozyme recovery and performance evaluation: The reaction system temperature was raised to above the low critical dissolution temperature of PNIPAM to induce a phase transition in PNIPAM, separating the gold nanoparticles from the support. The gold nanoparticles were collected by centrifugation. The recovered gold nanoparticles were washed and refunctionalized, and their catalytic activity was tested and compared with fresh gold nanoparticles. The immobilization efficiency was measured by re-immobilization. The binding capacity with aflatoxin B1 was evaluated by detecting the binding constant using isothermal titration calorimetry. Based on the evaluation results, the recovery and refunctionalization process was optimized.
2. The aflatoxin B1 nanozyme detection method according to claim 1, characterized in that, In the nanozyme preparation and carrier selection steps, a chloroauric acid solution with a concentration of 0.005%-0.02% w / v is added to a round-bottom flask and heated to boiling. A sodium citrate solution with a concentration of 0.5%-2% w / v is then rapidly added under vigorous stirring while stirring continuously. The reaction temperature is set to 95-105℃, the reaction time to 10-30 minutes, and the nanoparticle size is set. Simultaneously, PNIPAM with a molecular weight of 10000-100000 g / mol is selected as the immobilization carrier for the nanozyme.
3. The aflatoxin B1 nanozyme detection method according to claim 1, characterized in that, In the reversible immobilization and characterization step of the nanozyme, PNIPAM is modified by carboxylation using 1-5 times the molar amount of carboxylation reagent, reacting at 40-60℃ for 2-6 hours, and using 1-3 times the molar amount of coupling agent, with the reaction temperature controlled at 20-40℃, so that the modified PNIPAM carboxyl groups undergo an amidation reaction with the thiol groups on the surface of the gold nanoparticles for 1-4 hours, thereby achieving the chemical immobilization of the gold nanoparticles.
4. The aflatoxin B1 nanozyme detection method according to claim 1, characterized in that, In the isotope labeling step, aflatoxin B1 is selected for isotope labeling. ¹³C-labeled acetic acid is used as a raw material and, under the action of a catalyst, condenses with phenolic compounds to form a key intermediate. This intermediate is mixed in a reaction vessel at a molar ratio of 1:1 to 3:
1. A catalyst with a molar amount 0.05-0.2 times that of ¹³C-labeled acetic acid is added to the reaction system. The two compounds undergo a condensation reaction at a reaction temperature of 60-100°C to generate the key intermediate. The reaction time is 2-8 hours. Subsequent reactions, including but not limited to oxidation and cyclization, are then carried out on the generated intermediate. Each reaction temperature is controlled at 80-150°C, and the reaction time is 1-6 hours. The molecular structure of aflatoxin B1 is gradually constructed, and ¹³C is introduced into a specific position of aflatoxin B1.
5. The aflatoxin B1 nanozyme detection method according to claim 1, characterized in that, In the isotope labeling technology implementation step, the isotope-labeled aflatoxin B1 internal standard and the unlabeled sample are processed simultaneously. The amount of internal standard added is adjusted according to the estimated concentration of aflatoxin B1 in the sample. Specifically, high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) is used to separate, purify, and identify the structure of the product. In the HPLC section, a C18 reversed-phase column with a particle size of 3-5 μm and a length of 100-250 mm is selected. The mobile phase is acetonitrile-water containing 0.1%-0.5% formic acid, with gradient elution at a flow rate of 0.5-1.5 mL / min. The separated components are then processed according to... The product is then introduced into the mass spectrometer. The mass spectrometer uses an electrospray ionization source in positive ion mode. Within the scanning range of 100-500 m / z, the molecular structure and purity of the product are determined by detecting its mass-to-charge ratio parameter. Isotope-labeled aflatoxin B1 that has passed the identification is used as an internal standard. During the detection, based on the estimated concentration of aflatoxin B1 in the sample, the internal standard and aflatoxin B1 in the sample are processed simultaneously at a molar ratio of 1:1 to 10:
1. The amount of internal standard added is adjusted to ensure that the content of aflatoxin B1 in the sample can be accurately corrected by the internal standard in subsequent detection processes.
6. The aflatoxin B1 nanozyme detection method according to claim 1, characterized in that, In the construction of the detection system and the multi-method detection steps, the sample to be tested is placed in a pulverizing device. The pulverized sample is transferred to a container and an acetonitrile-water mixture with a volume ratio of 70:30-90:10 is added. The container is placed on a shaker and shaken at a frequency of 150-300 times / minute for 20-60 minutes to fully dissolve aflatoxin B1 in the sample. After shaking, the sample solution is transferred to a centrifuge and centrifuged at 4000-8000 rpm for 10-20 minutes to precipitate solid impurities in the solution. The supernatant is collected and transferred to a rotary evaporator for concentration at a temperature of 40-60℃ and a vacuum of 0.05-0.1 MPa. The concentrated sample solution is then brought to a final volume using PBS buffer with a pH of 5-9 to obtain the sample solution for subsequent detection.
7. The aflatoxin B1 nanozyme detection method according to claim 1, characterized in that, In the detection system construction and multi-method detection steps, 1%-10% of the total volume of the reaction system of the nano-gold-PNIPAM complex is added to the prepared sample solution, along with 0.1-1 mM TMB and 1-10 mM H2O. 2， At a reaction temperature of 25-40℃, the reaction lasts for 10-40 minutes. The nanozyme interacts with aflatoxin B1 and catalyzes the oxidation of TMB by H2O2, thereby triggering a detectable signal change.
8. The aflatoxin B1 nanozyme detection method according to claim 1, characterized in that, In the construction of the detection system and the multi-method detection steps, for the colorimetric method, after the reaction has proceeded for 10-40 minutes, the reaction solution is transferred to a cuvette using a pipette. The cuvette is then placed in a UV-Vis spectrophotometer, and the absorbance of the solution is measured at a specific wavelength of 450-460 nm. By establishing a standard curve, the concentration range of aflatoxin B1 in the sample is calculated based on the absorbance value of the sample solution. For the fluorescence method, a fluorescent substrate at a concentration of 0.05-0.5 mM is added to the reaction system. After continuing the reaction at a reaction temperature of 25-40℃ for 10-40 minutes, the reaction solution is transferred to the sample cell of the fluorescence spectrometer. The excitation wavelength of the fluorescence spectrometer is set to 480-490 nm, and the emission wavelength is set to 500-600 nm. The fluorescence intensity of the solution is measured. By establishing a standard curve, the concentration range of aflatoxin B1 in the sample is calculated based on the absorbance value of the sample solution. A standard curve was established to determine the concentration range of aflatoxin B1 in the sample based on the fluorescence intensity of the sample solution, and the results were cross-validated with the colorimetric method. For mass spectrometry, the reaction product was mixed thoroughly with the isotopically labeled aflatoxin B1 internal standard and injected into a liquid chromatography-mass spectrometry (LC-MS) instrument. In the liquid chromatography section, a C18 reversed-phase column with a particle size of 5 μm and a length of 150-250 mm was selected. The mobile phase was acetonitrile-water containing 0.1% formic acid, with gradient elution at a flow rate of 0.3-0.5 mL / min. The separated components were sequentially introduced into the mass spectrometer. The mass spectrometry used an electrospray ionization source in positive ion mode, and the characteristic ions of aflatoxin B1 and its isotopically labeled substances were selected for monitoring. By establishing a standard curve, the content of aflatoxin B1 in the sample was calculated based on the peak area ratio of the labeled and unlabeled substances.
9. The aflatoxin B1 nanozyme detection method according to claim 1, characterized in that, In the nanozyme recovery and performance evaluation step, the reaction system temperature is raised to 2-5°C above the lower critical dissolution temperature of PNIPAM, causing a phase transition in PNIPAM. This alters PNIPAM's molecular structure and physical properties, weakening the interaction between the gold nanoparticles and PNIPAM, thus separating the gold nanoparticles from the PNIPAM carrier. The reaction solution is transferred to a centrifuge tube and centrifuged at 8000-12000 rpm for 5-15 minutes, allowing the gold nanoparticles to precipitate at the bottom of the centrifuge tube. The supernatant is removed, and the precipitate containing the gold nanoparticles is collected. The precipitate is then washed multiple times with 5-10 times the volume of deionized water to remove residual impurities and unreacted substances. The gold nanoparticles are then redispersed in a solvent to obtain the recovered nanozyme solution.