Rapid MDA content detection method based on nanogold labeling
By preparing gold nanorod particles and functionalizing MDA antibodies, and utilizing the surface plasmon resonance absorption peak offset value of the gold nanorods, the problems of complex operation, high cost and low sensitivity of the existing MDA detection method were solved, and rapid and accurate MDA content detection was achieved.
Patent Information
- Application Number
- CN202510611866.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-13
- Publication Date
- 2025-09-05
AI Technical Summary
Existing MDA detection methods have problems such as complicated operation, expensive equipment, high detection cost, and low sensitivity, making it difficult to achieve rapid and accurate detection.
The long-diameter stable gold nanorod particles were prepared by the seed growth method based on the polymerization of gold nanorods. They were functionalized with thiol-MDA antibodies, and the standard curve was drawn using the surface plasmon resonance absorption peak shift value of the gold nanorods to achieve rapid detection of MDA content.
The method achieves rapid, sensitive and accurate detection of MDA content, overcomes the drawbacks of existing technologies, and provides a detection solution that is easy to operate and low in cost.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for rapid detection of MDA content based on nanogold labeling. Background Art
[0002] Malondialdehyde (MDA) is a lipid peroxidation product that is widely present in organisms. The level of MDA content can reflect the degree of lipid peroxidation in the organism and further evaluate the degree of cell damage. The detection of MDA content is of great significance in the fields of medicine, food, and environment. At present, the main MDA detection methods include spectrophotometry, high performance liquid chromatography, gas chromatography-mass spectrometry, etc. These methods have certain advantages in terms of sensitivity, accuracy, and detection speed, but have the following shortcomings: (1) Spectrophotometry is cumbersome to operate, easily affected by factors such as sample color and turbidity, and has low sensitivity. (2) High performance liquid chromatography equipment is expensive, the operation is complicated, and the detection cycle is long. (3) Gas chromatography-mass spectrometry requires professional equipment, has high requirements for sample pretreatment, and has high detection costs.
[0003] Gold nanoparticles (GNPs) are a type of metal nanomaterial with surface plasmon resonance properties. The surface of gold nanoparticles is rich in chemical activity and can specifically bind to biological molecules (such as proteins, nucleic acids, etc.) to form stable gold nanoparticle-labeled complexes. Gold nanoparticle labeling detection technology combines gold nanoparticles with target molecules (such as antibodies, antigens, etc.) to form gold nanoparticle-labeled complexes. When the labeled complex specifically binds to the target molecule in the sample to be tested, the surface plasmon resonance properties of the gold nanoparticles change, resulting in a change in the absorbance or luminescence intensity of the solution. By detecting changes in absorbance or luminescence intensity, rapid and sensitive detection of target molecules in the sample to be tested can be achieved.
[0004] The gold nanoparticle-labeled rapid MDA content detection method of the present invention has the advantages of high sensitivity, simple operation, and low cost, and is expected to play an important role in the fields of medicine, food, and environment. Summary of the Invention
[0005] The present invention aims to provide a method for rapid detection of MDA content based on nanogold labeling with high sensitivity.
[0006] In order to solve the above technical problems, the present invention adopts the following measures:
[0007] Using the seeded growth method of aggregated gold nanorods, we generate relatively stable gold nanorods. These are then functionalized with thiol-modified MDA antibodies. After mixing with a solution containing MDA, the MDA content in the solution is determined by measuring the surface plasmon resonance absorption peak shift of the gold nanorods and comparing it to a standard curve. The detection limit can reach 0.000005-300 nmol / L.
[0008] Specifically, the preparation of the gold nanorod particles includes the following steps: (1) Preparation of gold crystal seed solution Mix 625 μL of 2 mmol / L chloroauric acid, 1.88 mL of 0.2 mol / L CTAB, and 1.37 mL of pure water thoroughly. Add 450 μL of ice-cold NaBH₄ and mix vigorously for 2 minutes. The solution will turn from yellow to brown. Incubate at 28°C for 2 hours. (2) Preparation of growth medium 1) Take 11.875 mL of 0.2 mol / L CTAB, add 7.71 mL of pure water, and mix thoroughly. 2) Add 5 mL of 2 mmoL / L chloroauric acid, 150 μL of 10 mmoL / L AgNO3 and 160 μL of 100 mmoL / L AA, mix thoroughly, and the solution turns from yellow to colorless. Save for later use. (3) Growth and purification of gold nanorods. 1) Add 108 μL of gold crystal seed solution to the above growth medium, mix thoroughly, and incubate at 28°C for 12 h. 2) Centrifuge the prepared gold nanorod solution at 8500 rpm for 40 min and discard the supernatant. 3) Add 12.5 mL of ultrapure water, mix well, and dispense into 1.5 mL EP tubes. Centrifuge at 14,000 rpm for 10 min and discard the supernatant. 4) Add 1 mL of 0.5 mmol / L CTAB to each tube, mix well, and store at 28°C until ready for use.
[0009] Specifically, after the preparation of the gold nanorod particles is completed, the preparation quality of the gold nanorod particles needs to be measured. Specifically, 5 mL of the prepared gold nanorod particle solution is taken, and a scanning ultraviolet spectrophotometer is used to measure the plasma resonance absorption peak of the prepared gold nanorod particles in the range of 500-900 nm. The standard concentration is 750-800 nm, more preferably 770-790 nm, and most preferably 780 nm.
[0010] Specifically, the functionalization of gold nanorods includes the following steps: (1) Thiolation of MDA Antibody 0.2 mL of MDA antibody at different concentrations (1-30 μg / mL) was added to 0.8 mL of 0.5 mol / L PBS buffer containing EDTA, mixed thoroughly, and allowed to stand for 2 minutes. 30 μL of 5 mg / mL Traut's reagent was added and allowed to stand at 28°C for 1 hour. (2) Functionalization of gold nanorods Take 0.5 mL of the thiol-modified antibody and add it to 2 mL of the gold nanorod solution, mix well, then add 1 mL of PEG-6000 solution dropwise and let it stand at 28°C for 2 h.
[0011] Specifically, after the functionalization of the gold nanorods is completed, the absorption peak of the functionalized gold nanorods needs to be detected for the subsequent calculation of the red peak shift after the antibody and antigen bind, and then the MDA content is obtained. Specifically: 5 mL of functionalized gold nanorod particle solution is taken, and a scanning UV spectrophotometer is used to detect the plasma resonance absorption peak of the prepared gold nanorod particles in the range of 500-900 nm, and the red peak shift value is calculated. The shift value > 2 nm is recorded as the effective value.
[0012] Specifically, after the gold nanorods are functionalized, the MDA content can be determined, which includes the following steps: (1) MDA binding 100 μL of the target solution to be detected (containing MDA) was added, mixed with the functionalized gold nanorod solution and reacted for 2 minutes before performing absorption peak detection. (2) Detection of MDA standard curve 100 μL of MDA standard solution (0.01, 0.1, 1, 10, 100 nmol / mL) was added respectively, and the standard curve was drawn as shown below. Figure 2 As shown, after being mixed and reacted with the functionalized gold nanorod solution for 2 minutes, the absorption peak was detected, the red peak shift value was calculated, and a standard curve of the red peak shift value-MDA content was drawn. (3) Calculation of MDA content The MDA content in the target solution can be calculated by comparing the offset value of the absorption peak obtained in (1) with the standard curve in (2).
[0013] The beneficial effects of the present invention are: the present invention uses the convenient, rapid and highly accurate nano-gold rod method to detect MDA, an indicator for evaluating oxidative damage in the body, overcoming the shortcomings of existing MDA detection technology and providing a rapid breakthrough idea in the field of oxidative damage monitoring. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1These are the measurement results of gold nanorods functionalized with thiol-MDA antibodies at different concentrations: a: 1 μg / mL, b: 5 μg / mL, c: 10 μg / mL, d: 15 μg / mL, e: 20 μg / mL, f: 30 μg / mL.
[0015] Figure 2 It is the standard fitting curve of the red peak shift value of gold nanoparticle labeled MDA.
[0016] Figure 3 These are the measurement results after combining different concentrations of MDA standards with functionalized gold nanorods. DETAILED DESCRIPTION
[0017] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific figures:
[0018] 1 Materials and Methods
[0019] 1.1 Main reagents and instruments
[0020] Reagents: tetraethoxypropane; chloroauric acid; CTAB; NaBH4; AgNO3; AA; Traut's reagent; MDA human antibody.
[0021] Instruments: constant temperature centrifuge, magnetic stirrer, continuous ultraviolet spectrophotometer (500-900 nm), analytical balance, constant temperature box, pipette.
[0022] 1.2 Preparation of MDA standard solution
[0023] 10 mmol / L MDA standard solution: Accurately pipette 1.1 mL of 1,1,3,3-tetraethoxypropane into 50 mL of 0.1 mol / L HCl solution. Incubate on a magnetic stirrer at 50°C for 1 hour. Once the solution turns yellow, cool, extract with anhydrous ethanol, and purify by rotary evaporation to obtain MDA. Weigh 7.2063 g of purified MDA and dissolve it in 100 mL of PBS.
[0024] 1.3 Preparation of gold nanorods
[0025] 1.3.1 Gold Crystal Seed Liquid:
[0026] Mix 625 μL of 2 mmol / L chloroauric acid, 1.88 mL of 0.2 mol / L CTAB, and 1.37 mL of pure water thoroughly. Add 450 μL of ice-cold NaBH₄ and mix vigorously for 2 minutes. The solution will turn from yellow to brown. Incubate at 28°C for 2 hours.
[0027] 1.3.2 Growth medium preparation:
[0028] Take 11.875 mL of 0.2 mol / L CTAB, add 7.71 mL of pure water, and mix thoroughly; add 5 mL of 2 mmol / L chloroauric acid, 150 μL of 10 mmol / L AgNO3, and 160 μL of 100 mmol / L AA, mix thoroughly, and the solution will turn from yellow to colorless. Store at 28 °C for later use.
[0029] 1.3.3 Aggregation of gold nanorods:
[0030] Take 108 μL of gold crystal seed solution and add it to the above growth medium, mix thoroughly, and incubate at 28°C for 12 h.
[0031] 1.3.4 Gold nanorod purification:
[0032] The prepared gold nanorod solution was centrifuged at 8500 rpm for 40 min and the supernatant was discarded. 12.5 mL of ultrapure water was added, mixed, and divided into 1.5 mL EP tubes. The solution was centrifuged at 14000 rpm for 10 min and the supernatant was discarded. 1 mL of 0.5 mmol / L CTAB was added to each tube, mixed, and stored at 28°C for later use.
[0033] 1.4 Antibody thiolation
[0034] 0.2 mL of antibodies of varying concentrations were added to 0.8 mL of 0.5 mol / L PBS buffer containing EDTA, mixed thoroughly, and allowed to stand for 2 minutes. 30 μL of 5 mg / mL Traut's reagent was added and the mixture was incubated at 28°C for 1 hour.
[0035] 1.5 Functionalized gold nanoparticles
[0036] Take 0.5 mL of the thiol-modified antibody and add it to 2 mL of the gold nanorod solution, mix well, then add 1 mL of PEG6000 solution dropwise and let it stand at 28°C for 2 h.
[0037] 1.6 Preparation and determination of gold nanorods
[0038] The gold nanoparticles were scanned and measured using an ultraviolet spectrophotometer (Lambda 650, PerkinElmer, Germany) (wavelength: 900-500 nm).
[0039] 1.7MDA standard combined with gold nanoparticles
[0040] After the gold nanorods were bound to the MDA antibody, the gold nanorods were measured. After confirming that the antibody was labeled with the gold nanorods, 100 μL of MDA was added, mixed and reacted for 2 minutes, and then the measurement was repeated. Each concentration was measured three times and the average value was taken.
[0041] 2 Results
[0042] 2.1 Gold nanorod labeling of MDA
[0043] The results of UV spectrophotometer scanning showed that MDA antibody could bind to gold nanorods. When 1, 5, 10, 15, 20, and 30 μg / mL of antibody were used to bind to gold nanorods, the red peak shift values were 6.15, 7.40, 8.66, 8.6, 8.50, and 8.44 nm, respectively. The antibody at 10 μg / mL had the best binding effect. The subsequent experiments were conducted at a concentration of 10 μg / mL. Figure 1 ).
[0044] According to the results of red peak shift, the detection range of MDA target content detected by gold nanorod labeled MDA antibody is 0.000005-300nmoL / L, and the maximum red peak shift is 3.42nm ( Figure 3 ).
[0045] The above results show that this study used gold nanorods to label MDA antibodies for MDA content detection. The results showed that when the concentration of MDA antibody was 10μg / mL, the red peak offset value was high, and the detection range of MDA target was 0.000005-30nmoL / L. The normal human serum MDA content is within this range and can be detected.
[0046] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for rapid detection of MDA content based on gold nanoparticle labeling, characterized in that: Gold nanorods of specific size and characteristics are functionalized with thiolated MDA antibodies. The MDA content is determined by detecting the shift of the surface plasmon resonance absorption peak after mixing with a solution containing MDA.
2. The detection method according to claim 1, wherein the gold nanorod particles used are prepared by a polymerization gold nanorod seed growth method and have a specific plasmon resonance absorption peak range. 3 . The detection method according to claim 1 , wherein the concentration of the thiolated MDA antibody used is in the range of 1-30 μg / mL.
4. The absorption peak shift standard curve according to claim 1, wherein The MDA concentration range of the standard curve was 0.000005-30 nmoL / L.