A rapid SERS method for detecting homocysteine
By combining SERS technology with a reducing agent, rapid and accurate detection of homocysteine is achieved, solving the problems of slow speed, high cost and complicated operation of existing detection methods, and making it suitable for on-site testing.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-07
- Publication Date
- 2026-03-10
AI Technical Summary
Existing homocysteine detection methods suffer from slow detection speed, high cost, complex operation, and the need for professional skills, making it difficult to achieve rapid and low-cost on-site detection.
Homocysteine was reduced to a stable state using SERS technology combined with a reducing agent, mixed with Ag sol, and detected using a portable Raman spectrometer. Rapid quantitative analysis was achieved by fitting a standard curve.
It enables rapid, accurate, and low-cost homocysteine detection, making it suitable for ordinary people to operate, reducing detection costs and time requirements, and improving detection efficiency.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomarker detection technology, specifically relating to a rapid SERS method for detecting homocysteine. Background Technology
[0002] Homocysteine is a sulfur-containing amino acid and a metabolite of methionine, one of the 20 essential amino acids in the human body. Scientific research has found that homocysteine is an independent risk factor for cardiovascular and cerebrovascular diseases, increasing the risk of death from cerebrovascular disease by nearly 12 times in three-quarters of hypertensive patients. According to data from Peking University Hospital, the normal range for homocysteine levels in the blood is 6-17 μmol / L for men and 5-14 μmol / L for women. Exceeding this range is diagnosed as hyperhomocysteinemia. Hyperhomocysteinemia can cause fibrous plaques to form on the inner lining of arteries, damaging the elastic layer of blood vessels, leading to systemic atherosclerosis and thrombosis, thus increasing the incidence and mortality of cardiovascular and cerebrovascular diseases. Some nutritionists have suggested that for every 5 μmol / L increase in homocysteine levels above the normal range, the risk of cerebrovascular disease increases by approximately 1.59 times; conversely, a 3 μmol / L decrease reduces the risk by 24%. Therefore, analyzing and detecting homocysteine levels plays an important role in life science research and clinical diagnosis.
[0003] Currently, methods for detecting homocysteine include radioenzyme analysis, immunoassay, high-performance liquid chromatography (HPLC), mass spectrometry, capillary electrophoresis, and microfluidic paper-chip methods. Among these, radioenzyme analysis, while exhibiting good sensitivity and specificity, has a limited measurement range, cumbersome sample processing, and requires enzyme culture, protein precipitation, and neutralization reactions. Immunoassay utilizes immunological theory to determine sample content using antigens or antibodies, but the antigens and antibodies used are expensive, and the results suffer from poor repeatability and high false positive rates. While HPLC improves accuracy, its operation is cumbersome, requires large-scale equipment, and is costly, making it unsuitable for rapid detection. Given the shortcomings of these methods, developing a fast and cost-effective detection method is particularly important.
[0004] Surface-enhanced Raman spectroscopy (SERS) is a crucial spectroscopic technique with extremely high sensitivity, enabling detection at the single-molecule level. It has garnered increasing attention, particularly for the qualitative detection of trace substances and the rapid on-site detection of illicit additives. Therefore, since its discovery, SERS technology has been widely applied in numerous fields. Furthermore, portable Raman spectroscopy offers advantages such as portability and ease of operation. Combining SERS with portable Raman spectroscopy allows for rapid on-site sample detection. Currently, methods for detecting homocysteine using SERS have been reported. While these methods can detect homocysteine in the human biomolecule, they require highly specialized technical expertise, involve complex pretreatment processes, and are costly. Practical applications in the field of biomolecular detection still require a rapid, accurate, and low-cost detection method. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a rapid SERS method for detecting homocysteine. By using a reducing agent to reduce other forms of homocysteine to a single state, the total amount of homocysteine can be determined. Finally, it is mixed with reinforcing nanoparticles in a certain ratio and then subjected to SERS detection. The entire process is fast and the results are reliable.
[0006] SERS technology is a surface-sensitive technique that enhances Raman scattering by adsorbing molecules onto rough metal surfaces, with an enhancement factor of up to 10. 10 -10 14 This means that qualitative and quantitative detection of trace substances is possible. Therefore, due to these numerous advantages, SERS technology has been widely used in many fields since its discovery. Furthermore, compared to large instruments like chromatography, portable Raman spectroscopy is not only easy to carry and operate, but also allows for the rapid detection of samples when combined with SERS technology.
[0007] To achieve the above objectives, one of the technical solutions of the present invention is: a rapid SERS method for detecting homocysteine, specifically comprising the following steps:
[0008] S1: Reduction of homocysteine:
[0009] S11: Take standard homocysteine solutions of different concentrations, add reducing agent and mix thoroughly;
[0010] S12: Add buffer salt to the solution in step S11 to adjust the pH to weakly alkaline, mix thoroughly, let the solution stand, and separate to obtain the supernatant;
[0011] S2: Preparation of Ag sol;
[0012] S3: Standard SERS spectrum test: After the supernatant obtained in step S12 is mixed evenly with the Ag sol prepared in step S2, the SERS spectrum is obtained by SERS detection using a Raman spectrometer.
[0013] S4: Standard curve fitting: Plot the area of the characteristic peaks in the SERS spectrum obtained in step S3 against the logarithm of the standard concentration, and fit the curve to obtain the standard curve.
[0014] S5: Quantitative detection of SERS in the sample to be tested: After processing the sample in step S1, the SERS detection is performed using the method in step S3 to obtain the SERS spectrum of the sample to be tested. Then, the concentration of homocysteine in the sample is quantitatively analyzed based on the fitted standard curve.
[0015] In a preferred embodiment of the present invention, the volume ratio of homocysteine solution to reducing agent in step S11 is 5-15:1.
[0016] In a preferred embodiment of the present invention, the reducing agent in step S11 is one or more of sodium borohydride, 3-butylphosphine, and TCEP.
[0017] In a preferred embodiment of the present invention, the concentration of the reducing agent in step S11 is 5-24 g / L.
[0018] In a preferred embodiment of the present invention, the buffer salt in step S12 is one or more of MgSO4, NaCl, sodium citrate, disodium citrate, PSA, and PBS.
[0019] In a preferred embodiment of the present invention, the concentration of the buffer salt in step S12 is 0.5-3M.
[0020] In a preferred embodiment of the present invention, the pH adjustment in step S12 is 7-8.5.
[0021] In a preferred embodiment of the present invention, after adding the reducing agent in step S11, the mixture is vortexed for 2-5 minutes.
[0022] In a preferred embodiment of the present invention, after adding buffer salt in step S12, the mixture is vortexed for 1-3 minutes and then left to stand for 4-8 minutes.
[0023] In a preferred embodiment of the present invention, the Ag sol in step S2 is prepared by reducing AgNO3 with sodium citrate. The specific preparation method is as follows: 1 mL of 0.1 M AgNO3 solution is added to 100 mL of water, heated to boiling, and then 2.5 mL of 1% sodium citrate solution is added. The mixture is heated to boiling for 3 hours and then cooled to room temperature to obtain Ag sol with a particle size of 80-100 nm.
[0024] In a preferred embodiment of the present invention, the volume ratio of the supernatant to the Ag sol in step S3 is 1:0.4-0.6.
[0025] In a preferred embodiment of the present invention, the Raman spectrometer in step S3 is a portable Raman spectrometer with a laser wavelength of 785nm, a power of 500mW, and a collection time of 3-10s.
[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0027] 1. The detection method of the present invention can quickly and efficiently detect homocysteine in human biological small molecules. The sample to be tested is detected by SERS to obtain a spectrum, and the homocysteine concentration can be quickly obtained from the fitted standard curve, thereby effectively monitoring the status of human health. It is suitable for rapid detection of homocysteine.
[0028] 2. The detection method of the present invention is simple to operate, low in cost, and fast, and can realize portable detection of human biological health level without the need for large precision instruments;
[0029] 3. The detection method of the present invention does not require professional technicians to operate, and no professional knowledge is required for the testing personnel. Ordinary people can quickly master and operate the test, which greatly saves time and labor costs.
[0030] 4. The detection method of the present invention can be applied in the field of medical testing and measurement technology, and can provide a rapid and effective detection means for quality inspection departments, scientific research and testing institutions, hospitals, etc., which can improve detection efficiency, reduce detection costs, and is expected to be promoted to practical applications. Attached Figure Description
[0031] Figure 1 This is a flowchart of the detection method of the present invention;
[0032] Figure 2 Here is a scanning electron microscope (SEM) image of the Ag nanosol prepared in this invention.
[0033] Figure 3 Raman spectra of homocysteine in pure solid and liquid form;
[0034] Figure 4 This is a detection graph of homocysteine standard samples at different concentrations according to the present invention;
[0035] Figure 5 Logarithmic plots of homocysteine concentration curves at different concentrations.
[0036] Figure 6 SERS plots of three different concentrations of homocysteine were generated to simulate the actual test samples. Detailed Implementation
[0037] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments. The detection method flow provided by the present invention is as follows: Figure 1 As shown.
[0038] Preparation of Ag sol: This invention employs a one-pot method for Ag sol to enhance the Raman detection signal of homocysteine. The Ag sol is prepared by reducing AgNO3 with sodium citrate to synthesize Ag nanoparticles with a particle size of 80-100 nm. First, 1 mL of 0.1 M AgNO3 solution is added to 100 mL of water and heated to boiling. Then, 2.5 mL of 1% sodium citrate solution is added, and heating continues for 3 hours. After cooling to room temperature, the Ag sol is obtained for later use. Figure 2 The image shows the scanning electron microscope (SEM) characterization of Ag nanoparticles in Ag sol. It can be seen that the Ag particles have a uniform diameter of 80-100 nm.
[0039] Homocysteine reduction: Under certain physiological conditions, thiol-form homocysteine is activated by methanyl-tRNA synthetase (MetRS), and further converted into the five-membered ring thiolactone form (HTL) of activated homocysteine (Hcy-AMP). This indicates that homocysteine is unstable and easily oxidized. Therefore, adding a reducing agent is a crucial step. The total amount of homocysteine is measured to determine whether hyperhomocysteinemia is present. The reducing agent used here is one or more of sodium borohydride, 3-butylphosphine, and TCEP. After adding the reducing agent, the mixture is vortexed for 2-5 minutes. Raman spectra of pure solid and liquid homocysteine are shown below. Figure 3 As shown.
[0040] Add a buffer salt to the reduced solution to adjust the pH, vortex for 1-3 minutes, and then let it stand for 4-8 minutes. The buffer salt can be one or more of MgSO4, NaCl, sodium citrate, disodium citrate, PSA, and PBS. Adjust the pH to a slightly alkaline state, preferably pH 7-8.5.
[0041] SERS detection process: The final reaction product is mixed with the SERS-enhanced substrate Ag sol and directly used for Raman spectroscopy detection; Detection conditions: Portable Raman spectrometer, laser wavelength of 785nm, power of 500mW; integration time of 3-10s, integration times of 1.
[0042] Example 1
[0043] Preparation of Ag sol: This invention employs a one-pot method for Ag sol to enhance the Raman detection signal of homocysteine. The Ag sol is prepared by reducing AgNO3 with sodium citrate to synthesize Ag nanoparticles with a particle size of 80-100 nm. First, 1 mL of 0.1 M AgNO3 solution is added to 100 mL of water and heated to boiling. Then, 2.5 mL of 1% sodium citrate solution is added, and heating continues for 3 hours. After cooling to room temperature, the Ag sol is obtained for later use.
[0044] SERS curves of the products after reduction of homocysteine solutions of different concentrations were collected: Eight standard solutions containing different concentrations of homocysteine (0, 0.5, 1, 2, 4, 8, 10, and 20 mg / L) were prepared, with the 0 mg / L solution serving as a blank control. 200 μL of each homocysteine solution was mixed with 20 μL of reducing agent (10-50 g / L) and vortexed for 2-5 min. After adding buffer salt, vortexing was continued for 1-3 min, followed by a 3-min settling period. Then, 40 μL of Ag sol was added for SERS detection. Finally, 30 μL of buffer salt was added, and the mixture was evacuated several times for SERS measurement. The SERS spectra of the products after reduction of homocysteine at different concentrations were obtained, as shown below. Figure 4 As shown.
[0045] Fitting the standard curve: 710 cm⁻¹ after the reduction reaction -1 A standard curve was plotted by comparing the peak area of the characteristic peak with the logarithm of the homocysteine concentration in the standard sample. The peak area was plotted on the ordinate, and the logarithm of the homocysteine concentration (base 10) on the abscissa. The two variables showed a linear relationship, satisfying the equation: Y = 337339X - 935697, where Y is 710 cm⁻¹. -1 The peak area of the characteristic peak, where X is lg (homocysteine concentration).
[0046] SERS test simulating actual test samples: Different concentrations of homocysteine were added to serum samples that did not contain homocysteine to simulate actual test samples. After processing, Raman spectroscopy was performed. The concentration of homocysteine in the test sample could be obtained by fitting a standard curve and compared with the actual concentration.
[0047] Homocysteine was added to serum samples that did not contain homocysteine at concentrations of 1 mg / L, 5 mg / L, and 10 mg / L. The samples were then pretreated as described above, and finally, SERS detection was performed under the following conditions: laser wavelength 785 nm, power 500 mW, integration time 2 s, and one integration iteration. The results are shown below. Figure 6The SERS spectrum of the simulated actual sample with added homocysteine standard is shown. The average homocysteine concentrations measured in the three experiments were 0.848 mg / L, 4.185 mg / L, and 8.75 mg / L, respectively. The actual and measured homocysteine concentrations were 84.8%, 83.7%, and 87.5% of the values, respectively.
[0048] In actual testing, the sample is processed and then subjected to Raman spectroscopy. The concentration in the sample can be obtained by fitting a standard curve.
[0049] The detection method of this invention differs from other SERS methods. Other methods involve complex and time-consuming substrate preparation, are highly technical, or rely on aptamers, antigen-antibody specific binding, magnetic nanoparticle modification, or the preparation of 3D detection substrates. These methods are complex, use expensive antigens, antibodies, and aptamers, have poor reproducibility, and produce unstable results. Furthermore, detecting homocysteine using chromatography or mass spectrometry requires large-scale equipment, demanding specific experimental sites and environments, is expensive, and requires specialized technicians. The method of this invention is simple in concept and convenient to operate. By adding a reducing agent, it not only prevents homocysteine oxidation but also reduces other forms of homocysteine to achieve the goal of detecting total homocysteine content. This method offers convenient pretreatment, low cost, and speed, utilizing a portable Raman spectrometer, making it ideal for rapid on-site detection of SERS.
[0050] The above embodiments are merely optimized implementations of the present invention, used to exemplify the principles and effects of the present invention, and are not intended to limit the present invention. It should be noted that any modifications made to the above embodiments by those skilled in the art without departing from the spirit and scope of the present invention should also be considered within the protection scope of the present invention.
Claims
1. A SERS method for rapid detection of homocysteine, characterized by, The method comprises the following steps: S1: reduction of homocysteine; S11: taking a standard homocysteine solution with different concentrations, adding a reducing agent and mixing thoroughly; S12: adding a buffer salt to the solution in step S11 to adjust the pH to weak alkaline, mixing thoroughly and then standing the solution, and separating the supernatant; S2: preparation of Ag sol; S3: standard SERS spectrum test: mixing the supernatant obtained in step S12 with the Ag sol prepared in step S2 uniformly, and then using a Raman spectrometer to perform SERS detection to obtain a SERS spectrum; S4: standard curve fitting: plotting the area of a characteristic peak in the SERS spectrum obtained in step S3 against the logarithm of the concentration of the standard sample, and fitting to obtain a standard curve; S5: SERS quantitative detection of a sample to be tested: treating the sample to be tested by step S1, and then performing SERS detection by the method of step S3 to obtain a SERS spectrum of the sample to be tested, and then quantitatively analyzing the concentration of homocysteine in the sample according to the fitted standard curve. 2.The SERS method for rapid detection of homocysteine according to claim 1, characterized in that, The volume ratio of the homocysteine solution to the reducing agent in step S11 is 5-15:
1. 3.The SERS method for rapid detection of homocysteine according to claim 1, characterized in that, The reducing agent in step S11 is one or more of sodium borohydride, 3-butyl phosphine and TCEP. 4.The SERS method for rapid detection of homocysteine according to claim 1, characterized in that, The buffer salt in step S12 is one or more of MgSO4, NaCl, sodium citrate, disodium citrate, PSA and PBS. 5.The SERS method for rapid detection of homocysteine according to claim 1, characterized in that, The concentration of the reducing agent in step S11 is 5-24 g / L, and the concentration of the buffer salt in step S12 is 0.5-3 M. 6.The SERS method for rapid detection of homocysteine according to claim 1, characterized in that, After adding the reducing agent in step S11, vortex mixing is performed for 2-5 min, and after adding the buffer salt in step S12, vortex mixing is continued for 1-3 min, and the standing time is 4-8 min. 7.The SERS method for rapid detection of homocysteine according to claim 1, characterized in that, The pH adjustment in step S12 is 7-8.
5. 8.The SERS method for rapid detection of homocysteine according to claim 1, characterized in that, The Ag sol in step S2 is prepared by reducing AgNO3 with sodium citrate, and the synthesized Ag sol has a particle size of 80-100 nm. 9.The SERS method for rapid detection of homocysteine according to claim 1, characterized in that, The volume ratio of the supernatant to the Ag sol in step S3 is 1:0.4-0.
6. 10.The SERS method for rapid detection of homocysteine according to claim 1, characterized in that, The Raman spectrometer in step S3 is a portable Raman spectrometer, the laser wavelength is 785 nm, the power is 500 mW, and the collection time is 3-10 s.