Methotrexate Detection DNA Nanoparticle Chip, Its Preparation Method and Application

Abstract

This invention discloses a methotrexate detection DNA nanoparticle chip, its preparation method, and its application, belonging to the field of detection technology. The preparation method includes: centrifuging a silver nanoparticle suspension, adding an equal volume of 100 μM methotrexate aptamer solution, allowing it to stand for 1 hour, and then adjusting the volume to 80 times the original volume; adding 5 μL to a silicon wafer and drying at 45°C to form the chip. The detection method includes: purifying serum by acetonitrile precipitation, dichloromethane extraction, pH adjustment with sodium carbonate, centrifugation, and solid-phase extraction; adding 5 μL to the chip, drying, and then performing SERS detection at 684 cm⁻¹. ‑1 With 1322cm ‑1 The signal ratio is substituted into the standard curve for quantification. This invention combines stable hotspot construction and molecular capture capabilities, resulting in high detection sensitivity. The entire process takes only 15 minutes, making it suitable for rapid clinical monitoring of blood drug concentrations.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a methotrexate detection DNA nanoparticle chip, its preparation method, and its application. Background Technology

[0002] Methotrexate, an antifolate-like antitumor drug, is primarily used to treat malignant tumors such as leukemia, lymphoma, and osteosarcoma, as well as for the long-term control of autoimmune diseases such as rheumatoid arthritis and psoriasis. However, while high doses of methotrexate kill tumor cells in the body, they can also cause a series of side effects, including liver damage and bone marrow suppression. Therefore, strict dosage control is necessary when using methotrexate clinically to reduce the risk of its toxic side effects.

[0003] The clinical reference range for methotrexate concentration is as follows: 24 ≤ 10 μmol / L, 48 ≤ 1 μmol / L, and 72 ≤ 0.1-0.2 μmol / L after methotrexate administration. If methotrexate levels exceed the reference range, leucovorin calcium intervention should be administered promptly to reduce the toxic side effects of methotrexate. However, due to individual differences in metabolic pharmacokinetics, the absorption, metabolism, and excretion of drugs vary from person to person. Therefore, the blood concentration of methotrexate may differ between individuals at 24h, 48h, and 72h after administration. In such cases, monitoring the blood drug concentration in the patient's blood sample is crucial.

[0004] Currently, commonly used methods for monitoring blood drug concentrations include high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-mass spectrometry (LC-MS). However, these methods all have certain drawbacks. For example, the detection efficiency of HPLC is affected by the serum pretreatment method. Although HPLC has significant separation efficiency, it often requires complex pretreatment of blood samples, which is time-consuming. ELISA and LC-MS use expensive instruments, resulting in high costs, and usually require specialized technicians to operate. Therefore, developing a simple, rapid, sensitive, and low-cost new monitoring method is of great significance for the rapid monitoring of blood drug concentrations in clinical practice.

[0005] In recent years, surface-enhanced Raman spectroscopy (SERS) has developed rapidly, and an increasing number of studies have applied SERS to the detection of biomolecules at the tissue and cellular level in vivo. Therefore, developing a new method for monitoring blood drug concentrations using the technological advantages of SERS is promising. Furthermore, the molecular detection capability of SERS mainly depends on the stable gaps between nanoparticles. Therefore, controlling the formation of a stable detection region with stable gaps on the SERS substrate and trapping molecules within these gaps for SERS-based drug concentration monitoring is of great significance. Summary of the Invention

[0006] The purpose of this invention is to provide a DNA nanoparticle chip for methotrexate detection, its preparation method, and its application.

[0007] Currently, the detection of methotrexate blood concentrations using SERS technology does not fully encompass the entire concentration range of methotrexate. Furthermore, SERS substrate fabrication typically only involves forming stable nanoparticle gaps or increasing the substrate's ability to capture methotrexate molecules. A SERS substrate that can both form stable gaps and simultaneously capture methotrexate molecules within these gaps is lacking. This invention addresses this problem by proposing a SERS substrate with stable detection capabilities constructed using an aptamer. Since the methotrexate aptamer has a certain affinity for methotrexate, it can capture methotrexate molecules into the detection region, improving the SERS's ability to detect methotrexate. This enables rapid clinical blood drug concentration monitoring via SERS. This method features high sensitivity, rapid detection, and low cost.

[0008] This application first preprocesses methotrexate blood samples and simultaneously constructs a stable DNA nanoparticle chip capable of specifically capturing methotrexate molecules. This chip is then used to detect the concentration of methotrexate in the blood. This method can quantify the drug concentration in a patient's serum in just 15 minutes.

[0009] To solve the above-mentioned technical problems, the specific technical solution of the present invention is as follows:

[0010] A method for preparing a methotrexate detection DNA nanoparticle chip includes the following steps:

[0011] (1) Centrifuge the silver nanoparticle suspension, discard the supernatant, and obtain the precipitate;

[0012] (2) Add an equal volume of 100 μM methotrexate aptamer solution to the precipitate and let it stand for 1 hour;

[0013] (3) Add ultrapure water to make up to 80 times the original volume of silver nanoparticles to obtain a silver nanoparticle colloidal solution.

[0014] (4) The colloidal solution is dropped onto a silicon wafer and dried at 45°C to form a DNA nanoparticle chip.

[0015] In the above technical solution, the silver nanoparticles have a spherical structure, and the suspension concentration is 6×10⁻⁶. 8 The number of cells / mL was 7500-8000 r / min for 10 min.

[0016] In the above technical solution, the ratio of the total volume after stabilization in step (3) to the original volume of the silver nanoparticles is 80:1.

[0017] In the above technical solution, the amount of colloidal solution added in step (4) is 5 μL.

[0018] In the above technical solution, the concentration of the methotrexate aptamer is 100 μM.

[0019] A DNA nanoparticle chip prepared by the above method.

[0020] An application of the above-mentioned DNA nanoparticle chip in detecting methotrexate concentration in serum includes:

[0021] (1) Serum pretreatment: Add an equal volume of acetonitrile and dichloromethane, adjust the pH with sodium carbonate, centrifuge and take the supernatant, and purify by solid phase extraction;

[0022] (2) Add the purified sample onto the chip and dry it;

[0023] (3) SERS detection, calculation of methotrexate characteristic peak (684 cm⁻¹) -1 ) and aptamer characteristic peak (1322 cm) -1 Signal-to-weight ratio;

[0024] (4) Substitute into the standard curve to calculate the concentration.

[0025] In the above technical solution, the centrifugation speed in step (1) is 13000 r / min and the time is 10 min.

[0026] In the above technical solution, the sample drop volume in step (2) is 5 μL and the drying temperature is 45℃.

[0027] In the above technical solution, the Raman spectrometer parameters in step (3) are: excitation wavelength 785 nm, power 30 mW, and integration time 500 ms.

[0028] Beneficial effects:

[0029] (1) The present invention reduces the interference of other substances in the blood during SERS detection through a two-step process.

[0030] (2) This invention constructs a novel SERS substrate, namely a DNA nanoparticle chip, which can both construct a stable detection region and capture drug molecules within the detection region, thereby improving the sensitivity and stability of SERS for monitoring blood drug concentration. Furthermore, the entire process, from sample collection to quantification of blood drug concentration, takes only 15 minutes. It is suitable for rapid clinical monitoring of blood drug concentration. Attached Figure Description

[0031] Figure 1This represents the linear relationship for detecting methotrexate molecular standard solutions containing different concentrations. The horizontal axis represents the concentration of methotrexate, and the vertical axis represents the ratio of methotrexate signal to aptamer signal.

[0032] Figure 2 This is the relative standard deviation for testing a 5M methotrexate molecular standard solution.

[0033] Figure 3 This represents the linear relationship between the detection of methotrexate molecules in standard serum with different concentrations. The horizontal axis represents the concentration of methotrexate, and the vertical axis represents the ratio of methotrexate signal to aptamer signal.

[0034] Figure 4 This is the SERS spectrum of a serum sample. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the scope of protection of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0036] Example 1:

[0037] The preparation and detection method of DNA nanoparticle chip for methotrexate detection includes the following steps:

[0038] 1. Pretreatment of methotrexate serum samples

[0039] Patient serum was collected and an equal volume of acetonitrile was added to precipitate proteins. Dichloromethane was added to extract the acetonitrile, and sodium carbonate was added to adjust the pH. After centrifugation at 13,000 rpm for 10 min, the supernatant was collected. Solid-phase extraction was performed on the supernatant to obtain the purified methotrexate sample.

[0040] 2. Methotrexate aptamer-based DNA nanoparticle chips with stable detection regions

[0041] (1) Take a concentration of 6×10 8 A solution of spherical silver nanoparticles / ml and sodium citrate was centrifuged at 7500 r / min for 10 min at room temperature. The supernatant was discarded, and an equal volume of 100 μM methotrexate aptamer was added to the precipitate. After standing for 1 h, ddH2O was added to bring the volume up to 80 times the volume of the silver nanoparticles.

[0042] (2) Take 5 μl of the above solution and drop it onto the silicon wafer. After drying in an environment of 45°C, a DNA nanoparticle chip is formed on the silicon wafer.

[0043] (3) Take 5 μl of the pretreated sample and drop it onto the DNA nanoparticle chip.

[0044] (4) Place the silicon wafer with the tissue disruption solution added to it in an environment of 45°C to dry again.

[0045] 3. SERS detection and data output

[0046] (1) After the sample is dried, SERS detection is performed using a small Raman spectrometer (NR785E10W-Plus). The excitation wavelength of the Raman spectrometer is about 785nm, the detection power is 30mw, and the integration time is 500ms to obtain the SERS spectrum of the blood sample.

[0047] (2) The ratio of the methotrexate signal detected in the SERS spectrum to the signal of the chip's own aptamer is fitted to the serum methotrexate quantification curve to calculate the concentration of methotrexate in the blood sample.

[0048] This invention utilizes a methotrexate aptamer to construct a highly efficient and sensitive SERS substrate. Simultaneously, the aptamer itself possesses an affinity for drug molecules, enabling drug molecule capture and enhancing the SERS substrate's ability to detect drug molecules. This improves the sensitivity and stability of SERS for quantifying drug molecule concentrations in blood.

[0049] Example 2:

[0050] This example demonstrates the linearity of standard solutions, the precision of standard solutions, the linearity of spiked serum, and the precision of spiked serum. The results are as follows:

[0051] Figure 1 This diagram illustrates the linear relationship for detecting methotrexate molecular standard solutions containing different concentrations. The horizontal axis represents the concentration of methotrexate, and the vertical axis represents the ratio of methotrexate signal to aptamer signal. The linear range and limit of detection meet clinical requirements, and the signal intensity is directly proportional to the concentration, validating the feasibility of the detection principle.

[0052] Figure 2 This is the relative standard deviation (RSD) for the detection of a 5M methotrexate molecular standard solution. The RSD is 3.22%, which meets the accuracy requirements for quantitative detection.

[0053] Figure 3 This represents the linear relationship between the detection of methotrexate in standard serum with different concentrations of the molecule. The x-axis represents the concentration of methotrexate, and the y-axis represents the ratio of methotrexate signal to aptamer signal. The aptamer maintains its specific capture ability in different matrix concentrations.

[0054] Figure 4 This is the SERS spectrum of a serum sample.

[0055] Example 3:

[0056] Patient Wang X, male, 62 years old, underwent methotrexate treatment in March 2025. Blood drug concentration was measured 24 hours after methotrexate administration, and the methotrexate concentration was determined to be within the safe range.

[0057] (1) In order to determine the blood concentration of methotrexate after administration, 200 μl of serum sample was taken from the patient 24 h after administration;

[0058] (2) Add acetonitrile, 0.1M sodium carbonate and dichloromethane to the serum to be tested in sequence and centrifuge at 13000 rpm for 10 min to obtain the sample supernatant;

[0059] (3) Take 200 μl of supernatant and add it to the solid phase microextraction column, and elute with methanol to obtain the sample solution for SERS detection;

[0060] (4) Take a concentration of 6×10 8 The solution of spherical silver nanoparticles per ml was centrifuged at 7500 rpm for 10 min, the supernatant was removed, an equal volume of aptamer solution was added and diluted to 80 times the volume of the nanoparticles, and the mixture was mixed to obtain the DNA nanoparticle solution.

[0061] (5) Take 5 μl of the above solution and drop it onto a clean silicon wafer, then dry it at 45°C to form a DNA nanoparticle chip;

[0062] (6) Take 5 μl of the extracted sample solution and drop it onto the DNA nanoparticle chip and dry it at 45℃ before performing SERS detection;

[0063] (7) Collect SERS spectra of serum samples, such as Figure 4 Statistical analysis and calculation of methotrexate 684cm -1 The signal and the 1322cm carried by the aptamer -1 The ratio of the signals, and substitute it into the appendix. Figure 3 The concentration of methotrexate in the patient's serum was calculated from the linear curve.

[0064] (8) If the patient’s final blood drug concentration is less than 10 μmol / L, then the concentration of methotrexate in the patient’s blood is confirmed to be within the safe range.

[0065] Example 4:

[0066] Patient Wang X, male, 62 years old, underwent methotrexate treatment in March 2025. Blood drug concentration was measured 48 hours after methotrexate administration, and the methotrexate concentration was determined to be within the safe range.

[0067] (1) In order to determine the blood concentration of methotrexate after administration, 200 μl of serum sample was taken from the patient 48 h after administration;

[0068] (2) Add acetonitrile, 0.1M sodium carbonate and dichloromethane to the serum to be tested in sequence and centrifuge at 13000 rpm for 10 min to obtain the sample supernatant;

[0069] (3) Take 200 μl of supernatant and add it to the solid phase microextraction column, and elute with methanol to obtain the sample solution for SERS detection;

[0070] (4) Take a concentration of 6×10 8 The solution of spherical silver nanoparticles per ml was centrifuged at 7500 rpm for 10 min, the supernatant was removed, an equal volume of aptamer solution was added and diluted to 80 times the volume of the nanoparticles, and the mixture was mixed to obtain the DNA nanoparticle solution.

[0071] (5) Take 5 μl of the above solution and drop it onto a clean silicon wafer, then dry it at 45°C to form a DNA nanoparticle chip;

[0072] (6) Take 5 μl of the extracted sample solution and drop it onto the DNA nanoparticle chip and dry it at 45℃ before performing SERS detection;

[0073] (7) Collect SERS spectra of serum samples, such as Figure 4 Statistical analysis and calculation of methotrexate 684cm -1 The signal and the 1322cm carried by the aptamer -1 The ratio of the signals, and substitute it into the appendix. Figure 3 The concentration of methotrexate in the patient's serum was calculated from the linear curve.

[0074] (8) If the patient’s final blood drug concentration is less than 1 μmol / L, then the concentration of methotrexate in the patient’s blood is confirmed to be within the safe range.

[0075] Example 5:

[0076] Patient Wang X, male, 62 years old, underwent methotrexate treatment in March 2025. Blood drug concentration was measured 72 hours after methotrexate administration, and the methotrexate concentration was determined to be within the safe range.

[0077] (1) In order to determine the blood concentration of methotrexate after administration, 200 μl of serum sample was taken from the patient 72 h after administration;

[0078] (2) Add acetonitrile, 0.1M sodium carbonate and dichloromethane to the serum to be tested in sequence and centrifuge at 13000 rpm for 10 min to obtain the sample supernatant;

[0079] (3) Take 200 μl of supernatant and add it to the solid phase microextraction column, and elute with methanol to obtain the sample solution for SERS detection;

[0080] (4) Take a concentration of 6×10 8The solution of spherical silver nanoparticles per ml was centrifuged at 7500 rpm for 10 min, the supernatant was removed, an equal volume of aptamer solution was added and diluted to 80 times the volume of the nanoparticles, and the mixture was mixed to obtain the DNA nanoparticle solution.

[0081] (5) Take 5 μl of the above solution and drop it onto a clean silicon wafer, then dry it at 45°C to form a DNA nanoparticle chip;

[0082] (6) Take 5 μl of the extracted sample solution and drop it onto the DNA nanoparticle chip and dry it at 45℃ before performing SERS detection;

[0083] (7) Collect SERS spectra of serum samples, such as Figure 4 Statistical analysis and calculation of methotrexate 684cm -1 The signal and the 1322cm carried by the aptamer -1 The ratio of the signals, and substitute it into the appendix. Figure 3 The concentration of methotrexate in the patient's serum was calculated from the linear curve.

[0084] (8) If the patient’s final blood drug concentration is less than 0.1 μmol / L, then the concentration of methotrexate in the patient’s blood is confirmed to be within the safe range.

[0085] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing a methotrexate detection DNA nanoparticle chip, characterized in that, Includes the following steps: (1) Centrifuge the silver nanoparticle suspension, discard the supernatant, and obtain the precipitate; (2) Add an equal volume of 100 μM methotrexate aptamer solution to the precipitate and let it stand for 1 hour; (3) Add ultrapure water to make up to 80 times the original volume of silver nanoparticles to obtain a silver nanoparticle colloidal solution. (4) The colloidal solution is dropped onto a silicon wafer and dried at 45°C to form a DNA nanoparticle chip. When the DNA nanoparticle chip is used for SERS detection, the methotrexate characteristic peak at 684 cm⁻¹ is calculated. -1 The characteristic peak of the aptamer is 1322 cm⁻¹. -1 Signal-to-weight ratio.

2. The preparation method according to claim 1, characterized in that, The silver nanoparticles have a spherical structure, and the suspension concentration is 6×10⁻⁶. 8 The number of cells / mL was 7500-8000 r / min for 10 min.

3. The preparation method according to claim 1, characterized in that, In step (4), the amount of colloidal solution added is 5 μL.

4. A DNA nanoparticle chip prepared by the preparation method according to any one of claims 1-3.

5. The application of the DNA nanoparticle chip according to claim 4 in the detection of methotrexate concentration in serum, characterized in that, include: (1) Serum pretreatment: Add an equal volume of acetonitrile and dichloromethane, adjust the pH with sodium carbonate, centrifuge and take the supernatant, and purify by solid phase extraction; (2) Add the purified sample onto the chip and dry it; (3) SERS detection, calculation of the characteristic peak of methotrexate at 684 cm⁻¹ -1 The characteristic peak of the aptamer is 1322 cm⁻¹. -1 Signal-to-weight ratio; (4) Substitute into the standard curve to calculate the concentration.

6. The application according to claim 5, characterized in that, In step (1), the centrifugation speed is 13000 r / min and the time is 10 min.

7. The application according to claim 5, characterized in that, In step (2), the sample drop volume is 5 μL and the drying temperature is 45℃.

8. The application according to claim 5, characterized in that, In step (3), the Raman spectrometer parameters are: excitation wavelength 785 nm, power 30 mW, and integration time 500 ms.

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