DNA nanoparticle chip for detecting methotrexate as well as preparation method and application of DNA nanoparticle chip

Abstract

The invention discloses a DNA nanoparticle chip for methotrexate detection and a preparation method and application thereof, and belongs to the technical field of detection.The preparation method comprises the steps that after a silver nanoparticle suspension is centrifuged, a 100 mu M methotrexate aptamer solution with the same volume is added, standing is conducted for 1 h, and the volume is fixed to 80 times of the volume; and dropwise adding 5mu L of the solution to a silicon wafer, and drying at 45 DEG C to form the chip. The detection method comprises the following steps: after serum is subjected to acetonitrile precipitation, dichloromethane extraction, sodium carbonate pH adjustment, centrifugation and solid-phase extraction purification, taking 5 [mu] L, dropwise adding to the chip, drying, carrying out SERS detection, and substituting a signal ratio of 684 cm <-1 > to 1322 cm <-1 > into a standard curve for quantification. The method has stable hotspot construction and molecular capture capabilities, is high in detection sensitivity, only needs 15 minutes in the whole process, and is suitable for rapid monitoring of clinical blood concentration.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and in particular relates to a DNA nanoparticle chip for detecting methotrexate, a preparation method thereof, and an application thereof. Background Art

[0002] Methotrexate, an antifolate anti-tumor drug, is primarily used to treat malignant tumors such as leukemia, lymphoma, and osteosarcoma, as well as for the long-term management 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 required in clinical use of methotrexate to reduce the risk of toxic side effects.

[0003] The reference range of methotrexate concentration in clinical practice is: after taking methotrexate, 24 ≤ 10 μmol / L, 48 ≤ 1 μmol / L, and 72 ≤ 0.1-0.2 μmol / L. Once the methotrexate concentration is higher than the reference range, calcium folinate should be given in a timely manner to reduce the toxic side effects of methotrexate. However, during the use of drugs in clinical practice, due to differences in metabolic dynamics between individuals, the absorption, metabolism, and excretion capabilities of different people vary. Therefore, at 24h, 48h, 72h, and other times after methotrexate administration, the methotrexate concentration in the blood of individuals varies. At this time, it is crucial to monitor the blood drug concentration of the patient's blood samples.

[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, HPLC detection is affected by serum pretreatment methods. Although HPLC separation is effective, it often requires complex and time-consuming pretreatment of blood samples. ELISA and LC-MS use expensive instruments, are relatively costly, and typically require specialized technicians to operate. Therefore, the development of a simple, rapid, sensitive, and low-cost new monitoring method is of great significance for rapid clinical monitoring of blood drug concentrations.

[0005] Surface-enhanced Raman spectroscopy (SERS) has rapidly developed in recent years, with a growing number of studies applying SERS to the detection of biomacromolecules at the tissue and cellular levels in vivo. Therefore, leveraging the technological advantages of SERS to develop a new method for monitoring blood drug concentrations holds great promise. However, SERS's ability to detect molecules depends crucially on the stable gaps between nanoparticles. Therefore, controlling the formation of a stable detection region with a stable gap on the SERS substrate and trapping molecules within this gap is crucial for monitoring blood drug concentrations using SERS. Summary of the Invention

[0006] The purpose of the present invention is to provide a DNA nanoparticle chip for detecting methotrexate, a preparation method thereof and an application thereof.

[0007] At present, the detection of methotrexate blood concentration using SERS technology has not yet fully covered the entire concentration range of methotrexate. And the production of SERS substrates usually only includes forming stable nanoparticle gaps or increasing the substrate's ability to capture methotrexate molecules. There is a lack of a SERS substrate that can form a stable gap and capture methotrexate molecules into the gap. Based on this problem, the present invention proposes a SERS substrate with a stable detection effect constructed by an aptamer. At the same time, because the methotrexate aptamer has a certain affinity for methotrexate, it can capture methotrexate molecules into the detection area, thereby improving the SERS detection ability for methotrexate. Clinical rapid blood drug concentration monitoring is achieved through SERS. This method has the characteristics of high sensitivity, rapid detection, and low cost.

[0008] This application first pre-treats a methotrexate blood sample and constructs a DNA nanoparticle chip that stably and specifically captures methotrexate molecules. This chip is then used to measure methotrexate concentrations in the blood. This method can quantify the drug concentration in a patient's serum in just 15 minutes.

[0009] In order to solve the above technical problems, the specific technical solutions of the present invention are as follows:

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

[0011] (1) centrifuging the silver nanoparticle suspension and discarding the supernatant to obtain a 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) adding ultrapure water to a volume of 80 times the original volume of the 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 are spherical in structure and the concentration of the suspension is 6×10 8 The centrifugation conditions were 7500-8000 r / min and 10 min.

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

[0017] In the above technical solution, the amount of the 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 preparation method.

[0020] An application of the DNA nanoparticle chip in detecting the concentration of methotrexate in serum comprises:

[0021] (1) Serum pretreatment: add equal volumes of acetonitrile and dichloromethane, adjust the pH with sodium carbonate, centrifuge, collect 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 the characteristic peak of methotrexate (684 cm -1 ) and the characteristic peak of the aptamer (1322 cm -1 ) signal ratio;

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

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

[0026] In the above technical solution, the sample addition amount in step (2) is 5 μL, and the drying temperature is 45°C.

[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 treatment.

[0030] (2) This invention constructs a novel SERS substrate, namely a DNA nanoparticle chip, that can both create a stable detection region and capture drug molecules within the detection region, thereby improving the sensitivity and stability of SERS for monitoring blood drug concentrations. Furthermore, the entire process, from sampling to quantification of blood drug concentrations, takes only 15 minutes. This makes it suitable for rapid clinical monitoring of blood drug concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] Figure 2 It is the relative standard deviation of the test on 5M methotrexate molecular standard solution.

[0033] Figure 3 This is the linear relationship for testing serum spiked with different concentrations of methotrexate molecules. The horizontal axis represents the methotrexate concentration, and the vertical axis represents the ratio of the methotrexate signal to the aptamer signal.

[0034] Figure 4 is the SERS spectrum of the serum sample. DETAILED DESCRIPTION

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

[0036] Example 1:

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

[0038] 1. Pretreatment of Methotrexate Serum Samples

[0039] Patient serum was added with an equal volume of acetonitrile to precipitate proteins. The acetonitrile was extracted with dichloromethane, and the pH was adjusted with sodium carbonate. The supernatant was centrifuged at 13,000 rpm for 10 minutes and the upper aqueous phase was collected. Solid-phase extraction was performed on the upper aqueous phase to obtain the purified methotrexate sample.

[0040] 2. Construction of DNA nanoparticle chips with stable detection areas for methotrexate aptamers

[0041] (1) Take the concentration as 6×10 8 A spherical silver nanoparticle-sodium citrate solution with a volume of 100 μM silver nanoparticles / ml 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 make the volume 80 times that of the silver particles.

[0042] (2) Take 5 μl of the above solution and drop it on the silicon wafer. Place it in a 45°C environment and dry it to form a DNA nanoparticle chip on the silicon wafer.

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

[0044] (4) Place the silicon wafer with the tissue fragmentation solution to be tested in a 45°C environment to dry again.

[0045] 3. SERS detection and data output

[0046] (1) After the sample was dried, a small Raman spectrometer (NR785E10W-Plus) was used for SERS detection. The excitation wavelength of the Raman spectrometer was about 785 nm, the detection power was 30 mW, and the integration time was 500 ms 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 into the serum methotrexate quantitative curve to calculate the methotrexate concentration in the blood sample.

[0048] The present invention uses methotrexate aptamers to construct a highly efficient and sensitive SERS substrate. Furthermore, the aptamers themselves have an affinity for drug molecules and can capture them, enhancing the SERS substrate's ability to detect drug molecules. This improves the sensitivity and stability of SERS for quantifying drug concentrations in blood.

[0049] Example 2:

[0050] This example is used to demonstrate the linearity of the standard solution, the precision of the standard, the linearity of the spiked serum, and the precision of the spiked serum. The results are as follows:

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

[0052] Figure 2 This is the relative standard deviation of a 5M methotrexate molecular standard solution. RSD = 3.22%, meeting the quantitative detection accuracy requirements.

[0053] Figure 3 This is a linear relationship for detection of serum spiked with different concentrations of methotrexate. The horizontal axis represents the methotrexate concentration, and the vertical axis represents the ratio of the methotrexate signal to the aptamer signal. The aptamer maintains its specific capture ability in various matrix concentrations.

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

[0055] Example 3:

[0056] Patient Wang X, male, 62 years old, received methotrexate treatment in March 2025. A blood drug concentration test was performed 24 hours after the administration of methotrexate, which determined that the methotrexate concentration was within a safe range.

[0057] (1) To determine the blood concentration of methotrexate after administration, a 200 μl serum sample was obtained from the patient 24 h after administration;

[0058] (2) Acetonitrile, 0.1 M sodium carbonate, and dichloromethane were added to the serum to be tested in sequence and centrifuged at 13,000 rpm for 10 min to obtain the sample supernatant;

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

[0060] (4) Take the concentration as 6×10 8 A spherical silver nanoparticle solution of 1000 spherical silver nanoparticles / 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 solution was mixed to obtain a DNA nanoparticle solution;

[0061] (5) 5 μl of the above solution was dropped onto a clean silicon wafer and dried at 45°C to form a DNA nanoparticle chip;

[0062] (6) 5 μl of the extracted sample solution was dropped onto the DNA nanoparticle chip and dried at 45 °C before SERS detection.

[0063] (7) Collect the SERS spectra of the serum samples, such as Figure 4 , count and calculate methotrexate 684cm -1 The signal and the 1322 cm carried by the aptamer -1 The ratio of the signal and substitute it into the attached Figure 3 The methotrexate concentration 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, it is confirmed that the methotrexate concentration in the patient's blood is within the safe range.

[0065] Example 4:

[0066] Patient Wang X, male, 62 years old, received methotrexate treatment in March 2025. A blood drug concentration test was performed 48 hours after methotrexate administration, and it was determined that the methotrexate concentration was within a safe range.

[0067] (1) To determine the blood concentration of methotrexate after administration, a 200 μl serum sample was obtained from the patient 48 h after administration;

[0068] (2) Acetonitrile, 0.1 M sodium carbonate, and dichloromethane were added to the serum to be tested in sequence and centrifuged at 13,000 rpm for 10 min to obtain the sample supernatant;

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

[0070] (4) Take the concentration as 6×10 8 A spherical silver nanoparticle solution of 1000 spherical silver nanoparticles / 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 solution was mixed to obtain a DNA nanoparticle solution;

[0071] (5) 5 μl of the above solution was dropped onto a clean silicon wafer and dried at 45°C to form a DNA nanoparticle chip;

[0072] (6) 5 μl of the extracted sample solution was dropped onto the DNA nanoparticle chip and dried at 45 °C before SERS detection.

[0073] (7) Collect the SERS spectra of the serum samples, such as Figure 4 , count and calculate methotrexate 684cm -1 The signal and the 1322 cm carried by the aptamer -1 The ratio of the signal and substitute it into the attached Figure 3 The methotrexate concentration 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, it is confirmed that the methotrexate concentration in the patient's blood is within the safe range.

[0075] Example 5:

[0076] Patient Wang X, male, 62 years old, received methotrexate treatment in March 2025. A blood drug concentration test was performed 72 hours after methotrexate administration, which determined that the methotrexate concentration was within a safe range.

[0077] (1) To determine the blood concentration of methotrexate after administration, a 200 μl serum sample was obtained from the patient 72 h after administration;

[0078] (2) Acetonitrile, 0.1 M sodium carbonate, and dichloromethane were added to the serum to be tested in sequence and centrifuged at 13,000 rpm for 10 min to obtain the sample supernatant;

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

[0080] (4) Take the concentration as 6×10 8A spherical silver nanoparticle solution of 1000 spherical silver nanoparticles / 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 solution was mixed to obtain a DNA nanoparticle solution;

[0081] (5) 5 μl of the above solution was dropped onto a clean silicon wafer and dried at 45°C to form a DNA nanoparticle chip;

[0082] (6) 5 μl of the extracted sample solution was dropped onto the DNA nanoparticle chip and dried at 45 °C before SERS detection.

[0083] (7) Collect the SERS spectra of the serum samples, such as Figure 4 , count and calculate methotrexate 684cm -1 The signal and the 1322 cm carried by the aptamer -1 The ratio of the signal and substitute it into the attached Figure 3 The methotrexate concentration 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, it is confirmed that the methotrexate concentration in the patient's blood is within the safe range.

[0085] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present 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 the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

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

2. The preparation method according to claim 1, characterized in that The silver nanoparticles are spherical in structure and the concentration of the suspension is 6×10 8 The centrifugation conditions were 7500-8000 r / min and 10 min.

3. The preparation method according to claim 1, characterized in that The ratio of the total volume after constant volume in step (3) to the original volume of the silver nanoparticles is 80:

1.

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

5. The preparation method according to claim 1, characterized in that The concentration of the methotrexate aptamer was 100 μM.

6. A DNA nanoparticle chip prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the DNA nanoparticle chip according to claim 6 in detecting the concentration of methotrexate in serum, characterized in that: include: (1) Serum pretreatment: add equal volumes of acetonitrile and dichloromethane, adjust the pH with sodium carbonate, centrifuge, collect 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 (684 cm -1 ) and the characteristic peak of the aptamer (1322 cm -1 ) signal ratio; (4) Substitute the standard curve to calculate the concentration.

8. The use according to claim 7, characterized in that The centrifugal speed in step (1) is 13000 r / min and the time is 10 min.

9. The use according to claim 7, characterized in that In step (2), the sample addition amount is 5 μL and the drying temperature is 45°C.

10. The use according to claim 7, characterized in that The Raman spectrometer parameters in step (3) are: excitation wavelength 785 nm, power 30 mW, and integration time 500 ms.

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