Aptamer sensor for rapid detection of methotrexate, sensor preparation method and detection method

Abstract

This invention discloses an aptamer sensor for detecting methotrexate, a sensor preparation method, and a detection method. The aptamer sensor includes a magnetic carrier with streptavidin immobilized on its surface; a biotin-labeled aptamer immobilized on the magnetic carrier via biotin-streptavidin interaction; the aptamer being a DNA sequence specifically capable of binding to methotrexate; and a sucrase-labeled complementary strand that partially pairs with the aptamer. Upon binding to methotrexate, the aptamer undergoes a conformational change, releasing the sucrase-labeled complementary strand. This invention can sensitively detect methotrexate with a detection limit as low as 0.1 μmol / L, covering the critical concentration range (0.1-200 μmol / L) required for clinical therapeutic drug monitoring. Furthermore, the detection operation is convenient, does not rely on large equipment, and is low in cost.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, and in particular to a method for the immediate detection of methotrexate. Background Technology

[0002] High-dose methotrexate (HD-MTX) refers to methotrexate (MTX) administered at doses greater than 500 mg / m², and is commonly used as first-line chemotherapy for diseases such as acute lymphoblastic leukemia, non-Hodgkin's lymphoma, and osteosarcoma. However, this treatment method faces serious safety challenges in clinical application. Clinical studies have shown that due to individual metabolic differences, some patients may experience delayed drug excretion, leading to MTX accumulation in the body, which can then cause acute kidney injury, bone marrow suppression, and multiple organ failure.

[0003] To address these toxic risks, calcium folinate (CF) rescue therapy has emerged. CF can bypass dihydrofolate reductase, which is blocked by methotrexate (MTX), and restore tetrahydrofolate synthesis, thereby reducing the toxic effects of MTX on normal cells. However, the successful implementation of CF rescue therapy is highly dependent on precise monitoring of MTX blood concentrations. Clinical practice shows that the timing and dosage of CF administration must be dynamically adjusted according to MTX concentrations. For example, when the C24h concentration is >50 μmol / L, the CF dose needs to be doubled; while a C48h concentration >1 μmol / L indicates a need to prolong the rescue time. It is particularly important to note that premature initiation of CF rescue or overdose may significantly increase the risk of tumor recurrence. This stringent requirement for treatment precision makes therapeutic drug monitoring (TDM) a crucial component of HD-MTX treatment regimens.

[0004] However, in reality, due to insufficient clinical implementation of methotrexate blood concentration monitoring, many primary healthcare institutions are unable to standardize HD-MTX treatment. Lacking TDM (Therapeutic Drug Management) technology support, primary care physicians are forced to adopt alternative management strategies, including extending hydration time, increasing leucovorin dosage, and prolonging hospital stays. Simultaneously, they intensify serum creatinine monitoring to reduce the risk of serious adverse reactions. However, these alternative measures may not only affect treatment efficacy but also increase the medical burden on patients.

[0005] The survey results show that the main reason for the insufficient clinical adoption of MTX TDM is the limitation of existing detection technologies. Currently, commonly used clinical methods for MTX detection include immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS / MS). While LC-MS / MS is considered the gold standard due to its high sensitivity and accuracy, its equipment cost exceeds 2 million yuan and requires operation by specialized technicians. Immunoassay is also expensive (385 yuan per test), and its sensitivity (0.1 μmol / L) and specificity are limited, making it susceptible to interference from metabolites. These technological limitations hinder the widespread adoption of MTX TDM in resource-constrained medical institutions. Summary of the Invention

[0006] In view of this, the present invention provides a method for real-time detection of methotrexate based on nucleic acid aptamer-blood glucose meter signal transduction, so as to solve the technical problem of making methotrexate blood drug concentration monitoring simple to operate, highly sensitive, and not dependent on large instruments.

[0007] This invention relates to an aptamer sensor for detecting methotrexate, comprising:

[0008] A magnetic carrier with streptavidin immobilized on its surface;

[0009] Biotin-labeled aptamers, which are immobilized on the magnetic carrier via biotin-streptavidin interactions, are DNA sequences capable of specifically binding to methotrexate.

[0010] The complementary strand labeled with sucrase binds to the aptamer portion in a complementary pairing.

[0011] The aptamer undergoes a conformational change upon binding with methotrexate, releasing the sucrase-labeled complementary strand.

[0012] Furthermore, the aptamer is a DNA sequence as shown in SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0013] Furthermore, the magnetic carrier is a superparamagnetic nanoparticle.

[0014] Furthermore, the superparamagnetic nanoparticles have a core-shell structure, with the core being Fe3O4 nanoparticles and the shell containing silicon dioxide.

[0015] This invention also discloses a method for preparing the above-mentioned aptamer sensor, comprising: coating the surface of magnetic nanoparticles with a silica shell to obtain core-shell magnetic nanoparticles; amylating the core-shell magnetic nanoparticles with 3-aminopropyltriethoxysilane to obtain aminated magnetic nanoparticles; aldehyde-modifying the aminated magnetic nanoparticles with glutaraldehyde to obtain aldehyde-modified magnetic nanoparticles; reacting streptavidin with the aldehyde groups on the surface of the magnetic nanoparticles to obtain a streptavidin-modified magnetic carrier; immobilizing a biotin-labeled aptamer on the magnetic carrier through biotin-streptavidin interaction; and binding a sucrase-labeled complementary chain to the aptamer through complementary pairing to obtain an aptamer sensor.

[0016] Furthermore, when the biotin-labeled aptamer was immobilized on the magnetic support via biotin-streptavidin interaction, the saturated adsorption capacity of the aptamer on the magnetic support was 0.4 nmol / mg.

[0017] The complementary strand labeled with sucrase was bound to the aptamer through complementary pairing. The molar ratio of the aptamer to the complementary strand labeled with sucrase was 1:5, and the reaction time was 20 min.

[0018] This invention also discloses a method for detecting methotrexate, comprising:

[0019] The sample to be tested is mixed with the aptamer sensor solution and incubated to allow methotrexate to bind to the aptamer and release the sucrase-labeled complementary strand.

[0020] An external magnetic field is applied to perform solid-liquid separation, and a supernatant containing a free sucrase-labeled complementary chain is obtained;

[0021] The supernatant was mixed with a sucrose-containing reaction solution to carry out an enzymatic reaction to generate glucose, and then the glucose concentration was detected.

[0022] The concentration of methotrexate in the sample was calculated based on the quantitative relationship between glucose concentration and methotrexate concentration.

[0023] Furthermore, the concentration of the aptamer sensor solution is 1 mg / mL, and the volume is 60 µL; the incubation time is 60 minutes; the enzymatic reaction conditions are 45 minutes at 37°C, and the sucrose concentration of the sucrose-containing reaction solution is 2 mol / L.

[0024] Furthermore, when the methotrexate concentration is in the range of 0.1-5 μmol / L, the quantitative relationship between the glucose concentration and the methotrexate concentration is as follows:

[0025] Y = 0.9347X + 3.1821

[0026] Where Y is the glucose concentration in mmol / L; X is the methotrexate concentration in μmol / L.

[0027] When the methotrexate concentration is in the range of 5-200 μmol / L, the quantitative relationship between the glucose concentration and the methotrexate concentration is as follows:

[0028] Y = 2.1939 * lg X + 6.348

[0029] Where Y is the glucose concentration in mmol / L and X is the methotrexate concentration in μmol / L.

[0030] Furthermore, a blood glucose meter was used to detect the concentration of the glucose.

[0031] The beneficial effects of this invention are:

[0032] 1. Achieved high sensitivity and wide linear range detection: The technical solution of this invention can sensitively detect methotrexate (MTX) with a detection limit of 0.1 μmol / L, covering the key concentration range (0.1-200 μmol / L) required for therapeutic drug monitoring (TDM). Within the two concentration ranges of 0.1-5 μmol / L and 5-200 μmol / L, the detection signal (glucose concentration) and MTX concentration (or its logarithm) show a good linear relationship, ensuring the accuracy of quantitative analysis.

[0033] 2. Significantly improves the convenience and accessibility of detection: This invention transforms the complex MTX concentration detection into a routine glucose concentration reading. By specifically recognizing MTX through an aptamer and triggering the release of sucrase, which in turn catalyzes the conversion of sucrose into glucose, the glucose concentration is ultimately read using a widely available and easy-to-use blood glucose meter, thus allowing for the inference of MTX concentration. This eliminates the reliance on large, expensive specialized equipment (such as liquid chromatography-tandem mass spectrometry), solving the problems of high equipment costs, highly specialized operation, and difficulty in widespread adoption in primary healthcare institutions mentioned in the background section.

[0034] 3. Relatively low testing cost: The existing gold standard method, LC-MS / MS equipment, costs over 2 million yuan, and immunoassay testing is also expensive (385 yuan / test). This solution, however, uses an aptamer sensor combined with a standard blood glucose meter, with its main consumable being a self-made sensor. This is expected to significantly reduce the cost per test, making it more suitable for clinical promotion and widespread adoption.

[0035] 4. Standardized operating procedures and reasonable detection time: This invention provides optimized operating parameters, such as the amount of aptamer sensor used (60 μL, 1 mg / mL), the incubation time with MTX (60 minutes), and the reaction temperature and time with sucrose (37°C, 45 minutes). The entire detection process is clear and requires no complicated operations. The total time from sample processing to obtaining results is controlled within a few hours, making it suitable for point-of-care testing (POCT) scenarios.

[0036] 5. Possesses good specificity: This is demonstrated through specificity experiments (…). Figure 22 The results show that the aptamer sensor prepared in this invention has high selectivity for the target drug methotrexate (MTX), while the recognition signal of possible interfering substances such as folic acid, penicillin, vancomycin, omeprazole, and bovine serum albumin is very low, which effectively avoids the interference of other components in complex biological samples and ensures the reliability of the detection results.

[0037] 6. It has good stability: passing stability tests ( Figure 23 The results show that the aptamer sensor prepared by this invention did not undergo significant performance changes after being stored at 4°C for 14 days, indicating that it has good storage stability, ensuring the reliability of the detection results and meeting the need for flexible selection of sensor usage time in practical applications.

[0038] 7. Maintains good performance in complex biological samples: This was demonstrated through real-matrix application experiments ( Figure 24 and Figure 25 The results show that the aptamer sensor prepared in this invention exhibits a good linear relationship between blood glucose meter readings and MTX concentrations in 20% plasma within a concentration range of 0.1–5 μmol / L; and a good linear relationship between the logarithm of blood glucose meter readings and MTX concentrations within a concentration range of 5–200 μmol / L, with a detection limit of 0.1 μmol / L. This indicates that the sensor maintains good detection performance in complex biological samples, providing reliable technical support for clinical drug monitoring. Attached Figure Description

[0039] Figure 1 The synthesis process of streptavidin-modified magnetic carriers;

[0040] Figure 2 This describes the process of preparing an aptamer sensor from a magnetic carrier modified with streptavidin.

[0041] Figure 3 Electron microscopy images of Fe3O4, Fe3O4@SiO2-APTES, and SA-MBs nanoparticles.

[0042] Figure 4 Infrared spectral analysis diagrams of different materials.

[0043] Figure 5 X-ray diffraction characterization of different materials.

[0044] Figure 6 VSM plots of Fe3O4 (a) and SA-MBs (b).

[0045] Figure 7 This is the concentration-absorbance standard curve for streptavidin.

[0046] Figure 8 The UV absorption spectra of streptavidin solutions before (a) and after (b) the reaction are shown.

[0047] Figure 9 To detect the linear relationship of MTX in aptamers.

[0048] Figure 10 The variation of supernatant fluorescence intensity with aptamer concentration.

[0049] Figure 11 The change in supernatant fluorescence intensity over time.

[0050] Figure 12 To investigate the ratio of aptamer to complementary chain.

[0051] Figure 13 The binding time between the aptamer and the complementary chain was investigated.

[0052] Figure 14 This is a schematic diagram of the MTX detection principle for aptamer sensors.

[0053] Figure 15 To investigate the incubation time of aptamer sensors with methotrexate.

[0054] Figure 16 To investigate the reaction temperature between the aptamer sensor and sucrose.

[0055] Figure 17 To investigate the reaction time between the aptamer sensor and sucrose.

[0056] Figure 18 To investigate the reaction of sucrose solutions with aptamer sensors.

[0057] Figure 19 To investigate the amount of aptamer sensor used.

[0058] Figure 20 Linear relationship between different concentrations of MTX and blood glucose meter readings in buffer solution (0.1-5 μmol / L).

[0059] Figure 21 Linear relationship between different concentrations of MTX and blood glucose meter readings in buffer solution (5-200 μmol / L).

[0060] Figure 22 The results show the specificity of the aptamer sensor.

[0061] Figure 23 The results are from the stability experiment of the aptamer sensor.

[0062] Figure 24 Linear relationship between different concentrations of MTX and blood glucose meter readings in 20% plasma (0.1-5 μmol / L).

[0063] Figure 25 Linear relationship between different concentrations of MTX and blood glucose meter readings in 20% plasma (5-200 μmol / L). Detailed Implementation

[0064] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0065] In this embodiment, the aptamer sensor used for detecting methotrexate includes:

[0066] Magnetic carriers (MBs) with streptavidin immobilized on their surface;

[0067] Biotin-labeled aptamers, which are immobilized on the magnetic carrier via biotin-streptavidin interactions, are DNA sequences capable of specifically binding to methotrexate.

[0068] The complementary strand labeled with sucrase binds to the aptamer portion in a complementary pairing.

[0069] The aptamer undergoes a conformational change upon binding with methotrexate, releasing the sucrase-labeled complementary strand.

[0070] In this embodiment, the aptamer is a DNA sequence as shown in SEQ ID NO: 1, specifically: 5′-CTCTCGGGCGAACGCGGGATGTTTGGGGGACCCACGTTCGCCC-3′.

[0071] The magnetic carrier is a superparamagnetic nanoparticle, which has a core-shell structure. The core of the core-shell structure is an Fe3O4 nanoparticle, and the shell of the core-shell structure contains silicon dioxide.

[0072] The method for preparing the aptamer sensor includes: coating the surface of magnetic nanoparticles with a silica shell to obtain core-shell magnetic nanoparticles; modifying the core-shell magnetic nanoparticles with 3-aminopropyltriethoxysilane to obtain aminated magnetic nanoparticles; modifying the aminated magnetic nanoparticles with glutaraldehyde to obtain aldehyde-modified magnetic nanoparticles; reacting streptavidin with the aldehyde groups on the surface of the magnetic nanoparticles to obtain a streptavidin-modified magnetic carrier; immobilizing a biotin-labeled aptamer on the magnetic carrier through biotin-streptavidin interaction; and binding a sucrase-labeled complementary chain to the aptamer through complementary pairing to obtain the aptamer sensor. Figure 1 The synthesis process of streptavidin-modified magnetic carriers was demonstrated. Figure 2 Further demonstration Figure 2 The fabrication process of aptamer sensors.

[0073] As an improvement to the above embodiment, when the biotin-labeled aptamer is immobilized on the magnetic support via biotin-streptavidin interaction, the saturated adsorption capacity of the aptamer on the magnetic support is 0.4 nmol / mg. The sucrase-labeled complementary strand is then bound to the aptamer via complementary pairing, with a molar ratio of aptamer to sucrase-labeled complementary strand of 1:5 and a reaction time of 20 min.

[0074] Characterization of magnetic carrier materials:

[0075] The morphological characteristics of Fe3O4, Fe3O4@SiO2-APTES, and SA-MBs nanoparticles obtained during the preparation of aptamer sensors were observed by electron microscopy, such as... Figure 3 The electron micrographs of Fe3O4, Fe3O4@SiO2-APTES, and SA-MBs nanoparticles shown are provided. The Fe3O4 nanoparticles are monodisperse spherical particles with highly smooth surfaces and relatively uniform particle size, with a diameter of approximately 300 nm. Figure 3 (a and b in Figure 3). After modification with SiO2 and APTES, TEM observation clearly shows that the particles formed a distinct core-shell structure with a shell thickness of about 28 nm, indicating that SiO2 has successfully coated the Fe3O4 nanoparticles (c, d, and e in Figure 3). After modification with streptavidin, SEM shows that SA-MBs are relatively uniform in size and particle size. Figure 3 (f)

[0076] Infrared spectroscopy analysis of magnetic carriers:

[0077] The infrared characterization of the streptavidin-modified magnetic support is shown in Figure 4. (584 cm⁻¹) −1The peak values ​​at 799, 949, and 1081 cm⁻¹ are attributed to the stretching vibrations of Fe-O. −1 Characteristic absorption bands of the silica shell were observed at 1620 cm⁻¹, which are related to the tensile vibrations of Si-O, Si-OH, and Si-O-Si, respectively. −1 The left and right peaks are at 3416 cm. −1 The broad peaks nearby are due to the stretching and bending vibrations of N−H. Comparing with Figures (a) and (b), it is clear that Si−OH grafted with APTES reaches a peak at 949 cm⁻¹. −1 The intensity corresponding to the left and right vibration bands decreased to some extent, which is also evidence of the success of APTES grafting. At 2850 cm... −1 and 1709 cm −1 The peak values ​​at 1639 cm⁻¹ are due to the stretching vibrations of CH and C=O, respectively. −1 and 1539 cm −1 Characteristic bands of streptavidin (SA) were also observed. This indicates that streptavidin was successfully bonded to the surface of the magnetic material. This demonstrates the successful synthesis of the streptavidin-modified magnetic support.

[0078] X-ray diffraction characterization of magnetic carriers:

[0079] The X-ray diffraction characterization spectra of Fe3O4 (a), Fe3O4@SiO2-APTES-GA (b), and SA-MBs (c) are shown below. Figure 5 As shown. Within the range of 2θ = 10°–80°, the XRD patterns of Fe3O4 nanoparticles were observed. Six characteristic diffraction peaks of Fe3O4 nanoparticles were found at 2θ = 30.20°, 35.62°, 43.28°, 53.38°, 57.17°, and 62.79°, which were attributed to the (220), (311), (400), (422), (511), and (440) crystal planes of the Fe3O4 nanoparticles, respectively. This indicates that the above nanoparticles have good crystallinity.

[0080] Characterization of the magnetic properties of magnetic carriers:

[0081] The magnetic properties of Fe3O4 nanoparticles and SA-MBs were characterized using a vibrating sample magnetometer. The magnetization curves are shown below. Figure 6 As shown. When the external magnetic field is removed, the remanence of Fe3O4(a) and SA-MBs(b) is zero, which means that these particles possess superparamagnetism. The saturation magnetization of Fe3O4(a) and SA-MBs(b) is 72.2 emu g, respectively. −1 and 41.2 emu g −1This indicates that it has a high magnetic property, which can meet the magnetic separation requirements of subsequent experiments.

[0082] Streptavidin immobilization capacity of magnetic carriers:

[0083] Prepare streptavidin standard solutions of different concentrations, measure their absorbance at 562 nm using an ELISA reader, and plot a concentration-absorbance standard curve, as follows. Figure 7 As shown.

[0084] Aldehyde-modified magnetic nanoparticles were added to a streptavidin solution and incubated at room temperature. The absorbance of the streptavidin solution at 562 nm before and after the addition of the aldehyde-modified magnetic nanoparticles was measured using a microplate reader. The UV absorption spectra of the solutions before (a) and after (b) are shown below. Figure 8 As shown in the figure, the intensity of the UV absorption peak at 562 nm in the solution decreased after the reaction, indicating a decrease in the concentration of free streptavidin in the solution, meaning that some streptavidin was introduced onto the surface of the magnetic nanoparticles. Calculations based on the formula show that the amount of streptavidin fixed on the surface of the magnetic nanoparticles is 13.5 μg / mg.

[0085] Aptamers:

[0086] The K-type methotrexate aptamer described in this embodiment D The sequence ≈ 165 nM represents a high-affinity sequence, and the aptamer undergoes a significant conformational change upon binding to methotrexate (MTX), a characteristic that makes it suitable for constructing structure-transformation-based biosensors. Figure 9 As shown, the aptamer specifically binds to methotrexate and releases its complementary strand. Based on the inserted standard curve and its fitting equation Y = 0.1585x + 0.0113, where Y represents the fluorescence response signal ((F-F0) / F0) and x represents the MTX concentration (μmol / L), the high correlation coefficient (R² = 0.9909) indicates that the linear model has an extremely high fit and the data is reliable. This demonstrates that the selected aptamer meets the core requirements for a biosensor recognition element. Furthermore, it exhibits high sensitivity, responding to minute changes in MTX concentration; its sensitivity is proportional to concentration, allowing for accurate calculation of MTX content in unknown samples.

[0087] Investigation of the amount of aptamers immobilized on magnetic carriers:

[0088] Aptamer sequences labeled with biotin at the 3' end and 5' end with 5'6-FAM fluorescent dye were immobilized on SA-MBs via a biotin-streptavidin reaction. Figure 10When the labeled aptamer concentration was below 0.015 µM, the fluorescence intensity in the supernatant was low, indicating that the target aptamer was not saturated on SA-MBs. When the labeled aptamer concentration reached 0.02 µM, the fluorescence intensity in the supernatant increased sharply, indicating that the number of target aptamers captured on SA-MBs had reached saturation. Therefore, the saturation adsorption capacity of the aptamer on SA-MBs was 0.4 nmol / mg.

[0089] Selection of the fixation time of the aptamer on the magnetic carrier:

[0090] like Figure 11 As shown, when the aptamer sequence labeled with 5'6-FAM fluorescent pigment was added to the SA-MBs solution, the fluorescence intensity of the SA-MBs supernatant did not decrease significantly after 40 min of reaction. It can be concluded that the reaction was completed within 40 min. Therefore, the fixation time of the aptamer on the magnetic support was selected to be 40 min.

[0091] Selection of the ratio of sucrase-labeled complementary strand and aptamer reaction:

[0092] The ratios of complementary strands of aptamers and sucrase-labeled sucrase were investigated at 1:1, 1:3, 1:5, and 1:7. Figure 12 As shown, when the ratio is 1:5, the blood glucose meter signal value no longer increases. Therefore, the ratio of aptamer and sucrase-labeled complementary strand should be 1:5.

[0093] Selection of sucrase-labeled complementary strand and aptamer reaction time:

[0094] The binding time between the aptamer and the complementary strand labeled with sucrase was investigated. For example... Figure 13 As shown, the ratio of aptamer to sucrase-labeled complementary strand is 1:5. After 20 minutes of reaction, the blood glucose meter reading no longer increases. It can be concluded that the reaction is completed within 20 minutes. Therefore, the reaction time of sucrase-labeled complementary strand and aptamer is selected as 20 minutes.

[0095] The method for detecting methotrexate in this embodiment includes:

[0096] The sample to be tested is mixed with the aptamer sensor solution and incubated to allow methotrexate to bind to the aptamer and release a sucrase-labeled complementary strand. An external magnetic field is applied to perform solid-liquid separation, obtaining a supernatant containing the free sucrase-labeled complementary strand. This supernatant is mixed with a sucrose-containing reaction solution for an enzymatic reaction to generate glucose, and the glucose concentration is then measured using a blood glucose meter. Based on the quantitative relationship between glucose and methotrexate concentrations, the methotrexate concentration in the sample is calculated.

[0097] The concentration of the aptamer sensor solution is 1 mg / mL, and the volume is 60 µL; the incubation time is 60 minutes; the enzymatic reaction conditions are 45 minutes at 37°C, and the sucrose concentration of the sucrose-containing reaction solution is 2 mol / L.

[0098] When the methotrexate concentration is in the range of 0.1-5 μmol / L, the quantitative relationship between the glucose concentration and the methotrexate concentration is as follows:

[0099] Y = 0.9347X + 3.1821

[0100] Where Y is the glucose concentration in mmol / L; X is the methotrexate concentration in μmol / L.

[0101] When the methotrexate concentration is in the range of 5-200 μmol / L, the quantitative relationship between the glucose concentration and the methotrexate concentration is as follows:

[0102] Y = 2.1939 * lg X + 6.348

[0103] Where Y is the glucose concentration in mmol / L and X is the methotrexate concentration in μmol / L.

[0104] Selection of incubation time with the target methotrexate (MTX) during the detection process:

[0105] The aptamer sensor described above was reacted with MTX solutions containing 50 μmol / L and 100 μmol / L, and magnetic separation was performed after incubation for 0 min, 5 min, 15 min, 30 min, 60 min, and 100 min, respectively. Figure 15 As shown, the blood glucose meter signal value hardly increased after 1 h of incubation between the aptamer sensor and the target methotrexate (MTX). Therefore, the incubation time between the aptamer sensor and the target methotrexate (MTX) was selected.

[0106] During the testing process, the choice of reaction temperature with sucrose is as follows:

[0107] The aptamer sensor described above was reacted with solutions containing 50 μmol / L and 100 μmol / L MTX. After 1 h, magnetic separation was performed using a magnetic separator. 10 μL of the supernatant was then added to a 3.3 μL 2 mol / L Sucrose centrifuge tube, vortexed, and reacted at 0℃, 25℃, and 37℃ respectively. 5 μL of the reaction solution was then measured using a blood glucose meter. Figure 16As shown, the reaction signal value between the aptamer sensor and sucrase is highest at 37℃, therefore, the reaction temperature between the aptamer sensor and sucrose is selected as 37℃.

[0108] During the testing process, the choice of reaction time with sucrose:

[0109] The aptamer sensor described above was reacted with solutions containing 50 μmol / L and 100 μmol / L MTX. After 1 h, magnetic separation was performed using a magnetic separator. 10 μL of the supernatant was then added to a 3.3 μL 2 mol / L Sucrose centrifuge tube, vortexed, and incubated at 37°C. At 0 min, 5 min, 15 min, 30 min, 45 min, and 60 min, 5 μL of the reaction solution was measured using a blood glucose meter. Figure 17 As shown, the blood glucose meter signal gradually increases over time. To meet the detection requirements, the reaction time between the aptamer sensor and sucrose is set to 45 minutes.

[0110] Selection of the concentration of the sucrose solution used for testing:

[0111] The sucrose concentration, which is used by sucrase to catalyze the production of glucose, also plays a role in the final signal of the blood glucose meter. Therefore, examining the sucrose concentration is important. Figure 18 As shown, the blood glucose meter signal value was highest when using a 2 mol / L sucrose solution, so this condition was chosen for the experiment.

[0112] Selection of the amount of aptamer sensor used for detection:

[0113] The effect of different amounts of aptamer sensor solution on the signal value of the blood glucose meter was investigated. Figure 19 As shown, the signal value remained stable after using 60 µL of 1 mg / ml aptamer sensor solution, therefore 60 µL of aptamer sensor solution (1 mg / ml) was selected.

[0114] Aptamer sensor detection sensitivity evaluation:

[0115] 20 μL of MTX solutions of different concentrations were added to one portion of aptamer sensor solution (liquid was removed using an external magnetic field), gently vortexed to mix, and allowed to react for 1 h. The MBs solution after the reaction was separated using an external magnetic field, and 10 μL of the supernatant was added to a 3.3 μL centrifuge tube containing 2 mol / L sucrose. The mixture was vortexed to mix, and reacted at 37℃ for 45 min. 5 μL of the reaction solution was then used for blood glucose testing. The results are as follows: Figure 20 and Figure 21As shown, there is a good linear relationship between the blood glucose meter reading and the MTX concentration in the range of 0.1–5 μmol / L; and a good linear relationship between the blood glucose meter reading and the logarithm of the MTX concentration in the range of 5–200 μmol / L. The detection limit of MTX is 0.1 μmol / L.

[0116] Aptamer sensor detection sensitivity evaluation:

[0117] To investigate the specificity of the aptamer sensor, folic acid, penicillin, vancomycin, omeprazole, and bovine serum albumin were selected as interfering factors. Error bars were obtained through three parallel tests. The detection procedure was the same as that for MTX described above. The results are as follows. Figure 22 As shown in the figure, experiments indicate that the aptamer sensor has low specificity for other non-target compounds but good selectivity for the target drug MTX.

[0118] Stability assessment of aptamer sensors:

[0119] To meet the requirement of flexible timing when using the sensor, the aptamer sensor should maintain stability for a certain period during storage. A stability test was performed on the aptamer sensor, following the same testing steps as the MTX test described above. The results are as follows... Figure 23 As shown in the figure, the experiment indicates that the sensor's performance did not change significantly after being stored at 4°C for 14 days.

[0120] Applications of aptamer sensors in real matrices:

[0121] The application of the aptamer sensor in a real matrix was investigated. MTX samples of different concentrations were prepared in 20% plasma, and the detection procedure was the same as that for MTX detection described above. Results are as follows: Figure 24 and Figure 25 As shown, a good linear relationship exists between the blood glucose meter reading and the MTX concentration in the range of 0.1–5 μmol / L; and a good linear relationship exists between the blood glucose meter reading and the logarithm of the MTX concentration in the range of 5–200 μmol / L. The detection limit of MTX is 0.1 μmol / L.

[0122] In summary, this embodiment provides a method for real-time detection of methotrexate blood concentration that is highly sensitive, specific, easy to operate, low in cost, and does not rely on large instruments. It offers an effective solution to the clinical medication safety and efficacy challenges caused by insufficient therapeutic drug monitoring (TDM) in current high-dose methotrexate chemotherapy, and is particularly suitable for application in resource-limited primary healthcare institutions.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An aptamer sensor for detecting methotrexate, characterized in that, include: A magnetic carrier with streptavidin immobilized on its surface; Biotin-labeled aptamers, which are immobilized on the magnetic carrier via biotin-streptavidin interactions, are DNA sequences capable of specifically binding to methotrexate. The complementary strand labeled with sucrase binds to the aptamer portion in a complementary pairing. The aptamer undergoes a conformational change upon binding with methotrexate, releasing the sucrase-labeled complementary strand.

2. The aptamer sensor according to claim 1, characterized in that, The aptamer is a DNA sequence as shown in SEQ ID NO: 1 or a functionally equivalent variant thereof.

3. The aptamer sensor according to claim 1, characterized in that, The magnetic carrier is a superparamagnetic nanoparticle.

4. The aptamer sensor according to claim 3, characterized in that, The superparamagnetic nanoparticles have a core-shell structure, with the core being Fe3O4 nanoparticles and the shell containing silicon dioxide.

5. A method for preparing an aptamer sensor according to any one of claims 1-4, characterized in that, include: A silica shell is coated on the surface of magnetic nanoparticles to obtain core-shell structured magnetic nanoparticles. The core-shell magnetic nanoparticles were aminated using 3-aminopropyltriethoxysilane to obtain aminated magnetic nanoparticles; the aminated magnetic nanoparticles were then aldehyde-modified using glutaraldehyde to obtain aldehyde-modified magnetic nanoparticles; streptavidin was reacted with the aldehyde groups on the surface of the magnetic nanoparticles to obtain a streptavidin-modified magnetic carrier; a biotin-labeled aptamer was immobilized on the magnetic carrier through biotin-streptavidin interaction; and a sucrase-labeled complementary chain was bound to the aptamer through complementary pairing to obtain an aptamer sensor.

6. The method for preparing the aptamer sensor according to claim 1, characterized in that: When the biotin-labeled aptamer was immobilized on the magnetic support via biotin-streptavidin interaction, the saturated adsorption capacity of the aptamer on the magnetic support was 0.4 nmol / mg. The complementary strand labeled with sucrase was bound to the aptamer through complementary pairing. The molar ratio of the aptamer to the complementary strand labeled with sucrase was 1:5, and the reaction time was 20 min.

7. A method for detecting methotrexate, characterized in that, include: The sample to be tested is mixed with the aptamer sensor solution and incubated to allow methotrexate to bind to the aptamer and release the sucrase-labeled complementary strand. An external magnetic field is applied to perform solid-liquid separation, and a supernatant containing a free sucrase-labeled complementary chain is obtained; The supernatant was mixed with a sucrose-containing reaction solution to carry out an enzymatic reaction to generate glucose, and then the glucose concentration was detected. The concentration of methotrexate in the sample was calculated based on the quantitative relationship between glucose concentration and methotrexate concentration.

8. The detection method according to claim 7, characterized in that, The concentration of the aptamer sensor solution is 1 mg / mL, and the volume is 60 µL; the incubation time is 60 minutes; the enzymatic reaction conditions are 45 minutes at 37°C, and the sucrose concentration of the sucrose-containing reaction solution is 2 mol / L.

9. The method for detecting methotrexate according to claim 8, characterized in that: When the methotrexate concentration is in the range of 0.1-5 μmol / L, the quantitative relationship between the glucose concentration and the methotrexate concentration is as follows: Y = 0.9347X + 3.1821 Where Y is the glucose concentration in mmol / L; X is the methotrexate concentration in μmol / L. When the methotrexate concentration is in the range of 5-200 μmol / L, the quantitative relationship between the glucose concentration and the methotrexate concentration is as follows: Y = 2.1939 * lg X + 6.348 Where Y is the glucose concentration in mmol / L and X is the methotrexate concentration in μmol / L.

10. The method for detecting methotrexate according to claim 7, characterized in that: The concentration of glucose was measured using a blood glucose meter.

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